register interest

Professor Krina T Zondervan

Research Area: Genetics and Genomics
Technology Exchange: Bioinformatics, Medical statistics, SNP typing, Statistical genetics and Transcript profiling
Scientific Themes: Genetics & Genomics and Clinical Trials & Epidemiology
Keywords: Endometriosis, Epidemiology, Infertility, Women's Health, Genetics and Pelvic pain
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My research group works on understanding the pathogenesis of women's health conditions - in particular endometriosis and related symptoms - through genomic, molecular, and environmental epidemiological research methods. We are based both at the Wellcome Trust Centre for Human Genetics (WTCHG) and the Nuffield Dept of Obstetrics & Gynaecology (NDOG), where I am co-Director of the Endometriosis CaRe centre.

Endometriosis is a common, poorly understood chronic inflammatory condition in women of reproductive age, involving the presence of endometrial-like cells in extra-uterine pelvic sites. The condition causes pain and sub-fertility; is estimated to affect 5-10% of women in their reproductive years (176 million women worldwide and 1.5 million in the UK); has limited diagnostic and treatment options; and has a major impact on health-related quality of life of sufferers and their families.

Heritable factors are involved in endometriosis risk. Identifying which DNA variants underlie this heritability will improve our understanding of pathogenesis and inform treatment development. With the International Endogene Consortium, we have discovered 7 of the now 9 known genetic risk variants for endometriosis, in genome-wide association studies involving >5,500 cases and >10,000 controls. Current work includes further extensions of our genetic discovery work, and understanding the functionality of the loci and their relevance to drug target and biomarker discovery through integrated analyses of rich phenotypic data and biological samples collected in our ENDOX study. I am Principal Investigator of WERF EPHect (Endometriosis Phenome and Biobanking Harmonisation Project), bringing together 30+ academic and 3 industrial partners to develop a global concensus on phenotypic and epidemiological data collection as well as standard operating protocols for collection, processing and long-term storage of biological samples for research into endometriosis (www.endometriosisfoundation.org/ephect).

Funding: The Wellcome Trust; MRC; NIH; World Endometriosis Research Foundation; European Public Health Programme.

Name Department Institution Country
Professor Grant Montgomery Dept of Molecular Epidemiology Queensland Institute for Medical Research, Brisbane Australia
Dr Stacey Missmer Harvard Medical School Harvard University United States
Professor Christian Becker Nuffield Dept of Obstetrics & Gynaecology University of Oxford United Kingdom
Professor Andrew P Morris Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Cecilia Lindgren Big Data Institute Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr Jeffrey Rogers Dept of Molecular and Human Genetics, Human Genome Sequencing Center Baylor College of Medicine, Houston United States
Professor Joseph Kemnitz Dept. of Cell & Regenerative Biology WNPRC, University of Wisconsin-Madison United States
Prof Stephen Kennedy (MPLS) Oxford University,
Professor Mark McCarthy Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Professor Tim Spector Department of Twin Research and Genetic Epidemiology Kings College, London United Kingdom
Professor Chris Holmes Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Peter Donnelly FRS Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Thomas D'Hooghe Centre for Reproductive Medicine University of Leuven Belgium
Professor Peter Rogers Department of Obstetrics and Gynaecology University of Melbourne Australia
Professor Paolo Vercellini Clinica Ostetrica e Ginecologica I University of Milano School of Medicine Italy
Professor Andres Salumets Dept of Obstetrics & Gynaecology University of Tartu Estonia
Saare M, Rekker K, Laisk-Podar T, Rahmioglu N, Zondervan K, Salumets A, Götte M, Peters M. 2017. Challenges in endometriosis miRNA studies - From tissue heterogeneity to disease specific miRNAs. Biochim Biophys Acta, 1863 (9), pp. 2282-2292. | Show Abstract | Read more

In order to uncover miRNA changes in endometriosis pathogenesis, both endometriotic lesions and endometrial biopsies, as well as stromal and epithelial cells isolated from these tissues have been investigated and a large number of dysregulated miRNAs have been reported. However, the concordance between the result of different studies has remained small. One potential explanation for limited overlap between the proposed disease-related miRNAs could be the heterogeneity in tissue composition, as some studies have compared highly heterogeneous whole-lesion biopsies with endometrial tissue, some have compared the endometrium from patients and controls, and some have used pure cell fractions isolated from lesions and endometrium. This review focuses on the results of published miRNA studies in endometriosis to reveal the potential impact of tissue heterogeneity on the discovery of disease-specific miRNA alterations in endometriosis. Additionally, functional studies that explore the roles of endometriosis-involved miRNAs are discussed.

Kukushkina V, Modhukur V, Suhorutšenko M, Peters M, Mägi R, Rahmioglu N, Velthut-Meikas A, Altmäe S, Esteban FJ, Vilo J et al. 2017. DNA methylation changes in endometrium and correlation with gene expression during the transition from pre-receptive to receptive phase. Sci Rep, 7 (1), pp. 3916. | Show Abstract | Read more

The inner uterine lining (endometrium) is a unique tissue going through remarkable changes each menstrual cycle. Endometrium has its characteristic DNA methylation profile, although not much is known about the endometrial methylome changes throughout the menstrual cycle. The impact of methylome changes on gene expression and thereby on the function of the tissue, including establishing receptivity to implanting embryo, is also unclear. Therefore, this study used genome-wide technologies to characterize the methylome and the correlation between DNA methylation and gene expression in endometrial biopsies collected from 17 healthy fertile-aged women from pre-receptive and receptive phase within one menstrual cycle. Our study showed that the overall methylome remains relatively stable during this stage of the menstrual cycle, with small-scale changes affecting 5% of the studied CpG sites (22,272 out of studied 437,022 CpGs, FDR < 0.05). Of differentially methylated CpG sites with the largest absolute changes in methylation level, approximately 30% correlated with gene expression measured by RNA sequencing, with negative correlations being more common in 5' UTR and positive correlations in the gene 'Body' region. According to our results, extracellular matrix organization and immune response are the pathways most affected by methylation changes during the transition from pre-receptive to receptive phase.

Sapkota Y, Steinthorsdottir V, Morris AP, Fassbender A, Rahmioglu N, De Vivo I, Buring JE, Zhang F, Edwards TL, Jones S et al. 2017. Meta-analysis identifies five novel loci associated with endometriosis highlighting key genes involved in hormone metabolism. Nat Commun, 8 pp. 15539. | Show Abstract | Read more

Endometriosis is a heritable hormone-dependent gynecological disorder, associated with severe pelvic pain and reduced fertility; however, its molecular mechanisms remain largely unknown. Here we perform a meta-analysis of 11 genome-wide association case-control data sets, totalling 17,045 endometriosis cases and 191,596 controls. In addition to replicating previously reported loci, we identify five novel loci significantly associated with endometriosis risk (P<5 × 10(-8)), implicating genes involved in sex steroid hormone pathways (FN1, CCDC170, ESR1, SYNE1 and FSHB). Conditional analysis identified five secondary association signals, including two at the ESR1 locus, resulting in 19 independent single nucleotide polymorphisms (SNPs) robustly associated with endometriosis, which together explain up to 5.19% of variance in endometriosis. These results highlight novel variants in or near specific genes with important roles in sex steroid hormone signalling and function, and offer unique opportunities for more targeted functional research efforts.

Gemmell LC, Webster KE, Kirtley S, Vincent K, Zondervan KT, Becker CM. 2017. The management of menopause in women with a history of endometriosis: a systematic review. Hum Reprod Update, 23 (4), pp. 481-500. | Show Abstract | Read more

BACKGROUND: Endometriosis is typically regarded as a premenopausal disease, resolving after natural or iatrogenic menopause due to declining oestrogen levels. Nonetheless, case reports over the years have highlighted the incidence of recurrent postmenopausal endometriosis. It is now clear that both recurrence and malignant transformation of endometriotic foci can occur in the postmenopausal period. Postmenopausal women are commonly treated with hormone replacement therapy (HRT) to treat climacteric symptoms and prevent bone loss; however, HRT may reactivate endometriosis and stimulate malignant transformation in women with a history of endometriosis. Given the uncertain risks of initiating HRT, it is difficult to determine the best menopausal management for this group of women. OBJECTIVE AND RATIONAL: The aim of this study was to systematically review the existing literature on management of menopausal symptoms in women with a history of endometriosis. We also aimed to evaluate the published literature on the risks associated with HRT in these women, and details regarding optimal formulations and timing (i.e. initiation and duration) of HRT. SEARCH METHODS: Four electronic databases (MEDLINE via OVID, Embase via OVID, PsycINFO via OVID and CINAHL via EbscoHost) were searched from database inception until June 2016, using a combination of relevant controlled vocabulary terms and free-text terms related to 'menopause' and 'endometriosis'. Inclusion criteria were: menopausal women with a history of endometriosis and menopausal treatment including HRT or other preparations. Case reports/series, observational studies and clinical trials were included. Narrative review articles, organizational guidelines and conference abstracts were excluded, as were studies that did not report on any form of menopausal management. Articles were assessed for risk of bias and quality using GRADE criteria. OUTCOMES: We present a synthesis of the existing case reports of endometriosis recurrence or malignant transformation in women undergoing treatment for menopausal symptoms. We highlight common presenting symptoms, potential risk factors and outcomes amongst the studies. Sparse high-quality evidence was identified, with few observational studies and only two randomized controlled trials. Given this paucity of data, no definitive conclusions can be drawn concerning risk. WIDER IMPLICATIONS: Due to the lack of high-quality studies, it remains unclear how to advise women with a history of endometriosis regarding the management of menopausal symptoms. The absolute risk of disease recurrence and malignant transformation cannot be quantified, and the impact of HRT use on these outcomes is not known. Multicentre randomized trials or large observational studies are urgently needed to inform clinicians and patients alike.

Telomeres Mendelian Randomization Collaboration, Haycock PC, Burgess S, Nounu A, Zheng J, Okoli GN, Bowden J, Wade KH, Timpson NJ, Evans DM et al. 2017. Association Between Telomere Length and Risk of Cancer and Non-Neoplastic Diseases: A Mendelian Randomization Study. JAMA Oncol, 3 (5), pp. 636-651. | Show Abstract | Read more

Importance: The causal direction and magnitude of the association between telomere length and incidence of cancer and non-neoplastic diseases is uncertain owing to the susceptibility of observational studies to confounding and reverse causation. Objective: To conduct a Mendelian randomization study, using germline genetic variants as instrumental variables, to appraise the causal relevance of telomere length for risk of cancer and non-neoplastic diseases. Data Sources: Genomewide association studies (GWAS) published up to January 15, 2015. Study Selection: GWAS of noncommunicable diseases that assayed germline genetic variation and did not select cohort or control participants on the basis of preexisting diseases. Of 163 GWAS of noncommunicable diseases identified, summary data from 103 were available. Data Extraction and Synthesis: Summary association statistics for single nucleotide polymorphisms (SNPs) that are strongly associated with telomere length in the general population. Main Outcomes and Measures: Odds ratios (ORs) and 95% confidence intervals (CIs) for disease per standard deviation (SD) higher telomere length due to germline genetic variation. Results: Summary data were available for 35 cancers and 48 non-neoplastic diseases, corresponding to 420 081 cases (median cases, 2526 per disease) and 1 093 105 controls (median, 6789 per disease). Increased telomere length due to germline genetic variation was generally associated with increased risk for site-specific cancers. The strongest associations (ORs [95% CIs] per 1-SD change in genetically increased telomere length) were observed for glioma, 5.27 (3.15-8.81); serous low-malignant-potential ovarian cancer, 4.35 (2.39-7.94); lung adenocarcinoma, 3.19 (2.40-4.22); neuroblastoma, 2.98 (1.92-4.62); bladder cancer, 2.19 (1.32-3.66); melanoma, 1.87 (1.55-2.26); testicular cancer, 1.76 (1.02-3.04); kidney cancer, 1.55 (1.08-2.23); and endometrial cancer, 1.31 (1.07-1.61). Associations were stronger for rarer cancers and at tissue sites with lower rates of stem cell division. There was generally little evidence of association between genetically increased telomere length and risk of psychiatric, autoimmune, inflammatory, diabetic, and other non-neoplastic diseases, except for coronary heart disease (OR, 0.78 [95% CI, 0.67-0.90]), abdominal aortic aneurysm (OR, 0.63 [95% CI, 0.49-0.81]), celiac disease (OR, 0.42 [95% CI, 0.28-0.61]) and interstitial lung disease (OR, 0.09 [95% CI, 0.05-0.15]). Conclusions and Relevance: It is likely that longer telomeres increase risk for several cancers but reduce risk for some non-neoplastic diseases, including cardiovascular diseases.

Uimari O, Rahmioglu N, Nyholt DR, Vincent K, Missmer SA, Becker C, Morris AP, Montgomery GW, Zondervan KT. 2017. Genome-wide genetic analyses highlight mitogen-activated protein kinase (MAPK) signaling in the pathogenesis of endometriosis. Hum Reprod, 32 (4), pp. 780-793. | Show Abstract | Read more

Study question: Do genome-wide association study (GWAS) data for endometriosis provide insight into novel biological pathways associated with its pathogenesis? Summary answer: GWAS analysis uncovered multiple pathways that are statistically enriched for genetic association signals, analysis of Stage A disease highlighted a novel variant in MAP3K4, while top pathways significantly associated with all endometriosis and Stage A disease included several mitogen-activated protein kinase (MAPK)-related pathways. What is known already: Endometriosis is a complex disease with an estimated heritability of 50%. To date, GWAS revealed 10 genomic regions associated with endometriosis, explaining <4% of heritability, while half of the heritability is estimated to be due to common risk variants. Pathway analyses combine the evidence of single variants into gene-based measures, leveraging the aggregate effect of variants in genes and uncovering biological pathways involved in disease pathogenesis. Study design size, duration: Pathway analysis was conducted utilizing the International Endogene Consortium GWAS data, comprising 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry with genotype data imputed up to 1000 Genomes Phase three reference panel. GWAS was performed for all endometriosis cases and for Stage A (revised American Fertility Society (rAFS) I/II, n = 1686) and B (rAFS III/IV, n = 1364) cases separately. The identified significant pathways were compared with pathways previously investigated in the literature through candidate association studies. Participants/materials, setting, methods: The most comprehensive biological pathway databases, MSigDB (including BioCarta, KEGG, PID, SA, SIG, ST and GO) and PANTHER were utilized to test for enrichment of genetic variants associated with endometriosis. Statistical enrichment analysis was performed using the MAGENTA (Meta-Analysis Gene-set Enrichment of variaNT Associations) software. Main results and the role of chance: The first genome-wide association analysis for Stage A endometriosis revealed a novel locus, rs144240142 (P = 6.45 × 10-8, OR = 1.71, 95% CI = 1.23-2.37), an intronic single-nucleotide polymorphism (SNP) within MAP3K4. This SNP was not associated with Stage B disease (P = 0.086). MAP3K4 was also shown to be differentially expressed in eutopic endometrium between Stage A endometriosis cases and controls (P = 3.8 × 10-4), but not with Stage B disease (P = 0.26). A total of 14 pathways enriched with genetic endometriosis associations were identified (false discovery rate (FDR)-P < 0.05). The pathways associated with any endometriosis were Grb2-Sos provides linkage to MAPK signaling for integrins pathway (P = 2.8 × 10-5, FDR-P = 3.0 × 10-3), Wnt signaling (P = 0.026, FDR-P = 0.026) and p130Cas linkage to MAPK signaling for integrins pathway (P = 6.0 × 10-4, FDR-P = 0.029); with Stage A endometriosis: extracellular signal-regulated kinase (ERK)1 ERK2 MAPK (P = 5.0 × 10-4, FDR-P = 5.0 × 10-4) and with Stage B endometriosis: two overlapping pathways that related to extracellular matrix biology-Core matrisome (P = 1.4 × 10-3, FDR-P = 0.013) and ECM glycoproteins (P = 1.8 × 10-3, FDR-P = 7.1 × 10-3). Genes arising from endometriosis candidate gene studies performed to date were enriched for Interleukin signaling pathway (P = 2.3 × 10-12), Apoptosis signaling pathway (P = 9.7 × 10-9) and Gonadotropin releasing hormone receptor pathway (P = 1.2 × 10-6); however, these pathways did not feature in the results based on GWAS data. Large scale data: Not applicable. Limitations, reasons for caution: The analysis is restricted to (i) variants in/near genes that can be assigned to pathways, excluding intergenic variants; (ii) the gene-based pathway definition as registered in the databases; (iii) women of European ancestry. Wider implications of the findings: The top ranked pathways associated with overall and Stage A endometriosis in particular involve integrin-mediated MAPK activation and intracellular ERK/MAPK acting downstream in the MAPK cascade, both acting in the control of cell division, gene expression, cell movement and survival. Other top enriched pathways in Stage B disease include ECM glycoprotein pathways important for extracellular structure and biochemical support. The results highlight the need for increased efforts to understand the functional role of these pathways in endometriosis pathogenesis, including the investigation of the biological effects of the genetic variants on downstream molecular processes in tissue relevant to endometriosis. Additionally, our results offer further support for the hypothesis of at least partially distinct causal pathophysiology for minimal/mild (rAFS I/II) vs. moderate/severe (rAFS III/IV) endometriosis. Study funding/competing interest(s): The genome-wide association data and Wellcome Trust Case Control Consortium (WTCCC) were generated through funding from the Wellcome Trust (WT084766/Z/08/Z, 076113 and 085475) and the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498). N.R. was funded by a grant from the Medical Research Council UK (MR/K011480/1). A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant WT098017). All authors declare there are no conflicts of interest.

Barban N, Jansen R, de Vlaming R, Vaez A, Mandemakers JJ, Tropf FC, Shen X, Wilson JF, Chasman DI, Nolte IM et al. 2016. Genome-wide analysis identifies 12 loci influencing human reproductive behavior. Nat Genet, 48 (12), pp. 1462-1472. | Show Abstract | Read more

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits.

Borghese B, Zondervan KT, Abrao MS, Chapron C, Vaiman D. 2017. Recent insights on the genetics and epigenetics of endometriosis. Clin Genet, 91 (2), pp. 254-264. | Show Abstract | Read more

Endometriosis is a gynecologic disease affecting up to 10% of the women and a major cause of pain and infertility. It is characterized by the implantation of functional endometrial tissue at ectopic positions generally within the peritoneum. This complex disease has an important genetic component with a heritability estimated at around 50%. This review aims at providing recent insights into the genetic bases of endometriosis, and presents a detailed overview of evidence of epigenetic alterations specific to this disease. In the future, these alterations may constitute therapeutic targets for pharmacological compounds able to modify the epigenetic code.

Uimari O, Auvinen J, Jokelainen J, Puukka K, Ruokonen A, Järvelin MR, Piltonen T, Keinänen-Kiukaanniemi S, Zondervan K, Järvelä I et al. 2016. Uterine fibroids and cardiovascular risk. Hum Reprod, 31 (12), pp. 2689-2703. | Show Abstract | Read more

STUDY QUESTION: Are uterine fibroids associated with increased cardiovascular risk? SUMMARY ANSWER: This study reports an association between increased serum lipids and metabolic syndrome with an increased risk of uterine fibroids. WHAT IS KNOWN ALREADY: Recent studies suggest similarities in biological disease mechanisms and risk factors for fibroids and atherosclerosis: obesity, hypertension and abnormal serum lipids. These findings are awaiting confirmation that a population-based follow-up study could offer with extensive health examination data collection linked with a national hospital discharge register. STUDY DESIGN, SIZE, DURATION: The Northern Finland Birth Cohort (NFBC1966) is a population-based long-term follow-up study including all children with estimated date of delivery in 1966 in the Northern Finland area. The data were collected from national registries, postal questionnaires and clinical health examinations. The study population for this study comprised all females included in the NFBC1966 that underwent an extensive clinical health examination at age 46 years (n = 3635). PARTICIPANTS/MATERIALS, SETTING, METHODS: All females included in the NFBC1966 who were alive and traceable (n = 5118) were invited for the 46-year follow-up study; 3268 (63.9%) responded, returned the postal questionnaire and attended the clinical examination. Uterine fibroid cases were identified through the national hospital discharge register that has data on disease diagnoses based on WHO ICD-codes. Uterine fibroid codes, ICD-9: 218 and ICD-10: D25 were used for case identification. Self-reported fibroid cases were identified through the postal questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 729 fibroid cases were identified, including 293 based on hospital discharge registries. With adjustment for BMI, parity, education and current use of exogenous hormones the risk of prevalent fibroids rose significantly for every 1 mmol/l increase in LDL (OR = 1.13, 95% CI: 1.02-1.26 for all cases) and triglycerides (OR = 1.27, 95% CI: 1.09-1.49 for all cases). Metabolic syndrome associated with hospital discharge-based fibroid diagnosis (OR = 1.48, 95% CI: 1.09-2.01). Additionally every 1 unit increase in waist-hip ratio associated with fibroids (OR = 1.32, 95% CI: 1.10-1.57). LIMITATIONS, REASONS FOR CAUTION: The case ascertainment may present some limitations. There was likely an under-identification of cases and misclassification of some cases as controls; this would have diluted the effects of reported associations. The data analysed were cross-sectional and therefore cause and effect for the associations observed cannot be distinguished. WIDER IMPLICATIONS OF THE FINDINGS: Increased serum lipids and metabolic syndrome are associated with increased risk of uterine fibroids. Along with central obesity these findings add to an increased risk for cardiovascular disease among women with fibroids. These observations may suggest that there are shared predisposing factors underlying both uterine fibroids and adverse metabolic and cardiac disease risk, or that metabolic factors have a role in biological mechanisms underlying fibroid development. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Academy of Finland, University Hospital Oulu, University of Oulu, Finland, Northern Finland Health Care Foundation, Duodecim Foundation, ERDF European Regional Development Fund-Well-being and health: Research in the Northern Finland Birth Cohort 1966. The authors declare no conflict of interest.

Horikoshi M, Beaumont RN, Day FR, Warrington NM, Kooijman MN, Fernandez-Tajes J, Feenstra B, van Zuydam NR, Gaulton KJ, Grarup N et al. 2016. Genome-wide associations for birth weight and correlations with adult disease. Nature, 538 (7624), pp. 248-252. | Show Abstract | Read more

Birth weight (BW) has been shown to be influenced by both fetal and maternal factors and in observational studies is reproducibly associated with future risk of adult metabolic diseases including type 2 diabetes (T2D) and cardiovascular disease. These life-course associations have often been attributed to the impact of an adverse early life environment. Here, we performed a multi-ancestry genome-wide association study (GWAS) meta-analysis of BW in 153,781 individuals, identifying 60 loci where fetal genotype was associated with BW (P < 5 × 10(-8)). Overall, approximately 15% of variance in BW was captured by assays of fetal genetic variation. Using genetic association alone, we found strong inverse genetic correlations between BW and systolic blood pressure (Rg = -0.22, P = 5.5 × 10(-13)), T2D (Rg = -0.27, P = 1.1 × 10(-6)) and coronary artery disease (Rg = -0.30, P = 6.5 × 10(-9)). In addition, using large -cohort datasets, we demonstrated that genetic factors were the major contributor to the negative covariance between BW and future cardiometabolic risk. Pathway analyses indicated that the protein products of genes within BW-associated regions were enriched for diverse processes including insulin signalling, glucose homeostasis, glycogen biosynthesis and chromatin remodelling. There was also enrichment of associations with BW in known imprinted regions (P = 1.9 × 10(-4)). We demonstrate that life-course associations between early growth phenotypes and adult cardiometabolic disease are in part the result of shared genetic effects and identify some of the pathways through which these causal genetic effects are mediated.

Zondervan KT, Rahmioglu N, Morris AP, Nyholt DR, Montgomery GW, Becker CM, Missmer SA. 2016. Beyond Endometriosis Genome-Wide Association Study: From Genomics to Phenomics to the Patient. Semin Reprod Med, 34 (4), pp. 242-254. | Show Abstract | Read more

Endometriosis is a heritable, complex chronic inflammatory disease, for which much of the causal pathogenic mechanism remains unknown. Genome-wide association studies (GWAS) to date have identified 12 single nucleotide polymorphisms at 10 independent genetic loci associated with endometriosis. Most of these were more strongly associated with revised American Fertility Society stage III/IV, rather than stage I/II. The loci are almost all located in intergenic regions that are known to play a role in the regulation of expression of target genes yet to be identified. To identify the target genes and pathways perturbed by the implicated variants, studies are required involving functional genomic annotation of the surrounding chromosomal regions, in terms of transcription factor binding, epigenetic modification (e.g., DNA methylation and histone modification) sites, as well as their correlation with RNA transcription. These studies need to be conducted in tissue types relevant to endometriosis-in particular, endometrium. In addition, to allow biologically and clinically relevant interpretation of molecular profiling data, they need to be combined and correlated with detailed, systematically collected phenotypic information (surgical and clinical). The WERF Endometriosis Phenome and Biobanking Harmonisation Project is a global standardization initiative that has produced consensus data and sample collection protocols for endometriosis research. These now pave the way for collaborative studies integrating phenomic with genomic data, to identify informative subtypes of endometriosis that will enhance understanding of the pathogenic mechanisms of the disease and discovery of novel, targeted treatments.

Rogers PA, Adamson GD, Al-Jefout M, Becker CM, D'Hooghe TM, Dunselman GA, Fazleabas A, Giudice LC, Horne AW, Hull ML et al. 2017. Research Priorities for Endometriosis. Reprod Sci, 24 (2), pp. 202-226. | Show Abstract | Read more

The 3rd International Consensus Workshop on Research Priorities in Endometriosis was held in São Paulo on May 4, 2014, following the 12th World Congress on Endometriosis. The workshop was attended by 60 participants from 19 countries and was divided into 5 main sessions covering pathogenesis/pathophysiology, symptoms, diagnosis/classification/prognosis, disease/symptom management, and research policy. This research priorities consensus statement builds on earlier efforts to develop research directions for endometriosis. Of the 56 research recommendations from the 2011 meeting in Montpellier, a total of 41 remained unchanged, 13 were updated, and 2 were deemed to be completed. Fifty-three new research recommendations were made at the 2014 meeting in Sao Paulo, which in addition to the 13 updated recommendations resulted in a total of 66 new recommendations for research. The research recommendations published herein, as well as those from the 2 previous papers from international consensus workshops, are an attempt to promote high-quality research in endometriosis by identifying and agreeing on key issues that require investigation. New areas included in the 2014 recommendations include infertility, patient stratification, and research in emerging nations, in addition to an increased focus on translational research. A revised and updated set of research priorities that builds on this document will be developed at the 13th World Congress on Endometriosis to be held on May 17-20, 2017, in Vancouver, British Columbia, Canada.

Saare M, Modhukur V, Suhorutshenko M, Rajashekar B, Rekker K, Sõritsa D, Karro H, Soplepmann P, Sõritsa A, Lindgren CM et al. 2016. The influence of menstrual cycle and endometriosis on endometrial methylome. Clin Epigenetics, 8 (1), pp. 2. | Show Abstract | Read more

BACKGROUND: Alterations in endometrial DNA methylation profile have been proposed as one potential mechanism initiating the development of endometriosis. However, the normal endometrial methylome is influenced by the cyclic hormonal changes, and the menstrual cycle phase-dependent epigenetic signature should be considered when studying endometrial disorders. So far, no studies have been performed to evaluate the menstrual cycle influences and endometriosis-specific endometrial methylation pattern at the same time. RESULTS: Infinium HumanMethylation 450K BeadChip arrays were used to explore DNA methylation profiles of endometrial tissues from various menstrual cycle phases from 31 patients with endometriosis and 24 healthy women. The DNA methylation profile of patients and controls was highly similar and only 28 differentially methylated regions (DMRs) between patients and controls were found. However, the overall magnitude of the methylation differences between patients and controls was rather small (Δβ ranging from -0.01 to -0.16 and from 0.01 to 0.08, respectively, for hypo- and hypermethylated CpGs). Unsupervised hierarchical clustering of the methylation data divided endometrial samples based on the menstrual cycle phase rather than diseased/non-diseased status. Further analysis revealed a number of menstrual cycle phase-specific epigenetic changes with largest changes occurring during the late-secretory and menstrual phases when substantial rearrangements of endometrial tissue take place. Comparison of cycle phase- and endometriosis-specific methylation profile changes revealed that 13 out of 28 endometriosis-specific DMRs were present in both datasets. CONCLUSIONS: The results of our study accentuate the importance of considering normal cyclic epigenetic changes in studies investigating endometrium-related disease-specific methylation patterns.

Lu Y, Cuellar-Partida G, Painter JN, Nyholt DR, Australian Ovarian Cancer Study, International Endogene Consortium (IEC), Morris AP, Fasching PA, Hein A, Burghaus S et al. 2015. Shared genetics underlying epidemiological association between endometriosis and ovarian cancer. Hum Mol Genet, 24 (20), pp. 5955-5964. | Show Abstract | Read more

Epidemiological studies have demonstrated associations between endometriosis and certain histotypes of ovarian cancer, including clear cell, low-grade serous and endometrioid carcinomas. We aimed to determine whether the observed associations might be due to shared genetic aetiology. To address this, we used two endometriosis datasets genotyped on common arrays with full-genome coverage (3194 cases and 7060 controls) and a large ovarian cancer dataset genotyped on the customized Illumina Infinium iSelect (iCOGS) arrays (10 065 cases and 21 663 controls). Previous work has suggested that a large number of genetic variants contribute to endometriosis and ovarian cancer (all histotypes combined) susceptibility. Here, using the iCOGS data, we confirmed polygenic architecture for most histotypes of ovarian cancer. This led us to evaluate if the polygenic effects are shared across diseases. We found evidence for shared genetic risks between endometriosis and all histotypes of ovarian cancer, except for the intestinal mucinous type. Clear cell carcinoma showed the strongest genetic correlation with endometriosis (0.51, 95% CI = 0.18-0.84). Endometrioid and low-grade serous carcinomas had similar correlation coefficients (0.48, 95% CI = 0.07-0.89 and 0.40, 95% CI = 0.05-0.75, respectively). High-grade serous carcinoma, which often arises from the fallopian tubes, showed a weaker genetic correlation with endometriosis (0.25, 95% CI = 0.11-0.39), despite the absence of a known epidemiological association. These results suggest that the epidemiological association between endometriosis and ovarian adenocarcinoma may be attributable to shared genetic susceptibility loci.

Fawole AO, Bello FA, Ogunbode O, Odukogbe AT, Nkwocha GC, Nnoaham KE, Zondervan KT, Akintan A, Abdus-Salam RA, Okunlola MA. 2015. Endometriosis and associated symptoms among Nigerian women. Int J Gynaecol Obstet, 130 (2), pp. 190-194. | Show Abstract | Read more

OBJECTIVE: To determine the prevalence of endometriosis and identify associated symptoms among Nigerian women. METHODS: A cross-sectional study was conducted at a center in Ibadan, Nigeria, between October 2008 and December 2010. All women aged 18-45 years scheduled for their first diagnostic laparoscopy for gynecologic indications were enrolled. Participants completed a previously validated self-administered questionnaire. Endometriosis was diagnosed on the basis of visual evidence. RESULTS: Among 239 women analyzed, 115 (48.1%) had endometriotic lesions. Endometriosis was more common among women reporting dysmenorrhea and pelvic pain than among those not reporting these symptoms (20/28 [71.4%] vs 95/211 [45.0%]; P=0.009). Women who reported dysmenorrhea were significantly more likely to have endometriosis than were those without dysmenorrhea (90/171 [52.6%] vs 25/68 [36.8%]; P=0.027). The risk of endometriosis was not significantly increased in women with one pain symptom (odds ratio [OR]1.69; 95% confidence interval [CI] 0.67-4.27), but was significantly increased in women with two (OR 2.70; 95% CI 1.13-6.52) or three (OR 4.87; 95% CI 1.88-12.82) pain symptoms (χ(2)trend=15.5; P<0.001). In a multivariate logistic regression model, only pain other than dysmenorrhea or dyspareunia independently predicted endometriosis (P=0.017). CONCLUSION: Endometriosis is fairly common among Nigerian women. Efforts to increase the awareness of endometriosis among the public, researchers, and clinicians are needed.

Sapkota Y, Attia J, Gordon SD, Henders AK, Holliday EG, Rahmioglu N, MacGregor S, Martin NG, McEvoy M, Morris AP et al. 2015. Genetic burden associated with varying degrees of disease severity in endometriosis. Mol Hum Reprod, 21 (7), pp. 594-602. | Show Abstract | Read more

Endometriosis is primarily characterized by the presence of tissue resembling endometrium outside the uterine cavity and is usually diagnosed by laparoscopy. The most commonly used classification of disease, the revised American Fertility Society (rAFS) system to grade endometriosis into different stages based on disease severity (I to IV), has been questioned as it does not correlate well with underlying symptoms, posing issues in diagnosis and choice of treatment. Using two independent European genome-wide association (GWA) datasets and top-level classification of the endometriosis cases based on rAFS [minimal or mild (Stage A) and moderate-to-severe (Stage B) disease], we previously showed that Stage B endometriosis has greater contribution of common genetic variation to its aetiology than Stage A disease. Herein, we extend our previous analysis to four endometriosis stages [minimal (Stage I), mild (Stage II), moderate (Stage III) and severe (Stage IV) disease] based on the rAFS classification system and compared the genetic burden across stages. Our results indicate that genetic burden increases from minimal to severe endometriosis. For the minimal disease, genetic factors may contribute to a lesser extent than other disease categories. Mild and moderate endometriosis appeared genetically similar, making it difficult to tease them apart. Consistent with our previous reports, moderate and severe endometriosis showed greater genetic burden than minimal or mild disease. Overall, our results provide new insights into the genetic architecture of endometriosis and further investigation in larger samples may help to understand better the aetiology of varying degrees of endometriosis, enabling improved diagnostic and treatment modalities.

Rahmioglu N, Drong A, Lockstone H, Lindgren CM, Becker CM, Zondervan KT. 2015. Variability of Genome-Wide Gene Expression and DNA Methylation Profiles Across Tissue Samples From Women With and Without Endometriosis REPRODUCTIVE SCIENCES, 22 pp. 209A-209A.

Shungin D, Winkler TW, Croteau-Chonka DC, Ferreira T, Locke AE, Mägi R, Strawbridge RJ, Pers TH, Fischer K, Justice AE et al. 2015. New genetic loci link adipose and insulin biology to body fat distribution. Nature, 518 (7538), pp. 187-196. | Show Abstract | Read more

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

Locke AE, Kahali B, Berndt SI, Justice AE, Pers TH, Day FR, Powell C, Vedantam S, Buchkovich ML, Yang J et al. 2015. Genetic studies of body mass index yield new insights for obesity biology. Nature, 518 (7538), pp. 197-206. | Show Abstract | Read more

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10(-8)), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ∼2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

Rahmioglu N, Montgomery GW, Zondervan KT. 2015. Genetics of endometriosis. Womens Health (Lond), 11 (5), pp. 577-586. | Show Abstract | Read more

Endometriosis is a heritable complex disorder that is influenced by multiple genetic and environmental factors. Identification of these genetic factors will aid a better understanding of the underlying biology of the disease. In this article, we describe different methods of studying genetic variation of endometriosis, summarize results from genetic studies performed to date and provide recommendations for future studies to uncover additional factors contributing to the heritable component of endometriosis.

Allum F, Shao X, Guénard F, Simon MM, Busche S, Caron M, Lambourne J, Lessard J, Tandre K, Hedman ÅK et al. 2015. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants. Nat Commun, 6 pp. 7211. | Show Abstract | Read more

Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

Sapkota Y, Low SK, Attia J, Gordon SD, Henders AK, Holliday EG, MacGregor S, Martin NG, McEvoy M, Morris AP et al. 2015. Association between endometriosis and the interleukin 1A (IL1A) locus. Hum Reprod, 30 (1), pp. 239-248. | Show Abstract | Read more

STUDY QUESTION: Are single-nucleotide polymorphisms (SNPs) at the interleukin 1A (IL1A) gene locus associated with endometriosis risk? SUMMARY ANSWER: We found evidence for strong association between IL1A SNPs and endometriosis risk. WHAT IS KNOWN ALREADY: Genetic factors contribute substantially to the complex aetiology of endometriosis and the disease has an estimated heritability of ∼51%. We, and others, have conducted genome-wide association (GWA) studies for endometriosis, which identified a total of nine independent risk loci. Recently, two small Japanese studies reported eight SNPs (rs6542095, rs11677416, rs3783550, rs3783525, rs3783553, rs2856836, rs1304037 and rs17561) at the IL1A gene locus as suggestively associated with endometriosis risk. There is also evidence of a link between inflammation and endometriosis. STUDY DESIGN, SIZE, DURATION: We sought to further investigate the eight IL1A SNPs for association with endometriosis using an independent sample of 3908 endometriosis cases and 8568 controls of European and Japanese ancestry. The study was conducted between October 2013 and July 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: By leveraging GWA data from our previous multi-ethnic GWA meta-analysis for endometriosis, we imputed variants in the IL1A region, using a recent 1000 Genomes reference panel. After combining summary statistics for the eight SNPs from our European and Japanese imputed data with the published results, a fixed-effect meta-analysis was performed. An additional meta-analysis restricted to endometriosis cases with moderate-to-severe (revised American Fertility Society stage 3 or 4) disease versus controls was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: All eight IL1A SNPs successfully replicated at P < 0.014 in the European imputed data with concordant direction and similar size to the effects reported in the original Japanese studies. Of these, three SNPs (rs6542095, rs3783550 and rs3783525) also showed association with endometriosis at a nominal P < 0.05 in our independent Japanese sample. Fixed-effect meta-analysis of the eight SNPs for moderate-to-severe endometriosis produced a genome-wide significant association for rs6542095 (odds ratio = 1.21; 95% confidence interval = 1.13-1.29; P = 3.43 × 10(-8)). LIMITATIONS, REASONS FOR CAUTION: The meta-analysis for moderate-to-severe endometriosis included results of moderate-to-severe endometriosis cases from our European data sets and all endometriosis cases from the Japanese data sets, as disease stage information was not available for endometriosis cases in the Japanese data sets. WIDER IMPLICATIONS OF THE FINDINGS: SNP rs6542095 is located ∼2.3 kb downstream of the IL1A gene and ∼6.9 kb upstream of cytoskeleton-associated protein 2-like (CKAP2L) gene. The IL1A gene encodes the IL1a protein, a member of the interleukin 1 cytokine family which is involved in various immune responses and inflammatory processes. These results provide important replication in an independent Japanese sample and, for the first time, association of the IL1A locus in endometriosis patients of European ancestry. SNPs within the IL1A locus may regulate other genes, but if IL1A is the target, our results provide supporting evidence for a link between inflammatory responses and the pathogenesis of endometriosis. STUDY FUNDING/COMPETING INTERESTS: The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Rahmioglu N, Macgregor S, Drong AW, Hedman ÅK, Harris HR, Randall JC, Prokopenko I, International Endogene Consortium (IEC), The GIANT Consortium, Nyholt DR, Morris AP et al. 2015. Genome-wide enrichment analysis between endometriosis and obesity-related traits reveals novel susceptibility loci. Hum Mol Genet, 24 (4), pp. 1185-1199. | Show Abstract | Read more

Endometriosis is a chronic inflammatory condition in women that results in pelvic pain and subfertility, and has been associated with decreased body mass index (BMI). Genetic variants contributing to the heritable component have started to emerge from genome-wide association studies (GWAS), although the majority remain unknown. Unexpectedly, we observed an intergenic locus on 7p15.2 that was genome-wide significantly associated with both endometriosis and fat distribution (waist-to-hip ratio adjusted for BMI; WHRadjBMI) in an independent meta-GWAS of European ancestry individuals. This led us to investigate the potential overlap in genetic variants underlying the aetiology of endometriosis, WHRadjBMI and BMI using GWAS data. Our analyses demonstrated significant enrichment of common variants between fat distribution and endometriosis (P = 3.7 × 10(-3)), which was stronger when we restricted the investigation to more severe (Stage B) cases (P = 4.5 × 10(-4)). However, no genetic enrichment was observed between endometriosis and BMI (P = 0.79). In addition to 7p15.2, we identify four more variants with statistically significant evidence of involvement in both endometriosis and WHRadjBMI (in/near KIFAP3, CAB39L, WNT4, GRB14); two of these, KIFAP3 and CAB39L, are novel associations for both traits. KIFAP3, WNT4 and 7p15.2 are associated with the WNT signalling pathway; formal pathway analysis confirmed a statistically significant (P = 6.41 × 10(-4)) overrepresentation of shared associations in developmental processes/WNT signalling between the two traits. Our results demonstrate an example of potential biological pleiotropy that was hitherto unknown, and represent an opportunity for functional follow-up of loci and further cross-phenotype comparisons to assess how fat distribution and endometriosis pathogenesis research fields can inform each other.

Vitonis AF, Vincent K, Rahmioglu N, Fassbender A, Buck Louis GM, Hummelshoj L, Giudice LC, Stratton P, Adamson GD, Becker CM et al. 2014. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: II. Clinical and covariate phenotype data collection in endometriosis research. Fertil Steril, 102 (5), pp. 1223-1232. | Show Abstract | Read more

OBJECTIVE: To harmonize the collection of nonsurgical clinical and epidemiologic data relevant to endometriosis research, allowing large-scale collaboration. DESIGN: An international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries on five continents. SETTING: In 2013, two workshops followed by global consultation, bringing together 54 leaders in endometriosis research. PATIENTS: None. INTERVENTION(S): Development of a self-administered endometriosis patient questionnaire (EPQ), based on [1] systematic comparison of questionnaires from eight centers that collect data from endometriosis cases (and controls/comparison women) on a medium to large scale (publication on >100 cases); [2] literature evidence; and [3] several global consultation rounds. MAIN OUTCOME MEASURE(S): Standard recommended and minimum required questionnaires to capture detailed clinical and covariate data. RESULT(S): The standard recommended (EPHect EPQ-S) and minimum required (EPHect EPQ-M) questionnaires contain questions on pelvic pain, subfertility and menstrual/reproductive history, hormone/medication use, medical history, and personal information. CONCLUSION(S): The EPQ captures the basic set of patient characteristics and exposures considered by the WERF EPHect Working Group to be most critical for the advancement of endometriosis research, but is also relevant to other female conditions with similar risk factors and/or symptomatology. The instruments will be reviewed based on feedback from investigators, and-after a first review after 1 year-triannually through systematic follow-up surveys. Updated versions will be made available through http://endometriosisfoundation.org/ephect.

Fassbender A, Rahmioglu N, Vitonis AF, Viganò P, Giudice LC, D'Hooghe TM, Hummelshoj L, Adamson GD, Becker CM, Missmer SA et al. 2014. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project: IV. Tissue collection, processing, and storage in endometriosis research. Fertil Steril, 102 (5), pp. 1244-1253. | Show Abstract | Read more

OBJECTIVE: To harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of human tissues relevant to endometriosis. DESIGN: An international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries on five continents. SETTING: In 2013, two workshops were conducted followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing from around the world. PATIENT(S): None. INTERVENTION(S): Consensus SOPs were based on: 1) systematic comparison of SOPs from 24 global centers collecting tissue samples from women with and without endometriosis on a medium or large scale (publication on >100 cases); 2) literature evidence where available, or consultation with laboratory experts otherwise; and 3) several global consultation rounds. MAIN OUTCOME MEASURE(S): Standard recommended and minimum required SOPs for tissue collection, processing, and storage in endometriosis research. RESULT(S): We developed "recommended standard" and "minimum required" SOPs for the collection, processing, and storage of ectopic and eutopic endometrium, peritoneum, and myometrium, and a biospecimen data collection form necessary for interpretation of sample-derived results. CONCLUSION(S): The EPHect SOPs allow endometriosis research centers to decrease variability in tissue-based results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other gynecologic conditions involving endometrium, myometrium, and peritoneum. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback and through systematic triannual follow-up. Updated versions will be made available at: http://endometriosisfoundation.org/ephect.

Rahmioglu N, Fassbender A, Vitonis AF, Tworoger SS, Hummelshoj L, D'Hooghe TM, Adamson GD, Giudice LC, Becker CM, Zondervan KT et al. 2014. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: III. Fluid biospecimen collection, processing, and storage in endometriosis research. Fertil Steril, 102 (5), pp. 1233-1243. | Show Abstract | Read more

OBJECTIVE: To harmonize standard operating procedures (SOPs) and standardize the recording of associated data for collection, processing, and storage of fluid biospecimens relevant to endometriosis. DESIGN: An international collaboration involving 34 clinical/academic centers and 3 industry collaborators from 16 countries on 5 continents. SETTING: In 2013, 2 workshops were conducted, followed by global consultation, bringing together 54 leaders in endometriosis research and sample processing worldwide. PATIENT(S): None. INTERVENTION(S): Consensus SOPs were based on: [1] systematic comparison of SOPs from 18 global centers collecting fluid samples from women with and without endometriosis on a medium/large scale (publication on >100 cases), [2] literature evidence where available, or consultation with laboratory experts otherwise, and [3] several global consultation rounds. MAIN OUTCOME MEASURE(S): Standard recommended and minimum required SOPs for biofluid collection, processing, and storage in endometriosis research. RESULT(S): We developed recommended standard and minimum required SOPs for the collection, processing, and storage of plasma, serum, saliva, urine, endometrial/peritoneal fluid, and menstrual effluent, and a biospecimen data-collection form necessary for interpretation of sample-derived results. CONCLUSION(S): The Endometriosis Phenome and Biobanking Harmonisation Project SOPs allow endometriosis research centers to decrease variability in biofluid sample results, facilitating between-center comparisons and collaborations. The procedures are also relevant to research into other female conditions involving biofluid samples subject to cyclic reproductive influences. The consensus SOPs are based on the best available evidence; areas with limited evidence are identified as requiring further pilot studies. The SOPs will be reviewed based on investigator feedback, and through systematic tri-annual follow-up. Updated versions will be made available at: endometriosisfoundation.org/ephect.

Becker CM, Laufer MR, Stratton P, Hummelshoj L, Missmer SA, Zondervan KT, Adamson GD, WERF EPHect Working Group. 2014. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project: I. Surgical phenotype data collection in endometriosis research. Fertil Steril, 102 (5), pp. 1213-1222. | Show Abstract | Read more

OBJECTIVE: To standardize the recording of surgical phenotypic information on endometriosis and related sample collections obtained at laparoscopy, allowing large-scale collaborative research into the condition. DESIGN: An international collaboration involving 34 clinical/academic centers and three industry collaborators from 16 countries. SETTING: Two workshops were conducted in 2013, bringing together 54 clinical, academic, and industry leaders in endometriosis research and management worldwide. PATIENT(S): None. INTERVENTION(S): A postsurgical scoring sheet containing general and gynecological patient and procedural information, extent of disease, the location and type of endometriotic lesion, and any other findings was developed during several rounds of review. Comments and any systematic surgical data collection tools used in the reviewers' centers were incorporated. MAIN OUTCOME MEASURE(S): The development of a standard recommended (SSF) and minimum required (MSF) form to collect data on the surgical phenotype of endometriosis. RESULT(S): SSF and MSF include detailed descriptions of lesions, modes of procedures and sample collection, comorbidities, and potential residual disease at the end of surgery, along with previously published instruments such as the revised American Society for Reproductive Medicine and Endometriosis Fertility Index classification tools for comparison and validation. CONCLUSION(S): This is the first multicenter, international collaboration between academic centers and industry addressing standardization of phenotypic data collection for a specific disease. The Endometriosis Phenome and Biobanking Harmonisation Project SSF and MSF are essential tools to increase our understanding of the pathogenesis of endometriosis by allowing large-scale collaborative research into the condition.

Ng MC, Shriner D, Chen BH, Li J, Chen WM, Guo X, Liu J, Bielinski SJ, Yanek LR, Nalls MA et al. 2014. Meta-analysis of genome-wide association studies in African Americans provides insights into the genetic architecture of type 2 diabetes. PLoS Genet, 10 (8), pp. e1004517. | Show Abstract | Read more

Type 2 diabetes (T2D) is more prevalent in African Americans than in Europeans. However, little is known about the genetic risk in African Americans despite the recent identification of more than 70 T2D loci primarily by genome-wide association studies (GWAS) in individuals of European ancestry. In order to investigate the genetic architecture of T2D in African Americans, the MEta-analysis of type 2 DIabetes in African Americans (MEDIA) Consortium examined 17 GWAS on T2D comprising 8,284 cases and 15,543 controls in African Americans in stage 1 analysis. Single nucleotide polymorphisms (SNPs) association analysis was conducted in each study under the additive model after adjustment for age, sex, study site, and principal components. Meta-analysis of approximately 2.6 million genotyped and imputed SNPs in all studies was conducted using an inverse variance-weighted fixed effect model. Replications were performed to follow up 21 loci in up to 6,061 cases and 5,483 controls in African Americans, and 8,130 cases and 38,987 controls of European ancestry. We identified three known loci (TCF7L2, HMGA2 and KCNQ1) and two novel loci (HLA-B and INS-IGF2) at genome-wide significance (4.15 × 10(-94)<P<5 × 10(-8), odds ratio (OR)  = 1.09 to 1.36). Fine-mapping revealed that 88 of 158 previously identified T2D or glucose homeostasis loci demonstrated nominal to highly significant association (2.2 × 10(-23) < locus-wide P<0.05). These novel and previously identified loci yielded a sibling relative risk of 1.19, explaining 17.5% of the phenotypic variance of T2D on the liability scale in African Americans. Overall, this study identified two novel susceptibility loci for T2D in African Americans. A substantial number of previously reported loci are transferable to African Americans after accounting for linkage disequilibrium, enabling fine mapping of causal variants in trans-ethnic meta-analysis studies.

Pinnick KE, Nicholson G, Manolopoulos KN, McQuaid SE, Valet P, Frayn KN, Denton N, Min JL, Zondervan KT, Fleckner J et al. 2014. Distinct developmental profile of lower-body adipose tissue defines resistance against obesity-associated metabolic complications. Diabetes, 63 (11), pp. 3785-3797. | Show Abstract | Read more

Upper- and lower-body fat depots exhibit opposing associations with obesity-related metabolic disease. We defined the relationship between DEXA-quantified fat depots and diabetes/cardiovascular risk factors in a healthy population-based cohort (n = 3,399). Gynoid fat mass correlated negatively with insulin resistance after total fat mass adjustment, whereas the opposite was seen for abdominal fat. Paired transcriptomic analysis of gluteal subcutaneous adipose tissue (GSAT) and abdominal subcutaneous adipose tissue (ASAT) was performed across the BMI spectrum (n = 49; 21.4-45.5 kg/m(2)). In both depots, energy-generating metabolic genes were negatively associated and inflammatory genes were positively associated with obesity. However, associations were significantly weaker in GSAT. At the systemic level, arteriovenous release of the proinflammatory cytokine interleukin-6 (n = 34) was lower from GSAT than ASAT. Isolated preadipocytes retained a depot-specific transcriptional "memory" of embryonic developmental genes and exhibited differential promoter DNA methylation of selected genes (HOTAIR, TBX5) between GSAT and ASAT. Short hairpin RNA-mediated silencing identified TBX5 as a regulator of preadipocyte proliferation and adipogenic differentiation in ASAT. In conclusion, intrinsic differences in the expression of developmental genes in regional adipocytes provide a mechanistic basis for diversity in adipose tissue (AT) function. The less inflammatory nature of lower-body AT offers insight into the opposing metabolic disease risk associations between upper- and lower-body obesity.

Brawn J, Morotti M, Zondervan KT, Becker CM, Vincent K. 2014. Central changes associated with chronic pelvic pain and endometriosis. Hum Reprod Update, 20 (5), pp. 737-747. | Show Abstract | Read more

BACKGROUND: Chronic pelvic pain (CPP) is a significant public health problem with 1 million affected women in the UK. Although many pathologies are associated with CPP, the pain experienced is often disproportionate to the extent of disease identified and frequently no pathology is found (chronic pelvic pain syndrome). The central nervous system (CNS) is central to the experience of pain and chronic pain conditions in general are associated with alterations in both the structure and function of the CNS. This review describes the available evidence for central changes in association with conditions presenting with CPP. METHODS: A detailed literature search was performed to identify relevant papers, however, this is not a systematic review. RESULTS: CPP is associated with central changes similar to those identified in other pain conditions. Specifically these include, alterations in the behavioural and central response to noxious stimulation, changes in brain structure (both increases and decreases in the volume of specific brain regions), altered activity of both the hypothalamic-pituitary-adrenal axis and the autonomic nervous system (ANS) and psychological distress. CONCLUSIONS: The evidence reviewed in this paper demonstrates that CPP is associated with significant central changes when compared with healthy pain-free women. Moreover, the presence of these changes has the potential to both exacerbate symptoms and to predispose these women to the development of additional chronic conditions. These findings support the use of adjunctive medication targeting the CNS in these women.

Morotti M, Vincent K, Brawn J, Zondervan KT, Becker CM. 2014. Peripheral changes in endometriosis-associated pain. Hum Reprod Update, 20 (5), pp. 717-736. | Show Abstract | Read more

BACKGROUND: Pain remains the cardinal symptom of endometriosis. However, to date, the underlying mechanisms are still only poorly understood. Increasing evidence points towards a close interaction between peripheral nerves, the peritoneal environment and the central nervous system in pain generation and processing. Recently, studies demonstrating nerve fibres and neurotrophic and angiogenic factors in endometriotic lesions and their vicinity have led to increased interest in peripheral changes in endometriosis-associated pain. This review focuses on the origin and function of these nerves and factors as well as possible peripheral mechanisms that may contribute to the generation and modulation of pain in women with endometriosis. METHODS: We conducted a systematic search using several databases (PubMed, MEDLINE, EMBASE and CINAHL) of publications from January 1977 to October 2013 to evaluate the possible roles of the peripheral nervous system in endometriosis pathophysiology and how it can contribute to endometriosis-associated pain. RESULTS: Endometriotic lesions and peritoneal fluid from women with endometriosis had pronounced neuroangiogenic properties with increased expression of new nerve fibres, a shift in the distribution of sensory and autonomic fibres in some locations, and up-regulation of several neurotrophins. In women suffering from deep infiltrating endometriosis and bowel endometriosis, in which the anatomical distribution of lesions is generally more closely related to pelvic pain symptoms, endometriotic lesions and surrounding tissues present higher nerve fibre densities compared with peritoneal lesions and endometriomas. More data are needed to fully confirm a direct correlation between fibre density in these locations and the amount of perceived pain. A better correlation between the presence of nerve fibres and pain symptoms seems to exist for eutopic endometrium. However, this appears not to be exclusive to endometriosis. No correlation between elevated neurotrophin levels and pain severity appears to exist, suggesting the involvement of other mediators in the modulation of pain. CONCLUSIONS: The increased expression of neurotrophic factors and nerve fibres in endometriotic lesions, eutopic endometrium and the peritoneum imply a role of such peripheral changes in the pathogenesis of endometriosis-associated pain. However, a clear link between these findings and pain in patients with endometriosis has so far not been demonstrated.

Painter JN, Nyholt DR, Krause L, Zhao ZZ, Chapman B, Zhang C, Medland S, Martin NG, Kennedy S, Treloar S et al. 2014. Common variants in the CYP2C19 gene are associated with susceptibility to endometriosis. Fertil Steril, 102 (2), pp. 496-502.e5. | Show Abstract | Read more

OBJECTIVE: To follow-up previous studies highlighting a possible role for cytochrome P450, family 2, subfamily C, 19 (CYP2C19) in susceptibility to endometriosis by searching for additional variants in the CYP2C19 gene that may be associated with the disease. DESIGN: Case-control study. SETTING: Academic research. SUBJECT(S): The cases comprised 2,271 women with surgically confirmed endometriosis; the controls comprised 939 women with self-report of no endometriosis and 1,770 unscreened population samples. INTERVENTION(S): Sequencing of the CYP2C19 region and follow-up of 80 single nucleotide polymorphisms (SNPs) in two case-control samples. MAIN OUTCOME MEASURE(S): Allele frequency differences between cases and controls. RESULT(S): Sequencing of the CYP2C19 gene region resulted in the detection of a large number of known and novel SNPs. Genotyping of 80 polymorphic SNPs in 901 endometriosis cases and 939 controls resulted in study-wide significant association signals for SNPs in moderate or complete linkage disequilibrium with rs4244285, a functional SNP in exon 5 that abrogates CYP2C19 function through the creation of an alternative splice site. Evidence of association was also detected for another functional SNP in the CYP2C19 promoter, rs12248560, which was highlighted in our previous study. CONCLUSION(S): Functional variants in CYP2C19 may contribute to endometriosis susceptibility in both familial and sporadic cases.

Rahmioglu N, Nyholt DR, Morris AP, Missmer SA, Montgomery GW, Zondervan KT. 2014. Genetic variants underlying risk of endometriosis: insights from meta-analysis of eight genome-wide association and replication datasets. Hum Reprod Update, 20 (5), pp. 702-716. | Show Abstract | Read more

BACKGROUND: Endometriosis is a heritable common gynaecological condition influenced by multiple genetic and environmental factors. Genome-wide association studies (GWASs) have proved successful in identifying common genetic variants of moderate effects for various complex diseases. To date, eight GWAS and replication studies from multiple populations have been published on endometriosis. In this review, we investigate the consistency and heterogeneity of the results across all the studies and their implications for an improved understanding of the aetiology of the condition. METHODS: Meta-analyses were conducted on four GWASs and four replication studies including a total of 11 506 cases and 32 678 controls, and on the subset of studies that investigated associations for revised American Fertility Society (rAFS) Stage III/IV including 2859 cases. The datasets included 9039 cases and 27 343 controls of European (Australia, Belgium, Italy, UK, USA) and 2467 cases and 5335 controls of Japanese ancestry. Fixed and Han and Elkin random-effects models, and heterogeneity statistics (Cochran's Q test), were used to investigate the evidence of the nine reported genome-wide significant loci across datasets and populations. RESULTS: Meta-analysis showed that seven out of nine loci had consistent directions of effect across studies and populations, and six out of nine remained genome-wide significant (P < 5 × 10(-8)), including rs12700667 on 7p15.2 (P = 1.6 × 10(-9)), rs7521902 near WNT4 (P = 1.8 × 10(-15)), rs10859871 near VEZT (P = 4.7 × 10(-15)), rs1537377 near CDKN2B-AS1 (P = 1.5 × 10(-8)), rs7739264 near ID4 (P = 6.2 × 10(-10)) and rs13394619 in GREB1 (P = 4.5 × 10(-8)). In addition to the six loci, two showed borderline genome-wide significant associations with Stage III/IV endometriosis, including rs1250248 in FN1 (P = 8 × 10(-8)) and rs4141819 on 2p14 (P = 9.2 × 10(-8)). Two independent inter-genic loci, rs4141819 and rs6734792 on chromosome 2, showed significant evidence of heterogeneity across datasets (P < 0.005). Eight of the nine loci had stronger effect sizes among Stage III/IV cases, implying that they are likely to be implicated in the development of moderate to severe, or ovarian, disease. While three out of nine loci were inter-genic, the remaining were in or near genes with known functions of biological relevance to endometriosis, varying from roles in developmental pathways to cellular growth/carcinogenesis. CONCLUSIONS: Our meta-analysis shows remarkable consistency in endometriosis GWAS results across studies, with little evidence of population-based heterogeneity. They also show that the phenotypic classifications used in GWAS to date have been limited. Stronger associations with Stage III/IV disease observed for most loci emphasize the importance for future studies to include detailed sub-phenotype information. Functional studies in relevant tissues are needed to understand the effect of the variants on downstream biological pathways.

Montgomery GW, Luong HTT, Jones L, Fung JNT, Painter JN, Zondervan KT, Nyholt DR, Rogers PA. 2014. Fine Mapping Genomic Regions Associated with Endometriosis Risk REPRODUCTIVE SCIENCES, 21 (3), pp. 312A-312A.

Rahmioglu N, Missmer SA, Montgomery GW, Zondervan KT. 2014. Meta-Analysis of GWAS Signals for Endometriosis to Date: Consistency and Heterogeneity across Eight Datasets Totaling More Than 11,000 Cases and 32,000 Controls REPRODUCTIVE SCIENCES, 21 (3), pp. 106A-107A.

Painter JN, Nyholt DR, Krause L, Zhao ZZ, Chapman B, Zhang C, Medland S, Martin NG, Kennedy S, Treloar S et al. 2014. Common variants in the CYP2C19 gene are associated with susceptibility to endometriosis Fertility and Sterility, 102 (2), | Show Abstract | Read more

Objective To follow-up previous studies highlighting a possible role for cytochrome P450, family 2, subfamily C, 19 (CYP2C19) in susceptibility to endometriosis by searching for additional variants in the CYP2C19 gene that may be associated with the disease. Design Case-control study. Setting Academic research. Subject(s) The cases comprised 2,271 women with surgically confirmed endometriosis; the controls comprised 939 women with self-report of no endometriosis and 1,770 unscreened population samples. Intervention(s) Sequencing of the CYP2C19 region and follow-up of 80 single nucleotide polymorphisms (SNPs) in two case-control samples. Main Outcome Measure(s) Allele frequency differences between cases and controls. Result(s) Sequencing of the CYP2C19 gene region resulted in the detection of a large number of known and novel SNPs. Genotyping of 80 polymorphic SNPs in 901 endometriosis cases and 939 controls resulted in study-wide significant association signals for SNPs in moderate or complete linkage disequilibrium with rs4244285, a functional SNP in exon 5 that abrogates CYP2C19 function through the creation of an alternative splice site. Evidence of association was also detected for another functional SNP in the CYP2C19 promoter, rs12248560, which was highlighted in our previous study. Conclusion(s) Functional variants in CYP2C19 may contribute to endometriosis susceptibility in both familial and sporadic cases. © 2014 by American Society for Reproductive Medicine.

Montgomery GW, Zondervan KT, Nyholt DR. 2014. The future for genetic studies in reproduction. Mol Hum Reprod, 20 (1), pp. 1-14. | Show Abstract | Read more

Genetic factors contribute to risk of many common diseases affecting reproduction and fertility. In recent years, methods for genome-wide association studies (GWAS) have revolutionized gene discovery for common traits and diseases. Results of GWAS are documented in the Catalog of Published Genome-Wide Association Studies at the National Human Genome Research Institute and report over 70 publications for 32 traits and diseases associated with reproduction. These include endometriosis, uterine fibroids, age at menarche and age at menopause. Results that pass appropriate stringent levels of significance are generally well replicated in independent studies. Examples of genetic variation affecting twinning rate, infertility, endometriosis and age at menarche demonstrate that the spectrum of disease-related variants for reproductive traits is similar to most other common diseases. GWAS 'hits' provide novel insights into biological pathways and the translational value of these studies lies in discovery of novel gene targets for biomarkers, drug development and greater understanding of environmental factors contributing to disease risk. Results also show that genetic data can help define sub-types of disease and co-morbidity with other traits and diseases. To date, many studies on reproductive traits have used relatively small samples. Future genetic marker studies in large samples with detailed phenotypic and clinical information will yield new insights into disease risk, disease classification and co-morbidity for many diseases associated with reproduction and infertility.

Lee SH, Harold D, Nyholt DR, ANZGene Consortium, International Endogene Consortium, Genetic and Environmental Risk for Alzheimer's disease Consortium, Goddard ME, Zondervan KT, Williams J, Montgomery GW et al. 2013. Estimation and partitioning of polygenic variation captured by common SNPs for Alzheimer's disease, multiple sclerosis and endometriosis. Hum Mol Genet, 22 (4), pp. 832-841. | Show Abstract | Read more

Common diseases such as endometriosis (ED), Alzheimer's disease (AD) and multiple sclerosis (MS) account for a significant proportion of the health care burden in many countries. Genome-wide association studies (GWASs) for these diseases have identified a number of individual genetic variants contributing to the risk of those diseases. However, the effect size for most variants is small and collectively the known variants explain only a small proportion of the estimated heritability. We used a linear mixed model to fit all single nucleotide polymorphisms (SNPs) simultaneously, and estimated genetic variances on the liability scale using SNPs from GWASs in unrelated individuals for these three diseases. For each of the three diseases, case and control samples were not all genotyped in the same laboratory. We demonstrate that a careful analysis can obtain robust estimates, but also that insufficient quality control (QC) of SNPs can lead to spurious results and that too stringent QC is likely to remove real genetic signals. Our estimates show that common SNPs on commercially available genotyping chips capture significant variation contributing to liability for all three diseases. The estimated proportion of total variation tagged by all SNPs was 0.26 (SE 0.04) for ED, 0.24 (SE 0.03) for AD and 0.30 (SE 0.03) for MS. Further, we partitioned the genetic variance explained into five categories by a minor allele frequency (MAF), by chromosomes and gene annotation. We provide strong evidence that a substantial proportion of variation in liability is explained by common SNPs, and thereby give insights into the genetic architecture of the diseases.

Nyholt DR, Low SK, Anderson CA, Painter JN, Uno S, Morris AP, MacGregor S, Gordon SD, Henders AK, Martin NG et al. 2012. Genome-wide association meta-analysis identifies new endometriosis risk loci. Nat Genet, 44 (12), pp. 1355-1359. | Show Abstract | Read more

We conducted a genome-wide association meta-analysis of 4,604 endometriosis cases and 9,393 controls of Japanese and European ancestry. We show that rs12700667 on chromosome 7p15.2, previously found to associate with disease in Europeans, replicates in Japanese (P = 3.6 × 10(-3)), and we confirm association of rs7521902 at 1p36.12 near WNT4. In addition, we establish an association of rs13394619 in GREB1 at 2p25.1 with endometriosis and identify a newly associated locus at 12q22 near VEZT (rs10859871). Excluding cases of European ancestry of minimal or unknown severity, we identified additional previously unknown loci at 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21.3 (rs1537377). All seven SNP effects were replicated in an independent cohort and associated at P <5 × 10(-8) in a combined analysis. Finally, we found a significant overlap in polygenic risk for endometriosis between the genome-wide association cohorts of European and Japanese descent (P = 8.8 × 10(-11)), indicating that many weakly associated SNPs represent true endometriosis risk loci and that risk prediction and future targeted disease therapy may be transferred across these populations.

Eggert SL, Huyck KL, Somasundaram P, Kavalla R, Stewart EA, Lu AT, Painter JN, Montgomery GW, Medland SE, Nyholt DR et al. 2012. Genome-wide linkage and association analyses implicate FASN in predisposition to Uterine Leiomyomata. Am J Hum Genet, 91 (4), pp. 621-628. | Show Abstract | Read more

Uterine leiomyomata (UL), the most prevalent pelvic tumors in women of reproductive age, pose a major public health problem given their high frequency, associated morbidities, and most common indication for hysterectomies. A genetic component to UL predisposition is supported by analyses of ethnic predisposition, twin studies, and familial aggregation. A genome-wide SNP linkage panel was genotyped and analyzed in 261 white UL-affected sister-pair families from the Finding Genes for Fibroids study. Two significant linkage regions were detected in 10p11 (LOD = 4.15) and 3p21 (LOD = 3.73), and five additional linkage regions were identified with LOD scores > 2.00 in 2q37, 5p13, 11p15, 12q14, and 17q25. Genome-wide association studies were performed in two independent cohorts of white women, and a meta-analysis was conducted. One SNP (rs4247357) was identified with a p value (p = 3.05 × 10(-8)) that reached genome-wide significance (odds ratio = 1.299). The candidate SNP is under a linkage peak and in a block of linkage disequilibrium in 17q25.3, which spans fatty acid synthase (FASN), coiled-coil-domain-containing 57 (CCDC57), and solute-carrier family 16, member 3 (SLC16A3). By tissue microarray immunohistochemistry, we found elevated (3-fold) FAS levels in UL-affected tissue compared to matched myometrial tissue. FAS transcripts and/or protein levels are upregulated in various neoplasms and implicated in tumor cell survival. FASN represents the initial UL risk allele identified in white women by a genome-wide, unbiased approach and opens a path to management and potential therapeutic intervention.

Luong HT, Nyholt DR, Painter JN, Chapman B, Kennedy S, Treloar SA, Zondervan KT, Montgomery GW. 2012. No evidence for genetic association with the let-7 microRNA-binding site or other common KRAS variants in risk of endometriosis. Hum Reprod, 27 (12), pp. 3616-3621. | Show Abstract | Read more

STUDY QUESTION: Is there a contribution of the minor allele at the KRAS single nucleotide polymorphism (SNP) rs61764370 in the let-7 microRNA-binding site to endometriosis risk? SUMMARY ANSWER: We found no evidence for association between endometriosis risk and rs61764370 or any other SNPs in KRAS. WHAT IS KNOWN ALREADY: The rs61764370 SNP in the 3' untranslated region of the KRAS gene is predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA, and was recently reported to be at a high frequency (31%) in 132 women of varying ancestry with endometriosis compared with frequencies in a database of population controls (up to 7.6% depending on ancestry), suggesting a strong effect of this KRAS SNP in the aetiology of endometriosis. STUDY DESIGN, SIZE AND DURATION: This was a case-control study with a total of 11 206 subjects. The study was performed between February 2012 and July 2012. PARTICIPANTS/MATERIALS, SETTINGAND METHODS: We first investigated a possible association between common markers in KRAS and endometriosis risk from our genome-wide association (GWA) data in 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry. Although rs61764370 was not genotyped on the GWA arrays, five SNPs typed in the study were highly correlated with this variant. The rs61764370 and two SNPs highly correlated with rs61764370 were then genotyped in 933 endometriosis cases and 952 controls using the Sequenom MassARRAY platform. MAIN RESULTS AND THE ROLE OF CHANCE: There was no evidence for an association between rs61764370 and endometriosis risk P = 0.411 and odds ratio = 1.10 (95% confidence intervals: 0.88-1.36). We also found no evidence for an association between the highly correlated SNP rs17387019 and endometriosis. Their minor allele frequencies in cases and controls were of 0.087-0.091 similar to the population frequency reported previously for this variant in controls. Analyses of endometriosis cases with revised American Fertility Society stage III/IV disease also showed no evidence for an association between these SNPs and endometriosis risk. LIMITATIONS AND REASONS FOR CAUTION: The GWA and genotyped data sets were not independent since individuals and cases from some families overlap. Controls in our GWA study were not screened for endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The key SNP, rs61764370, was genotyped in a subset of samples. Our results do not support the suggestion that carrying the minor allele at rs61764370 contributes to a significant number of endometriosis cases and rs61764370 is, therefore, unlikely to be a useful marker of endometriosis risk. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Grundberg E, Small KS, Hedman ÅK, Nica AC, Buil A, Keildson S, Bell JT, Yang TP, Meduri E, Barrett A et al. 2012. Mapping cis- and trans-regulatory effects across multiple tissues in twins. Nat Genet, 44 (10), pp. 1084-1089. | Show Abstract | Read more

Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many expression quantitative trait locus (eQTL) studies, typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis effect on expression cannot be accounted for by common cis variants, a finding that reveals the contribution of low-frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene, and we identify several replicating trans variants that act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.

Rahmioglu N, Missmer SA, Montgomery GW, Zondervan KT. 2012. Insights into Assessing the Genetics of Endometriosis. Curr Obstet Gynecol Rep, 1 (3), pp. 124-137. | Show Abstract | Read more

Endometriosis is a complex disease arising from the interplay between multiple genetic and environmental factors. The genetic variants potentially underlying the hereditary component of endometriosis have been widely investigated through hypothesis-driven candidate gene studies, an approach that generally has proven to be inherently difficult and problematic for a number of reasons. Recently, through major collaborative efforts in the endometriosis research field, hypothesis-free genome-wide approaches have started to provide new insights into potential pathways leading to development of endometriosis, as well as highlighting the phenotypic heterogeneity of the condition. This review summarizes the most recent studies investigating the genetic variation contributing to endometriosis, with a particular focus on genome-wide approaches, and discusses promising future directions of genetic research.

Nnoaham KE, Webster P, Kumbang J, Kennedy SH, Zondervan KT. 2012. Is early age at menarche a risk factor for endometriosis? A systematic review and meta-analysis of case-control studies. Fertil Steril, 98 (3), pp. 702-712.e6. | Show Abstract | Read more

OBJECTIVE: To review published studies evaluating early menarche and the risk of endometriosis. DESIGN: Systematic review and meta-analysis of case-control studies. SETTING: None. PATIENT(S): Eighteen case-control studies of age at menarche and risk of endometriosis including 3,805 women with endometriosis and 9,526 controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Medline and Embase databases were searched from 1980 to 2011 to locate relevant studies. Results of primary studies were expressed as effect sizes of the difference in mean age at menarche of women with and without endometriosis. Effect sizes were used in random effects meta-analysis. RESULT(S): Eighteen of 45 articles retrieved met the inclusion criteria. The pooled effect size in meta-analysis was 0.10 (95% confidence interval -0.01-0.21), and not significantly different from zero (no effect). Results were influenced by substantial heterogeneity between studies (I(2) = 72.5%), which was eliminated by restricting meta-analysis to studies with more rigorous control of confounders; this increased the pooled effect size to 0.15 (95% confidence interval 0.08-0.22), which was significantly different from zero. This represents a probability of 55% that a woman with endometriosis had earlier menarche than one without endometriosis if both were randomly chosen from a population. CONCLUSION(S): There is a small increased risk of endometriosis with early menarche. The potential for disease misclassification in primary studies suggests that this risk could be higher.

Nnoaham KE, Hummelshoj L, Kennedy SH, Jenkinson C, Zondervan KT, World Endometriosis Research Foundation Women's Health Symptom Survey Consortium. 2012. Developing symptom-based predictive models of endometriosis as a clinical screening tool: results from a multicenter study. Fertil Steril, 98 (3), pp. 692-701.e5. | Show Abstract | Read more

OBJECTIVE: To generate and validate symptom-based models to predict endometriosis among symptomatic women prior to undergoing their first laparoscopy. DESIGN: Prospective, observational, two-phase study, in which women completed a 25-item questionnaire prior to surgery. SETTING: Nineteen hospitals in 13 countries. PATIENT(S): Symptomatic women (n = 1,396) scheduled for laparoscopy without a previous surgical diagnosis of endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sensitivity and specificity of endometriosis diagnosis predicted by symptoms and patient characteristics from optimal models developed using multiple logistic regression analyses in one data set (phase I), and independently validated in a second data set (phase II) by receiver operating characteristic (ROC) curve analysis. RESULT(S): Three hundred sixty (46.7%) women in phase I and 364 (58.2%) in phase II were diagnosed with endometriosis at laparoscopy. Menstrual dyschezia (pain on opening bowels) and a history of benign ovarian cysts most strongly predicted both any and stage III and IV endometriosis in both phases. Prediction of any-stage endometriosis, although improved by ultrasound scan evidence of cyst/nodules, was relatively poor (area under the curve [AUC] = 68.3). Stage III and IV disease was predicted with good accuracy (AUC = 84.9, sensitivity of 82.3% and specificity 75.8% at an optimal cut-off of 0.24). CONCLUSION(S): Our symptom-based models predict any-stage endometriosis relatively poorly and stage III and IV disease with good accuracy. Predictive tools based on such models could help to prioritize women for surgical investigation in clinical practice and thus contribute to reducing time to diagnosis. We invite other researchers to validate the key models in additional populations.

Montgomery GW, Nyholt DR, Macgregor S, Painter JN, Luong HTT, Dinger ME, Morris AP, Treloar SA, Missmer SA, Rogers PAW, Zondervan KT. 2012. Identifying the Genes Associated with Endometriosis Risk on Chromosomes 1 and 7 REPRODUCTIVE SCIENCES, 19 (S3), pp. 141A-141A.

Min JL, Nicholson G, Halgrimsdottir I, Almstrup K, Petri A, Barrett A, Travers M, Rayner NW, Mägi R, Pettersson FH et al. 2012. Coexpression network analysis in abdominal and gluteal adipose tissue reveals regulatory genetic loci for metabolic syndrome and related phenotypes. PLoS Genet, 8 (2), pp. e1002505. | Show Abstract | Read more

Metabolic Syndrome (MetS) is highly prevalent and has considerable public health impact, but its underlying genetic factors remain elusive. To identify gene networks involved in MetS, we conducted whole-genome expression and genotype profiling on abdominal (ABD) and gluteal (GLU) adipose tissue, and whole blood (WB), from 29 MetS cases and 44 controls. Co-expression network analysis for each tissue independently identified nine, six, and zero MetS-associated modules of coexpressed genes in ABD, GLU, and WB, respectively. Of 8,992 probesets expressed in ABD or GLU, 685 (7.6%) were expressed in ABD and 51 (0.6%) in GLU only. Differential eigengene network analysis of 8,256 shared probesets detected 22 shared modules with high preservation across adipose depots (D(ABD-GLU) = 0.89), seven of which were associated with MetS (FDR P<0.01). The strongest associated module, significantly enriched for immune response-related processes, contained 94/620 (15%) genes with inter-depot differences. In an independent cohort of 145/141 twins with ABD and WB longitudinal expression data, median variability in ABD due to familiality was greater for MetS-associated versus un-associated modules (ABD: 0.48 versus 0.18, P = 0.08; GLU: 0.54 versus 0.20, P = 7.8×10(-4)). Cis-eQTL analysis of probesets associated with MetS (FDR P<0.01) and/or inter-depot differences (FDR P<0.01) provided evidence for 32 eQTLs. Corresponding eSNPs were tested for association with MetS-related phenotypes in two GWAS of >100,000 individuals; rs10282458, affecting expression of RARRES2 (encoding chemerin), was associated with body mass index (BMI) (P = 6.0×10(-4)); and rs2395185, affecting inter-depot differences of HLA-DRB1 expression, was associated with high-density lipoprotein (P = 8.7×10(-4)) and BMI-adjusted waist-to-hip ratio (P = 2.4×10(-4)). Since many genes and their interactions influence complex traits such as MetS, integrated analysis of genotypes and coexpression networks across multiple tissues relevant to clinical traits is an efficient strategy to identify novel associations.

Painter JN, Zondervan KT, Montgomery GW. 2012. Understanding the Pathogenesis of Endometriosis: Gene Mapping Studies pp. 54-64. | Show Abstract | Read more

Endometriosis is a common gynecological disease associated with severe pelvic pain and subfertility. Genetic variation contributes to disease risk and gene mapping studies offer an important approach to understanding the biology of endometriosis. Candidate gene studies have not identified any genes robustly associated with endometriosis, since most published studies had limited power to detect the modest effects expected for individual risk variants. Linkage studies for the disease identified several linkage peaks, but the gene(s) contributing to these linkage signals have not been identified. Genome-wide association studies provide a powerful approach to the discovery of genes or variants contributing to disease risk and several groups have now conducted genome-wide association studies for endometriosis. While these studies have highlighted a number of chromosomal areas harboring interesting candidate genes that should be investigated further, based on experience with other diseases we will need to collect further large and well-characterized samples for endometriosis to identify a comprehensive set of the variants contributing most to endometriosis risk. © 2012 Blackwell Publishing Ltd.

Cited:

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Nnoaham KE, Hummelshoj L, Kennedy SH, Jenkinson C, Zondervan KT. 2012. Developing symptom-based predictive models of endometriosis as a clinical screening tool: Results from a multicenter study Fertility and Sterility, 98 (3), | Show Abstract | Read more

Objective: To generate and validate symptom-based models to predict endometriosis among symptomatic women prior to undergoing their first laparoscopy. Design: Prospective, observational, two-phase study, in which women completed a 25-item questionnaire prior to surgery. Setting: Nineteen hospitals in 13 countries. Patient(s): Symptomatic women (n = 1,396) scheduled for laparoscopy without a previous surgical diagnosis of endometriosis. Intervention(s): None. Main Outcome Measure(s): Sensitivity and specificity of endometriosis diagnosis predicted by symptoms and patient characteristics from optimal models developed using multiple logistic regression analyses in one data set (phase I), and independently validated in a second data set (phase II) by receiver operating characteristic (ROC) curve analysis. Result(s): Three hundred sixty (46.7%) women in phase I and 364 (58.2%) in phase II were diagnosed with endometriosis at laparoscopy. Menstrual dyschezia (pain on opening bowels) and a history of benign ovarian cysts most strongly predicted both any and stage III and IV endometriosis in both phases. Prediction of any-stage endometriosis, although improved by ultrasound scan evidence of cyst/nodules, was relatively poor (area under the curve [AUC] = 68.3). Stage III and IV disease was predicted with good accuracy (AUC = 84.9, sensitivity of 82.3% and specificity 75.8% at an optimal cut-off of 0.24). Conclusion(s): Our symptom-based models predict any-stage endometriosis relatively poorly and stage III and IV disease with good accuracy. Predictive tools based on such models could help to prioritize women for surgical investigation in clinical practice and thus contribute to reducing time to diagnosis. We invite other researchers to validate the key models in additional populations. © 2012 by American Society for Reproductive Medicine.

Cited:

120

Scopus

Nyholt DR, Low SK, Anderson CA, Painter JN, Uno S, Morris AP, MacGregor S, Gordon SD, Henders AK, Martin NG et al. 2012. Genome-wide association meta-analysis identifies new endometriosis risk loci Nature Genetics, 44 (12), pp. 1355-1359. | Show Abstract | Read more

We conducted a genome-wide association meta-analysis of 4,604 endometriosis cases and 9,393 controls of Japanese and European ancestry. We show that rs12700667 on chromosome 7p15.2, previously found to associate with disease in Europeans, replicates in Japanese (P = 3.6 × 10-3), and we confirm association of rs7521902 at 1p36.12 near WNT4. In addition, we establish an association of rs13394619 in GREB1 at 2p25.1 with endometriosis and identify a newly associated locus at 12q22 near VEZT (rs10859871). Excluding cases of European ancestry of minimal or unknown severity, we identified additional previously unknown loci at 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21.3 (rs1537377). All seven SNP effects were replicated in an independent cohort and associated at P < 5 × 10-8 in a combined analysis. Finally, we found a significant overlap in polygenic risk for endometriosis between the genome-wide association cohorts of European and Japanese descent (P = 8.8 × 10-11), indicating that many weakly associated SNPs represent true endometriosis risk loci and that risk prediction and future targeted disease therapy may be transferred across these populations. © 2012 Nature America, Inc. All rights reserved.

Cited:

27

Scopus

Nnoaham KE, Webster P, Kumbang J, Kennedy SH, Zondervan KT. 2012. Is early age at menarche a risk factor for endometriosis? A systematic review and meta-analysis of case-control studies Fertility and Sterility, 98 (3), | Show Abstract | Read more

Objective: To review published studies evaluating early menarche and the risk of endometriosis. Design: Systematic review and meta-analysis of case-control studies. Setting: None. Patient(s): Eighteen case-control studies of age at menarche and risk of endometriosis including 3,805 women with endometriosis and 9,526 controls. Intervention(s): None. Main Outcome Measure(s): Medline and Embase databases were searched from 1980 to 2011 to locate relevant studies. Results of primary studies were expressed as effect sizes of the difference in mean age at menarche of women with and without endometriosis. Effect sizes were used in random effects meta-analysis. Result(s): Eighteen of 45 articles retrieved met the inclusion criteria. The pooled effect size in meta-analysis was 0.10 (95% confidence interval -0.01-0.21), and not significantly different from zero (no effect). Results were influenced by substantial heterogeneity between studies (I 2 = 72.5%), which was eliminated by restricting meta-analysis to studies with more rigorous control of confounders; this increased the pooled effect size to 0.15 (95% confidence interval 0.08-0.22), which was significantly different from zero. This represents a probability of 55% that a woman with endometriosis had earlier menarche than one without endometriosis if both were randomly chosen from a population. Conclusion(s): There is a small increased risk of endometriosis with early menarche. The potential for disease misclassification in primary studies suggests that this risk could be higher. © 2012 by American Society for Reproductive Medicine.

Dancet EA, Ameye L, Sermeus W, Welkenhuysen M, Nelen WL, Tully L, De Bie B, Veit J, Vedsted-Hansen H, Zondervan KT et al. 2011. The ENDOCARE questionnaire (ECQ): a valid and reliable instrument to measure the patient-centeredness of endometriosis care in Europe. Hum Reprod, 26 (11), pp. 2988-2999. | Show Abstract | Read more

BACKGROUND: Endometriosis is prevalent and women need high-quality care, which should be patient-centered. This study aimed to develop a valid and reliable patient-centeredness questionnaire, based on a defined concept of patient-centered endometriosis care (PCEC). METHODS: A literature review, focus groups (FGs) with patients and an expert panel defined PCEC with 10 dimensions. The ENDOCARE questionnaire (ECQ) was developed. FGs resulted in 43 specific statements covering the 10 dimensions of PCEC, for which the ECQ measured 'importance' and 'performance'. Medical and demographic questions and an open question were added. The Dutch ECQ questionnaire was piloted and reciprocally translated into English and Italian. Patients with endometriosis from Belgium, The Netherlands, Italy and the UK were invited to complete the ECQ online. Item analysis, inter-item analysis and confirmatory and exploratory factor analyses (EFA) and reliability analysis were performed. The theory-driven dimensions were adapted. RESULTS: The ECQ was completed by 541 patients. Based on item analysis, five statements were deleted. Factor analysis was performed on 322 questionnaires (only from respondents with a partner). Insights from the data-driven EFA suggested adaptations of the theory-driven dimensions. The reliability statistics of 9/10 adapted theory-driven dimensions were satisfactory and the root mean square error of approximation was good. CONCLUSIONS: This study resulted in a valid and reliable instrument to measure PCEC. For data presentation, the adapted theory-driven dimensions of PCEC are preferred over the data-driven factors. The ECQ may serve to benchmark patient-centeredness, conduct cross-cultural European research and set targets for improvement.

Nicholson G, Rantalainen M, Li JV, Maher AD, Malmodin D, Ahmadi KR, Faber JH, Barrett A, Min JL, Rayner NW et al. 2011. A genome-wide metabolic QTL analysis in Europeans implicates two loci shaped by recent positive selection. PLoS Genet, 7 (9), pp. e1002270. | Show Abstract | Read more

We have performed a metabolite quantitative trait locus (mQTL) study of the (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) metabolome in humans, building on recent targeted knowledge of genetic drivers of metabolic regulation. Urine and plasma samples were collected from two cohorts of individuals of European descent, with one cohort comprised of female twins donating samples longitudinally. Sample metabolite concentrations were quantified by (1)H NMR and tested for association with genome-wide single-nucleotide polymorphisms (SNPs). Four metabolites' concentrations exhibited significant, replicable association with SNP variation (8.6×10(-11)<p<2.8×10(-23)). Three of these-trimethylamine, 3-amino-isobutyrate, and an N-acetylated compound-were measured in urine. The other-dimethylamine-was measured in plasma. Trimethylamine and dimethylamine mapped to a single genetic region (hence we report a total of three implicated genomic regions). Two of the three hit regions lie within haplotype blocks (at 2p13.1 and 10q24.2) that carry the genetic signature of strong, recent, positive selection in European populations. Genes NAT8 and PYROXD2, both with relatively uncharacterized functional roles, are good candidates for mediating the corresponding mQTL associations. The study's longitudinal twin design allowed detailed variance-components analysis of the sources of population variation in metabolite levels. The mQTLs explained 40%-64% of biological population variation in the corresponding metabolites' concentrations. These effect sizes are stronger than those reported in a recent, targeted mQTL study of metabolites in serum using the targeted-metabolomics Biocrates platform. By re-analysing our plasma samples using the Biocrates platform, we replicated the mQTL findings of the previous study and discovered a previously uncharacterized yet substantial familial component of variation in metabolite levels in addition to the heritability contribution from the corresponding mQTL effects.

Nnoaham KE, Hummelshoj L, Webster P, d'Hooghe T, de Cicco Nardone F, de Cicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT, World Endometriosis Research Foundation Global Study of Women's Health consortium. 2011. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril, 96 (2), pp. 366-373.e8. | Show Abstract | Read more

OBJECTIVE: To assess the impact of endometriosis on health-related quality of life (HRQoL) and work productivity. DESIGN: Multicenter cross-sectional study with prospective recruitment. SETTING: Sixteen clinical centers in ten countries. PATIENT(S): A total of 1,418 premenopausal women, aged 18-45 years, without a previous surgical diagnosis of endometriosis, having laparoscopy to investigate symptoms or to be sterilized. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Diagnostic delay, HRQoL, and work productivity. RESULT(S): There was a delay of 6.7 years, principally in primary care, between onset of symptoms and a surgical diagnosis of endometriosis, which was longer in centers where women received predominantly state-funded health care (8.3 vs. 5.5 years). Delay was positively associated with the number of pelvic symptoms (chronic pelvic pain, dysmenorrhoea, dyspareunia, and heavy periods) and a higher body mass index. Physical HRQoL was significantly reduced in affected women compared with those with similar symptoms and no endometriosis. Each affected woman lost on average 10.8 hours (SD 12.2) of work weekly, mainly owing to reduced effectiveness while working. Loss of work productivity translated into significant costs per woman/week, from US$4 in Nigeria to US$456 in Italy. CONCLUSION(S): Endometriosis impairs HRQoL and work productivity across countries and ethnicities, yet women continue to experience diagnostic delays in primary care. A higher index of suspicion is needed to expedite specialist assessment of symptomatic women. Future research should seek to clarify pain mechanisms in relation to endometriosis severity.

Lin J, Zong L, Kennedy SH, Zondervan KT. 2011. Coding regions of INHBA, SFRP4 and HOXA10 are not implicated in familial endometriosis linked to chromosome 7p13-15. Mol Hum Reprod, 17 (10), pp. 605-611. | Show Abstract | Read more

Endometriosis is a common, chronic gynaecological disease affecting up to 10% of women in their reproductive years. Its aetiology still remains unclear, but evidence indicates genetic factors play a role. We previously identified a region of significant linkage on chromosome 7 in 52 families comprising at least three affected women, stretching ∼6.4 Mb. We screened coding regions and parts of the regulatory regions of three candidate genes with a known role in endometrial development and function-INHBA, SFRP4 and HOXA10-located under or very near the linkage peak, for potential causal mutations using Sanger sequencing. Sequencing was conducted in 47 cases from the 15 families contributing most to the linkage signal (Z(mean) ≥ 1). Minor allele frequencies (MAFs) of observed variants were compared with MAFs from two publicly available reference populations of European ancestry: 60 individuals in HapMap and 150 individuals in the 1000 Genomes Project. A total of 11 variants were found, 5 (45%) of which were common (MAF > 0.05) among the 15 case families and the reference populations (P-values for MAF difference: 0.88-1.00). The remaining six were rare and unlikely to be individually or cumulatively responsible for the linkage signal. The results indicate that the coding regions of these three genes do not harbour mutations responsible for linkage to endometriosis in these families.

Painter JN, Nyholt DR, Morris A, Zhao ZZ, Henders AK, Lambert A, Wallace L, Martin NG, Kennedy SH, Treloar SA et al. 2011. High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19. Fertil Steril, 95 (7), pp. 2236-2240. | Show Abstract | Read more

OBJECTIVE: To refine a previously reported linkage peak for endometriosis on chromosome 10q26, and conduct follow-up analyses and a fine-mapping association study across the region to identify new candidate genes for endometriosis. DESIGN: Case-control study. SETTING: Academic research. PATIENT(S): Cases=3,223 women with surgically confirmed endometriosis; controls=1,190 women without endometriosis and 7,060 population samples. INTERVENTION(S): Analysis of 11,984 single nucleotide polymorphisms on chromosome 10. MAIN OUTCOME MEASURE(S): Allele frequency differences between cases and controls. RESULT(S): Linkage analyses on families grouped by endometriosis symptoms (primarily subfertility) provided increased evidence for linkage (logarithm of odds score=3.62) near a previously reported linkage peak. Three independent association signals were found at 96.59 Mb (rs11592737), 105.63 Mb (rs1253130), and 124.25 Mb (rs2250804). Analyses including only samples from linkage families supported the association at all three regions. However, only rs11592737 in the cytochrome P450 subfamily C (CYP2C19) gene was replicated in an independent sample of 2,079 cases and 7,060 population controls. CONCLUSION(S): The role of the CYP2C19 gene in conferring risk for endometriosis warrants further investigation.

Painter JN, Anderson CA, Nyholt DR, Macgregor S, Lin J, Lee SH, Lambert A, Zhao ZZ, Roseman F, Guo Q et al. 2011. Genome-Wide Association Study Identifies a Locus at 7p15.2 Associated With Endometriosis OBSTETRICAL & GYNECOLOGICAL SURVEY, 66 (4), pp. 214-216. | Read more

Zondervan KT, Nnoaham KE, Hummelshoj L, Sivananthan S, Webster P, Jenkinson C, Kennedy SH. 2011. Clinical Symptoms as Predictors of a Surgical Diagnosis of Endometriosis REPRODUCTIVE SCIENCES, 18 (3), pp. 275A-275A.

Zondervan KT, Painter JN, Anderson CA, Nyholt DR, Macgregor S, Lee H, Visscher PM, Kraft P, Martin NG, Morris AP et al. 2011. Genome-Wide Association Study of Endometriosis Shows Differential Etiology by Stage and Identifies a Locus at 7p15.2 Associated with the Development of Moderate-Severe Disease REPRODUCTIVE SCIENCES, 18 (3), pp. 197A-197A.

Clarke GM, Anderson CA, Pettersson FH, Cardon LR, Morris AP, Zondervan KT. 2011. Basic statistical analysis in genetic case-control studies. Nat Protoc, 6 (2), pp. 121-133. | Show Abstract | Read more

This protocol describes how to perform basic statistical analysis in a population-based genetic association case-control study. The steps described involve the (i) appropriate selection of measures of association and relevance of disease models; (ii) appropriate selection of tests of association; (iii) visualization and interpretation of results; (iv) consideration of appropriate methods to control for multiple testing; and (v) replication strategies. Assuming no previous experience with software such as PLINK, R or Haploview, we describe how to use these popular tools for handling single-nucleotide polymorphism data in order to carry out tests of association and visualize and interpret results. This protocol assumes that data quality assessment and control has been performed, as described in a previous protocol, so that samples and markers deemed to have the potential to introduce bias to the study have been identified and removed. Study design, marker selection and quality control of case-control studies have also been discussed in earlier protocols. The protocol should take ~1 h to complete.

Zondervan KT. 2011. Genetic Association Study Design pp. 25-48. | Show Abstract | Read more

Association studies can be performed using any type of genetic polymorphism, but the one most commonly used is the single nucleotide polymorphism (SNP). This is because of its abundance across the genome, comprising 90% of all human variation. The aim of this chapter is to review various aspects of population-based study design, aimed at uncovering common genetic variations underlying complex traits. There is the realization that many of these variants lie ingene deserts and their biological function is not obvious. These variants tend to explain only very small proportions of overall heritability, leaving many wondering where the missing heritability can be found. The first type of genetic association study to be conducted, before the availability of dense SNP LD maps, was the candidate gene study. Most candidate gene studies have suffered from limitations in study design, including inappropriate case definition and control selection and-most importantly-vastly inadequate sample sizes. Despite early concerns about multiple testing issues and power, GWA studies have brought the gene mapping successes for complex traits, which candidate gene studies were largely unable to deliver. Following this, the chapter also presents the population-based study design. The most popular population-based study design for binary traits (whether focusing on candidate genes, regions, or the entire genome) is the case-control study. In a case-control study, a set of cases is identified and their genotyping information is compared to a set of suitable controls in order to find genetic variants associated with the trait under study. © 2011 Elsevier Inc. All rights reserved.

Strawbridge RJ, Dupuis J, Prokopenko I, Barker A, Ahlqvist E, Rybin D, Petrie JR, Travers ME, Bouatia-Naji N, Dimas AS et al. 2011. Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes. Diabetes, 60 (10), pp. 2624-2634. | Show Abstract | Read more

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.

Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, Steinthorsdottir V, Thorleifsson G, Zillikens MC, Speliotes EK, Mägi R et al. 2011. Erratum: Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution (Nature Genetics (2010) 42 (949-960)) Nature Genetics, 43 (11), pp. 1164. | Read more

Cited:

160

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Painter JN, Anderson CA, Nyholt DR, MacGregor S, Lin J, Lee SH, Lambert A, Zhao ZZ, Roseman F, Guo Q et al. 2011. Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis Nature Genetics, 43 (1), pp. 51-54. | Show Abstract | Read more

Endometriosis is a common gynecological disease associated with pelvic pain and subfertility. We conducted a genome-wide association study (GWAS) in 3,194 individuals with surgically confirmed endometriosis (cases) and 7,060 controls from Australia and the UK. Polygenic predictive modeling showed significantly increased genetic loading among 1,364 cases with moderate to severe endometriosis. The strongest association signal was on 7p15.2 (rs12700667) for 'all' endometriosis (P = 2.6 ×- 10- 7 , odds ratio (OR) = 1.22, 95% CI 1.13-1.32) and for moderate to severe disease (P = 1.5 ×- 10 -9 , OR = 1.38, 95% CI 1.24-1.53). We replicated rs12700667 in an independent cohort from the United States of 2,392 self-reported, surgically confirmed endometriosis cases and 2,271 controls (P = 1.2 ×- 10 -3 , OR = 1.17, 95% CI 1.06-1.28), resulting in a genome-wide significant P value of 1.4 ×- 10 -9 (OR = 1.20, 95% CI 1.13-1.27) for 'all' endometriosis in our combined datasets of 5,586 cases and 9,331 controls. rs12700667 is located in an intergenic region upstream of the plausible candidate genes NFE2L3 and HOXA10. © 2011 Nature America, Inc. All rights reserved.

Painter JN, Nyholt DR, Morris A, Zhao ZZ, Henders AK, Lambert A, Wallace L, Martin NG, Kennedy SH, Treloar SA et al. 2011. High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19 Fertility and Sterility, 95 (7), pp. 2236-2240. | Show Abstract | Read more

Objective: To refine a previously reported linkage peak for endometriosis on chromosome 10q26, and conduct follow-up analyses and a fine-mapping association study across the region to identify new candidate genes for endometriosis. Design: Case-control study. Setting: Academic research. Patient(s): Cases = 3,223 women with surgically confirmed endometriosis; controls = 1,190 women without endometriosis and 7,060 population samples. Intervention(s): Analysis of 11,984 single nucleotide polymorphisms on chromosome 10. Main Outcome Measure(s): Allele frequency differences between cases and controls. Result(s): Linkage analyses on families grouped by endometriosis symptoms (primarily subfertility) provided increased evidence for linkage (logarithm of odds score = 3.62) near a previously reported linkage peak. Three independent association signals were found at 96.59 Mb (rs11592737), 105.63 Mb (rs1253130), and 124.25 Mb (rs2250804). Analyses including only samples from linkage families supported the association at all three regions. However, only rs11592737 in the cytochrome P450 subfamily C (CYP2C19) gene was replicated in an independent sample of 2,079 cases and 7,060 population controls. Conclusion(s): The role of the CYP2C19 gene in conferring risk for endometriosis warrants further investigation. © 2011 by American Society for Reproductive Medicine.

Cited:

71

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Nicholson G, Rantalainen M, Maher AD, Li JV, Malmodin D, Ahmadi KR, Faber JH, Hallgrímsdóttir IB, Barrett A, Toft H et al. 2011. Human metabolic profiles are stably controlled by genetic and environmental variation Molecular Systems Biology, 7 | Show Abstract | Read more

1 H Nuclear Magnetic Resonance spectroscopy (1 H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top-down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non-identical twin pairs donated plasma and urine samples longitudinally. We acquired 1 H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common-environmental), individual-environmental, and longitudinally unstable components. We estimate t hat stable variation, comprising familial and individual-environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in 1 H NMR-detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker-discovery studies. We provide a power-calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect 1 H NMR-based biomarkers quantifying predisposition to disease. © 2011 EMBO and Macmillan Publishers Limited All rights reserved.

Nicholson G, Rantalainen M, Maher AD, Li JV, Malmodin D, Ahmadi KR, Faber JH, Hallgrímsdóttir IB, Barrett A, Toft H et al. 2011. Human metabolic profiles are stably controlled by genetic and environmental variation. Mol Syst Biol, 7 (1), pp. 525. | Show Abstract | Read more

¹H Nuclear Magnetic Resonance spectroscopy (¹H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top-down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non-identical twin pairs donated plasma and urine samples longitudinally. We acquired ¹H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common-environmental), individual-environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual-environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in ¹H NMR-detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker-discovery studies. We provide a power-calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect ¹H NMR-based biomarkers quantifying predisposition to disease.

Cited:

235

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Nnoaham KE, Hummelshoj L, Webster P, D'Hooghe T, De Cicco Nardone F, De Cicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT. 2011. Impact of endometriosis on quality of life and work productivity: A multicenter study across ten countries Fertility and Sterility, 96 (2), | Show Abstract | Read more

Objective: To assess the impact of endometriosis on health-related quality of life (HRQoL) and work productivity. Design: Multicenter cross-sectional study with prospective recruitment. Setting: Sixteen clinical centers in ten countries. Patient(s): A total of 1,418 premenopausal women, aged 18-45 years, without a previous surgical diagnosis of endometriosis, having laparoscopy to investigate symptoms or to be sterilized. Intervention(s): None. Main Outcome Measure(s): Diagnostic delay, HRQoL, and work productivity. Result(s): There was a delay of 6.7 years, principally in primary care, between onset of symptoms and a surgical diagnosis of endometriosis, which was longer in centers where women received predominantly state-funded health care (8.3 vs. 5.5 years). Delay was positively associated with the number of pelvic symptoms (chronic pelvic pain, dysmenorrhoea, dyspareunia, and heavy periods) and a higher body mass index. Physical HRQoL was significantly reduced in affected women compared with those with similar symptoms and no endometriosis. Each affected woman lost on average 10.8 hours (SD 12.2) of work weekly, mainly owing to reduced effectiveness while working. Loss of work productivity translated into significant costs per woman/week, from US$4 in Nigeria to US$456 in Italy. Conclusion(s): Endometriosis impairs HRQoL and work productivity across countries and ethnicities, yet women continue to experience diagnostic delays in primary care. A higher index of suspicion is needed to expedite specialist assessment of symptomatic women. Future research should seek to clarify pain mechanisms in relation to endometriosis severity. Copyright © 2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, Steinthorsdottir V, Thorleifsson G, Zillikens MC, Speliotes EK, Mägi R et al. 2011. Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution. Nat Genet, 43 (11), pp. 1164. | Read more

Rantalainen M, Herrera BM, Nicholson G, Bowden R, Wills QF, Min JL, Neville MJ, Barrett A, Allen M, Rayner NW et al. 2011. MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven. PLoS One, 6 (11), pp. e27338. | Show Abstract | Read more

To understand how miRNAs contribute to the molecular phenotype of adipose tissues and related traits, we performed global miRNA expression profiling in subcutaneous abdominal and gluteal adipose tissue of 70 human subjects and characterised which miRNAs were differentially expressed between these tissues. We found that 12% of the miRNAs were significantly differentially expressed between abdominal and gluteal adipose tissue (FDR adjusted p<0.05) in the primary study, of which 59 replicated in a follow-up study of 40 additional subjects. Further, 14 miRNAs were found to be associated with metabolic syndrome case-control status in abdominal tissue and three of these replicated (primary study: FDR adjusted p<0.05, replication: p<0.05 and directionally consistent effect). Genome-wide genotyping was performed in the 70 subjects to enable miRNA expression quantitative trait loci (eQTL) analysis. Candidate miRNA eQTLs were followed-up in the additional 40 subjects and six significant, independent cis-located miRNA eQTLs (primary study: p<0.001; replication: p<0.05 and directionally consistent effect) were identified. Finally, global mRNA expression profiling was performed in both tissues to enable association analysis between miRNA and target mRNA expression levels. We find 22% miRNAs in abdominal and 9% miRNAs in gluteal adipose tissue with expression levels significantly associated with the expression of corresponding target mRNAs (FDR adjusted p<0.05). Taken together, our results indicate a clear difference in the miRNA molecular phenotypic profile of abdominal and gluteal adipose tissue, that the expressions of some miRNAs are influenced by cis-located genetic variants and that miRNAs are associated with expression levels of their predicted mRNA targets.

Nica AC, Parts L, Glass D, Nisbet J, Barrett A, Sekowska M, Travers M, Potter S, Grundberg E, Small K et al. 2011. The architecture of gene regulatory variation across multiple human tissues: the MuTHER study. PLoS Genet, 7 (2), pp. e1002003. | Show Abstract | Read more

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.

Min JL, Taylor JM, Richards JB, Watts T, Pettersson FH, Broxholme J, Ahmadi KR, Surdulescu GL, Lowy E, Gieger C et al. 2011. The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits. PLoS One, 6 (7), pp. e22070. | Show Abstract | Read more

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097-16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

Painter JN, Anderson CA, Nyholt DR, Macgregor S, Lin J, Lee SH, Lambert A, Zhao ZZ, Roseman F, Guo Q et al. 2011. Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis. Nat Genet, 43 (1), pp. 51-54. | Show Abstract | Read more

Endometriosis is a common gynecological disease associated with pelvic pain and subfertility. We conducted a genome-wide association study (GWAS) in 3,194 individuals with surgically confirmed endometriosis (cases) and 7,060 controls from Australia and the UK. Polygenic predictive modeling showed significantly increased genetic loading among 1,364 cases with moderate to severe endometriosis. The strongest association signal was on 7p15.2 (rs12700667) for 'all' endometriosis (P = 2.6 × 10⁻⁷, odds ratio (OR) = 1.22, 95% CI 1.13-1.32) and for moderate to severe disease (P = 1.5 × 10⁻⁹, OR = 1.38, 95% CI 1.24-1.53). We replicated rs12700667 in an independent cohort from the United States of 2,392 self-reported, surgically confirmed endometriosis cases and 2,271 controls (P = 1.2 × 10⁻³, OR = 1.17, 95% CI 1.06-1.28), resulting in a genome-wide significant P value of 1.4 × 10⁻⁹ (OR = 1.20, 95% CI 1.13-1.27) for 'all' endometriosis in our combined datasets of 5,586 cases and 9,331 controls. rs12700667 is located in an intergenic region upstream of the plausible candidate genes NFE2L3 and HOXA10.

Lee SH, Nyholt DR, Macgregor S, Henders AK, Zondervan KT, Montgomery GW, Visscher PM. 2010. A simple and fast two-locus quality control test to detect false positives due to batch effects in genome-wide association studies. Genet Epidemiol, 34 (8), pp. 854-862. | Show Abstract | Read more

The impact of erroneous genotypes having passed standard quality control (QC) can be severe in genome-wide association studies, genotype imputation, and estimation of heritability and prediction of genetic risk based on single nucleotide polymorphisms (SNP). To detect such genotyping errors, a simple two-locus QC method, based on the difference in test statistic of association between single SNPs and pairs of SNPs, was developed and applied. The proposed approach could detect many problematic SNPs with statistical significance even when standard single SNP QC analyses fail to detect them in real data. Depending on the data set used, the number of erroneous SNPs that were not filtered out by standard single SNP QC but detected by the proposed approach varied from a few hundred to thousands. Using simulated data, it was shown that the proposed method was powerful and performed better than other tested existing methods. The power of the proposed approach to detect erroneous genotypes was ∼80% for a 3% error rate per SNP. This novel QC approach is easy to implement and computationally efficient, and can lead to a better quality of genotypes for subsequent genotype-phenotype investigations.

Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, Steinthorsdottir V, Thorleifsson G, Zillikens MC, Speliotes EK, Mägi R et al. 2010. Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution. Nat Genet, 42 (11), pp. 949-960. | Show Abstract | Read more

Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10⁻⁹ to P = 1.8 × 10⁻⁴⁰) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10⁻³ to P = 1.2 × 10⁻¹³). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.

Anderson CA, Pettersson FH, Clarke GM, Cardon LR, Morris AP, Zondervan KT. 2010. Data quality control in genetic case-control association studies. Nat Protoc, 5 (9), pp. 1564-1573. | Show Abstract | Read more

This protocol details the steps for data quality assessment and control that are typically carried out during case-control association studies. The steps described involve the identification and removal of DNA samples and markers that introduce bias. These critical steps are paramount to the success of a case-control study and are necessary before statistically testing for association. We describe how to use PLINK, a tool for handling SNP data, to perform assessments of failure rate per individual and per SNP and to assess the degree of relatedness between individuals. We also detail other quality-control procedures, including the use of SMARTPCA software for the identification of ancestral outliers. These platforms were selected because they are user-friendly, widely used and computationally efficient. Steps needed to detect and establish a disease association using case-control data are not discussed here. Issues concerning study design and marker selection in case-control studies have been discussed in our earlier protocols. This protocol, which is routinely used in our labs, should take approximately 8 h to complete.

Min JL, Barrett A, Watts T, Pettersson FH, Lockstone HE, Lindgren CM, Taylor JM, Allen M, Zondervan KT, McCarthy MI. 2010. Variability of gene expression profiles in human blood and lymphoblastoid cell lines. BMC Genomics, 11 (1), pp. 96. | Show Abstract | Read more

BACKGROUND: Readily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets. RESULTS: Gene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range rho = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P < 1*10(-16)) with higher variability (P < 1*10(-16)) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (rho = 0.83; rho = 0.79) of the expression levels of detected probes between LCLs and B-cell subsets were much lower compared to the two B-cell isolation methods (rho = 0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs. CONCLUSION: Gene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes, before application in large clinical studies.

Lin J, Kennedy SH, Svarovsky T, Rogers J, Kemnitz JW, Xu A, Zondervan KT. 2009. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil. Anal Biochem, 395 (2), pp. 265-267. | Show Abstract | Read more

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

Rogers PA, D'Hooghe TM, Fazleabas A, Gargett CE, Giudice LC, Montgomery GW, Rombauts L, Salamonsen LA, Zondervan KT. 2009. Priorities for endometriosis research: recommendations from an international consensus workshop. Reprod Sci, 16 (4), pp. 335-346. | Show Abstract | Read more

Endometriosis is an estrogen-dependent disorder where endometrial tissue forms lesions outside the uterus. Endometriosis affects an estimated 10% of women in the reproductive-age group, rising to 30% to 50% in patients with infertility and/or pain, with significant impact on their physical, mental, and social well-being. There is no known cure, and most current medical treatments are not suitable long term due to their side-effect profiles. Endometriosis has an estimated annual cost in the United States of $18.8 to $22 billion (2002 figures). Although endometriosis was first described more than 100 years ago, current knowledge of its pathogenesis, spontaneous evolution, and the pathophysiology of the related infertility and pelvic pain, remain unclear. A consensus workshop was convened following the 10th World Congress on Endometriosis to establish recommendations for priorities in endometriosis research. One major issue identified as impacting on the capacity to undertake endometriosis research is the need for multidisciplinary expertise. A total of 25 recommendations for research have been developed, grouped under 5 subheadings: (1) diagnosis, (2) classification and prognosis, (3) treatment and outcome, (4) epidemiology, and (5) pathophysiology. Endometriosis research is underfunded relative to other diseases with high health care burdens. This may be due to the practical difficulties of developing competitive research proposals on a complex and poorly understood disease, which affects only women. By producing this consensus international research priorities statement it is the hope of the workshop participants that researchers will be encouraged to develop new interdisciplinary research proposals that will attract increased funding support for work on endometriosis.

Pettersson FH, Anderson CA, Clarke GM, Barrett JC, Cardon LR, Morris AP, Zondervan KT. 2009. Marker selection for genetic case-control association studies. Nat Protoc, 4 (5), pp. 743-752. | Show Abstract | Read more

Association studies can focus on candidate gene(s), a particular genomic region, or adopt a genome-wide association approach, each of which has implications for marker selection. The strategy for marker selection will affect the statistical power of the study to detect a disease association and is a crucial element of study design. The abundant single nucleotide polymorphisms (SNPs) are the markers of choice in genetic case-control association studies. The genotypes of neighboring SNPs are often highly correlated ('in linkage disequilibrium', LD) within a population, which is utilized for selecting specific 'tagSNPs' to serve as proxies for other nearby SNPs in high LD. General guidelines for SNP selection in candidate genes/regions and genome-wide studies are provided in this protocol, along with illustrative examples. Publicly available web-based resources are utilized to browse and retrieve data, and software, such as Haploview and Goldsurfer2, is applied to investigate LD and to select tagSNPs.

Nnoaham KE, Sivananthan S, Hummelshoj L, Jenkinson C, Webster P, Kennedy SH, Zondervan KT. 2009. MULTI-CENTRE STUDIES OF THE GLOBAL IMPACT OF ENDOMETRIOSIS AND THE PREDICTIVE VALUE OF ASSOCIATED SYMPTOMS. J Endometr, 1 (1), pp. 36-45. | Show Abstract

INTRODUCTION: Endometriosis can be difficult to diagnose clinically and models that use symptoms to predict whether the disease is present or not are based on limited patient populations. Endometriosis also influences health-related quality of life, but little is known about its impact across the world. We therefore initiated two integrated multicentre studies to collect prospective, standardised, epidemiological data, to 1) examine the global impact of endometriosis and relative effect of risk-factors, and 2) develop a symptom-based diagnostic tool. METHODS: The Global Study of Women's Health (GSWH) and the Women's Health Symptom Survey (WHSS) prospectively recruit 18-45 year old women having a laparoscopy across 23 and 19 centres, respectively, worldwide. Women with a previous surgical diagnosis of endometriosis are excluded. Multi-lingual patient questionnaires and a surgical questionnaire, incorporating validated instruments, are used to collect the data. The GSWH aims to recruit >2,000 women by December 2009; the WHSS to recruit 1,000 women in each of the two model-generating and validation stages. RESULTS: A six-week pilot study in Oxford, UK, established the feasibility of the study protocols. Of 32 eligible women, 27 participated (response rate - 84.4%); 26% completed the questionnaire online. Endometriosis was found in 47.4%. Extrapolating the recruitment rates from the pilot study, the target sample sizes for the GWSH and WHSS were deemed feasible. CONCLUSIONS: Using standardised data collection, the GSWH and WHSS will provide insight into the global impact of endometriosis and develop a validated, symptom-based, diagnostic tool. They have the potential to provide the basis for future, longitudinal, follow-up studies and a collaborative Endometriosis Biobank implementing standardised collection of DNA and tissue samples.

Montgomery GW, Nyholt DR, Zhao ZZ, Treloar SA, Painter JN, Missmer SA, Kennedy SH, Zondervan KT. 2008. The search for genes contributing to endometriosis risk. Hum Reprod Update, 14 (5), pp. 447-457. | Show Abstract | Read more

BACKGROUND: Genetic variation contributes to the risk of developing endometriosis. This review summarizes gene mapping studies in endometriosis and the prospects of finding gene pathways contributing to disease using the latest genome-wide strategies. METHODS: To identify candidate-gene association studies of endometriosis, a systematic literature search was conducted in PubMed of publications up to 1 April 2008, using the search terms 'endometriosis' plus 'allele' or 'polymorphism' or 'gene'. Papers included were those with information on both case and control selection, showed allelic and/or genotypic results for named germ-line polymorphisms and were published in the English language. RESULTS: Genetic variants in 76 genes have been examined for association, but none shows convincing evidence of replication in multiple studies. There is evidence for genetic linkage to chromosomes 7 and 10, but the genes (or variants) in these regions contributing to disease risk have yet to be identified. Genome-wide association is a powerful method that has been successful in locating genetic variants contributing to a range of common diseases. Several groups are planning these studies in endometriosis. For this to be successful, the endometriosis research community must work together to genotype sufficient cases, using clearly defined disease classifications, and conduct the necessary replication studies in several thousands of cases and controls. CONCLUSIONS: Genes with convincing evidence for association with endometriosis are likely to be identified in large genome-wide studies. This will provide a starting point for functional and biological studies to develop better diagnosis and treatment for this debilitating disease.

Bhanoori M, Kameshwari DB, Zondervan KT, Deenadayal M, Kennedy S, Shivaji S. 2008. The endothelial nitric oxide synthase Glu298Asp polymorphism is not a risk factor for endometriosis in south Indian women. Eur J Obstet Gynecol Reprod Biol, 139 (1), pp. 53-58. | Show Abstract | Read more

OBJECTIVE: To investigate whether the eNOS gene influences the risk of developing endometriosis in south Indian women. STUDY DESIGN: The single nucleotide polymorphism, Glu298Asp, in exon7 of the eNOS gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic. The genotype frequencies of the polymorphism were compared, using polymerase chain reaction and sequencing analysis. The localization and expression of eNOS in the eutopic endometrium of five cases and four controls was also analyzed using immunohistochemistry and western blotting. RESULTS: No statistically significant differences were observed in the genotype distributions and allele frequencies (p=0.3) between the cases and controls according to codominant, dominant and recessive genotype models. The localization and expression of this protein were similar in the endometrium of cases and controls. CONCLUSION: In the present study we could neither observe a difference in the eNOS expression nor establish an association between the eNOS Glu298Asp exon 7 polymorphism in south Indian women with endometriosis.

Bennett DA, Xu P, Clarke R, Zondervan K, Parish S, Palmer A, Cardon L, Peto R, Lathrop M, Collins R, International Study of Infarct Survival Collaborators. 2008. The exon 1-8C/G SNP in the PSMA6 gene contributes only a small amount to the burden of myocardial infarction in 6946 cases and 2720 controls from a United Kingdom population. Eur J Hum Genet, 16 (4), pp. 480-486. | Show Abstract | Read more

The proteasome system is a proteolytic pathway that regulates the expression of genes involved in inflammation. Polymorphisms in the gene encoding subunit alpha type 6 (PSMA6)--in particular the rs1048990 exon 1-8C/G SNP--have been implicated with susceptibility to myocardial infarction (MI) in a Japanese study. We examined whether several polymorphisms in the PSMA6 gene were related to MI risk in 6946 nonfatal MI cases and 2720 unrelated controls in a UK population. The homozygous GG genotype for rs1048990 was much less frequent in this UK population than in the Japanese population (2.1 vs 8.9%), and was associated with an odds ratio (OR) for MI of 1.09 (95% confidence interval (CI): 0.98-1.21) per G allele in a co-dominant genetic model and 1.32 (95% CI: 0.90-1.93) in a recessive genetic model. Although not statistically significant, these results for this variant are still consistent with the Japanese hypothesis-generating study. Our findings, when taken together with four other studies (including the hypothesis-generating one), yielded a combined OR for MI of 1.15 (95% CI: 1.08-1.21) per G allele in a co-dominant model and 1.38 (95% CI: 1.22-1.57) for the GG genotype in a recessive model. Larger studies involving more than 10,000 disease cases would be required to further elucidate the role of this variant for susceptibility to MI. However, given the rarity of this variant in Caucasians, the attributable risk of rs1048990 for MI is unlikely to be great in western populations.

Sinaii N, Plumb K, Cotton L, Lambert A, Kennedy S, Zondervan K, Stratton P. 2008. Differences in characteristics among 1,000 women with endometriosis based on extent of disease. Fertil Steril, 89 (3), pp. 538-545. | Show Abstract | Read more

OBJECTIVE: To determine the relationship between disease severity and patient characteristics in endometriosis. DESIGN: Cross-sectional study of self-reported survey data. SETTING: Academic research setting. PATIENT(S): One thousand women in the Oxford Endometriosis Gene (OXEGENE) study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Participants were assigned to one of two groups with predominantly revised AFS stage I-II (group I, n = 423) or III-IV disease (group II, n = 517). Their characteristics were compared by disease extent. RESULT(S): Most participants were white (96%) and of reproductive age (81%). Women in group I were significantly younger on entering the study (39.9 +/- 0.5 vs. 44.5 +/- 0.4 years). Overall time to diagnosis did not differ between groups. The most common symptoms leading to a diagnosis were dysmenorrhea (79%) and pelvic pain (69%). In group II, subfertility (21.5% vs. 30.0%) and an ovarian mass (7.3% vs. 29.4%) more commonly led to a diagnosis, whereas dyspareunia (51.1% vs. 39.5%) was significantly more common in group I. Subfertility (41.5% vs. 53.4%) remained more common in group II throughout reproductive life, although birth and miscarriage rates were similar. CONCLUSION(S): Pelvic pain is common to all with endometriosis and those with more extensive disease report higher rates of subfertility. Remarkably, the time to diagnosis was similar among women.

Treloar SA, Zhao ZZ, Le L, Zondervan KT, Martin NG, Kennedy S, Nyholt DR, Montgomery GW. 2007. Variants in EMX2 and PTEN do not contribute to risk of endometriosis. Mol Hum Reprod, 13 (8), pp. 587-594. | Show Abstract | Read more

Endometriosis has a genetic component, and significant linkage has been found to a region on chromosome 10q. Two candidate genes, EMX2 and PTEN, implicated in both endometriosis and endometrial cancer, lie on chromosome 10q. We hypothesized that variation in EMX2 and/or PTEN could contribute to the risk of endometriosis and may account for some of the linkage signal on 10q. We genotyped single nucleotide polymorphisms (SNPs) in a case-control design to evaluate association between endometriosis and common variations in these two genes. The genotyping and statistical analysis were based on samples collected from Australian volunteers. The cases were 768 unrelated women with surgically confirmed endometriosis selected from affected sister pair (ASP) families participating in the Australian Genes behind Endometriosis Study. The controls were 768 female participants in twin studies who, based on screening questions, did not have a diagnosis of endometriosis. Genotypes of 22 SNPs in the EMX2 gene and 15 SNPs in the PTEN gene were the main outcome measures. Statistical analysis provided measures of linkage disequilibrium and association. Permutation testing showed no globally significant association between any SNPs or haplotypes and endometriosis for either gene. It is unlikely that the EMX2 or PTEN gene variants investigated contribute to risk for initiation and/or development of endometriosis.

Zondervan KT, Treloar SA, Lin J, Weeks DE, Nyholt DR, Mangion J, MacKay IJ, Cardon LR, Martin NG, Kennedy SH, Montgomery GW. 2007. Significant evidence of one or more susceptibility loci for endometriosis with near-Mendelian inheritance on chromosome 7p13-15. Hum Reprod, 22 (3), pp. 717-728. | Show Abstract | Read more

BACKGROUND: Endometriosis is a common disease with a heritable component. The collaborative International Endogene Study consists of two data sets (Oxford and Australia) comprising 1176 families with multiple affected. The aim was to investigate whether the apparent concentration of cases in a proportion of families could be explained by one or more rare variants with (near-)Mendelian autosomal inheritance. METHODS AND RESULTS: Linkage analyses (aimed at finding chromosomal regions harbouring disease-predisposing genes) were conducted in families with three or more affected (Oxford: n = 52; Australia: n = 196). In the Oxford data set, a non-parametric linkage score (Kong & Cox (K&C) Log of ODds (LOD)) of 3.52 was observed on chromosome 7p (genome-wide significance P = 0.011). A parametric MOD score (equal to maximum LOD maximized over 357 possible inheritance models) of 3.89 was found at 65.72 cM (D7S510) for a dominant model with reduced penetrance. After including the Australian data set, the non-parametric K&C LOD of the combined data set was 1.46 at 57.3 cM; the parametric analysis found an MOD score of 3.30 at D7S484 (empirical significance: P = 0.035) for a recessive model with high penetrance. Critical recombinant analysis narrowed the probable region of linkage down to overlapping 6.4 Mb and 11 Mb intervals containing 48 and 96 genes, respectively. CONCLUSIONS: This is the first report to suggest that there may be one or more high-penetrance susceptibility loci for endometriosis with (near-)Mendelian inheritance.

Zondervan KT, Cardon LR. 2007. Designing candidate gene and genome-wide case-control association studies. Nat Protoc, 2 (10), pp. 2492-2501. | Show Abstract | Read more

This protocol describes how to appropriately design a genetic association case-control study, either focusing on a candidate gene (CG) or region or implementing a genome-wide approach. The steps described involve: (i) defining the case phenotype in adequate detail; (ii) checking the heritability of the disease in question; (iii) considering whether a population-based study is the appropriate design for the research question; (iv) the appropriate selection of controls; (v) sample size calculations and (vi) giving due consideration to whether it is a de novo or replication study. General guidelines are given, as well as specific examples of a CG and a genome-wide association study into type 2 diabetes. Software and websites used in this protocol include the International HapMap Consortium website, Genetic Power Calculator, CaT, and SNPSpD. Running each of the programs takes only a few seconds; the rate-limiting steps involve thinking through the designs and parameters in the disease models.

Ikuhashi Y, Yoshida S, Kennedy S, Zondervan K, Takemura N, Deguchi M, Ohara N, Maruo T. 2007. Vascular endothelial growth factor +936 C/T polymorphism is associated with an increased risk of endometriosis in a Japanese population. Acta Obstet Gynecol Scand, 86 (11), pp. 1352-1358. | Show Abstract | Read more

BACKGROUND: Vascular endothelial growth factor (VEGF) concentration in endometriosis patients is higher than controls, in serum and ascites, suggesting that VEGF may play an important role in the pathogenesis of endometriosis. In this study, we investigated whether polymorphisms in the VEGF gene are associated with endometriosis in a Japanese population. METHODS: Genotyping of VEGF -460 C/T, +405 G/C and +936 C/T polymorphisms were performed in 147 endometriosis cases diagnosed by laparotomy or laparoscopy at a university hospital, and 181 controls, by polymerase chain reaction-restriction fragment length polymorphism analysis. We compared the genotype distribution and allele frequency of these 3 polymorphisms between endometriosis patients and controls. RESULTS: No significant differences in the frequency and genotype distribution of VEGF -460 C/T, +405 G/C and +936 C/T polymorphisms were found between the endometriosis patients (all disease stages) and controls. However, a positive association was found between stage III-IV disease and the VEGF +936 T allele (p=0.018). CONCLUSIONS: The VEGF +936 T allele is associated with an increased risk of stage III-IV endometriosis in a Japanese population.

Linsell L, Dawson J, Zondervan K, Rose P, Carr A, Randall T, Fitzpatrick R. 2006. Pain and overall health status in older people with hip and knee replacement: a population perspective. J Public Health (Oxf), 28 (3), pp. 267-273. | Show Abstract | Read more

OBJECTIVE: To investigate the health-related quality of life and presence of hip or knee pain according to whether or not people had had previous hip or knee arthroplasty. STUDY DESIGN AND SETTING: Cross-sectional survey representing randomly selected sample of 5500 elderly (65+) people. Pain prevalence rates obtained from standard screening questions. Standard pain severity ratings obtained for each hip and knee. RESULTS: People with a past arthroplasty had worse health status compared to other people (p < 0.001 for all but two SF-36 dimensions). Hip or knee pain was more prevalent amongst people with past hip or knee replacement than amongst those without (62.5% versus 36.5% respectively; following adjustment for age and sex: Mantel-Haenszel combined odds ratio = 2.90, 95% CI 2.30-3.68, p < 0.001). More replaced knee joints were symptomatic than replaced hip joints (OR = 1.62, p = 0.022). CONCLUSIONS: Elderly people with a past hip or knee arthroplasty have significantly greater health and social care needs than other people--especially those related to pain and mobility. This may reflect the generalised nature of the underlying disease process.

Grace V, Zondervan K. 2006. Chronic pelvic pain in women in New Zealand: comparative well-being, comorbidity, and impact on work and other activities. Health Care Women Int, 27 (7), pp. 585-599. | Show Abstract | Read more

This article reports the findings of a population-based study in New Zealand that investigated comparative well-being, comorbidity, and the impact of chronic pelvic pain (CPP) on activities. Chronic pelvic pain was defined as lower abdominal pain that is associated with neither the menstrual cycle nor sexual activity. A postal questionnaire was administered to a random sample resulting in a study group of 1,160. The negative impact of CPP on women's general well-being is significant. They were more likely than women without CPP to have other long-standing illnesses, other unspecified conditions involving pain or fatigue, and sleep patterns were more seriously disturbed. Pain restricted their activities. Comparisons with the limited data available from other studies are made.

Clarke R, Xu P, Bennett D, Lewington S, Zondervan K, Parish S, Palmer A, Clark S, Cardon L, Peto R et al. 2006. Lymphotoxin-alpha gene and risk of myocardial infarction in 6,928 cases and 2,712 controls in the ISIS case-control study. PLoS Genet, 2 (7), pp. e107. | Show Abstract | Read more

Lymphotoxin-alpha (LTA) is a pro-inflammatory cytokine that plays an important role in the immune system and local inflammatory response. LTA is expressed in atherosclerotic plaques and has been implicated in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Polymorphisms in the gene encoding lymphotoxin-alpha (LTA) on Chromosome 6p21 have been associated with susceptibility to CHD, but results in different studies appear to be conflicting. We examined the association of seven single nucleotide polymorphisms (SNPs) across the LTA gene, and their related haplotypes, with risk of myocardial infarction (MI) in the International Study of Infarct Survival (ISIS) case-control study involving 6,928 non-fatal MI cases and 2,712 unrelated controls. The seven SNPs (including the rs909253 and rs1041981 SNPs previously implicated in the risk of CHD) were in strong linkage disequilibrium with each other and contributed to six common haplotypes. Some of the haplotypes for LTA were associated with higher plasma concentrations of C-reactive protein (p = 0.004) and lower concentrations of albumin (p = 0.023). However, none of the SNPs or related haplotypes were significantly associated with risk of MI. The results of the ISIS study were considered in the context of six previously published studies that had assessed this association, and this meta-analysis found no significant association with CHD risk using a recessive model and only a modest association using a dominant model (with narrow confidence intervals around these risk estimates). Overall, these studies provide reliable evidence that these common polymorphisms for the LTA gene are not strongly associated with susceptibility to coronary disease.

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Clarke R, Xu P, Bennett D, Lewington S, Zondervan K, Parish S, Palmer A, Clark S, Cardon L, Peto R et al. 2006. Lymphotoxin-α gene and risk of myocardial infarction in 6,928 cases and 2,712 controls in the ISIS case-control study PLoS Genetics, 2 (7), pp. 0990-0996. | Show Abstract | Read more

Lymphotoxin-α (LTA) is a pro-inflammatory cytokine that plays an important role in the immune system and local inflammatory response. LTA is expressed in atherosclerotic plaques and has been implicated in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Polymorphisms in the gene encoding lymphotoxin-α (LTA) on Chromosome 6p21 have been associated with susceptibility to CHD, but results in different studies appear to be conflicting. We examined the association of seven single nucleotide polymorphisms (SNPs) across the LTA gene, and their related haplotypes, with risk of myocardial infarction (MI) in the International Study of Infarct Survival (ISIS) case-control study involving 6,928 non-fatal MI cases and 2,712 unrelated controls. The seven SNPs (including the rs909253 and rs1041981 SNPs previously implicated in the risk of CHD) were in strong linkage disequilibrium with each other and contributed to six common haplotypes. Some of the haplotypes for LTA were associated with higher plasma concentrations of C-reactive protein (p = 0.004) and lower concentrations of albumin (p = 0.023). However, none of the SNPs or related haplotypes were significantly associated with risk of MI. The results of the ISIS study were considered in the context of six previously published studies that had assessed this association, and this meta-analysis found no significant association with CHD risk using a recessive model and only a modest association using a dominant model (with narrow confidence intervals around these risk e stimates). Overall, these studies provide reliable evidence that these common polymorphisms for the LTA gene are not strongly associated with susceptibility to coronary disease. Copyright: © 2006 Clarke et al.

Linsell L, Dawson J, Zondervan K, Rose P, Randall T, Fitzpatrick R, Carr A. 2006. Prevalence and incidence of adults consulting for shoulder conditions in UK primary care; patterns of diagnosis and referral. Rheumatology (Oxford), 45 (2), pp. 215-221. | Show Abstract | Read more

OBJECTIVES: To estimate the national prevalence and incidence of adults consulting for a shoulder condition and to investigate patterns of diagnosis, treatment, consultation and referral 3 yr after initial presentation. METHODS: Prevalence and incidence rates were estimated for 658469 patients aged 18 and over in the year 2000 using a primary care database, the IMS Disease Analyzer-Mediplus UK. A cohort of 9215 incident cases was followed-up prospectively for 3 yr beyond the initial consultation. RESULTS: The annual prevalence and incidence of people consulting for a shoulder condition was 2.36% [95% confidence interval (CI) 2.32-2.40%] and 1.47% (95% CI 1.44-1.50%), respectively. Prevalence increased linearly with age whilst incidence peaked at around 50 yr then remained static at around 2%. Around half of the incident cases consulted once only, while 13.6% were still consulting with a shoulder problem during the third year of follow-up. During the 3 yr following initial presentation, 22.4% of patients were referred to secondary care, 30.8% were prescribed non-steroidal anti-inflammatory drugs and 10.6% were given an injection by their general practitioner (GP). GPs tended to use a limited number of generalized codes when recording a diagnosis; just five of 426 possible Read codes relating to shoulder conditions accounted for 74.6% of the diagnoses of new cases recorded by GPs. CONCLUSIONS: The prevalence of people consulting for shoulder problems in primary care is substantially lower than community-based estimates of shoulder pain. Most referrals occur within 3 months of initial presentation, but only a minority of patients are referred to orthopaedic specialists or rheumatologists. GPs may lack confidence in applying precise diagnoses to shoulder conditions.

Dawson J, Linsell L, Doll H, Zondervan K, Rose P, Carr A, Randall T, Fitzpatrick R. 2005. Assessment of the Lequesne index of severity for osteoarthritis of the hip in an elderly population. Osteoarthritis Cartilage, 13 (10), pp. 854-860. | Show Abstract | Read more

OBJECTIVE: To assess the measurement properties of the Lequesne index of severity for osteoarthritis of the hip (LISOH) together with its overall usefulness with reference to the original stated aims. METHOD: Postal questionnaire was sent to a random sample of 5500 Oxfordshire residents, aged 65 and above. Respondents with hip symptoms at baseline (but without verification of a diagnosis) were sent an identical follow-up questionnaire 12 months later. The questionnaire included a general health section, including the Short Form-36 survey, and a hip section which began with a screening question about hip pain. Respondents who reported having a prolonged episode of hip pain were asked to complete the LISOH. RESULTS: At baseline, response rate of 66.3% (3341/5039) was obtained from eligible participants; 19.2% (610/3175) of respondents reported having hip pain. Internal reliability (Cronbach's alpha) was 0.84 (95% CI: 0.81-0.86) for all 11 items of the LISOH; however, factor analysis identified two factors (sub-scales): "function and mobility" and "pain and discomfort". Rasch analysis revealed that the two factors were only unidimensional when applied to sub-groups of respondents. Convergent validity of the LISOH was questionable, as the "function and mobility" factor was more highly correlated with SF-36 bodily pain score than was the "pain and discomfort" factor. The assessment of sensitivity over time was problematic due to changing patterns of symptomatic weight-bearing joints over time. CONCLUSIONS: The current study identifies major limitations with the LISOH--particularly if used as a single composite measure.

Bhanoori M, Arvind Babu K, Pavankumar Reddy NG, Lakshmi Rao K, Zondervan K, Deenadayal M, Kennedy S, Shivaji S. 2005. The vascular endothelial growth factor (VEGF) +405G>C 5'-untranslated region polymorphism and increased risk of endometriosis in South Indian women: a case control study. Hum Reprod, 20 (7), pp. 1844-1849. | Show Abstract | Read more

BACKGROUND: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis and vascular permeability, is known to play a key role in the pathophysiology of endometriosis. METHODS AND RESULTS: The single nucleotide polymorphisms, -460C>T and +405G>C, in the 5'-untranslated region of the VEGF gene were tested for association in a case-control study of 215 affected women and 210 women with no evidence of disease. All the women were of South Indian origin and ascertained from the same infertility clinic. The genotype and allele frequencies of the -460C>T polymorphism did not differ significantly between cases and controls. In contrast, the genotype (P = 0.002) and allele (P = 0.001) frequencies of the +405G>C polymorphism showed a significant difference between cases and controls. The +405 GG genotype was found more often in patients with an endometrioma >3 cm compared to controls. The frequency of the -460T/+405C haplotype (P = 0.016) was significantly lower in affected women compared to controls. CONCLUSIONS: The -460T/+405C haplotype in the VEGF gene, which is associated with lower promoter activity, was significantly less common in women with endometriosis than in controls. These data suggest that the +405G allele may influence the likelihood of a woman developing the disease.

Dawson J, Linsell L, Zondervan K, Rose P, Carr A, Randall T, Fitzpatrick R. 2005. Impact of persistent hip or knee pain on overall health status in elderly people: a longitudinal population study. Arthritis Rheum, 53 (3), pp. 368-374. | Show Abstract | Read more

OBJECTIVE: To investigate hip or knee symptoms in older persons from a longitudinal, population perspective, and to determine the impact of persistent hip or knee pain on general health status over time. METHODS: A postal questionnaire was sent to a random sample of 5,500 individuals ages > or = 65 years containing the Short Form 36 (SF-36) general health survey, Lequesne hip and knee indices, and a hip/knee pain severity item. Respondents reporting hip or knee symptoms at baseline received an identical questionnaire 12 months later. Respondents were classified into a persistent pain group with either hip or knee pain at both baseline and followup, and a non-persistent pain group who reported hip or knee pain at baseline but no pain at followup. RESULTS: At baseline, 1,305 (40.7%) of 3,210 eligible respondents reported hip or knee pain. At 1 year, 1,072 (82.1%) of 1,305 individuals responded, of whom 820 (76.5%) remained symptomatic (the persistent group). In multivariate analysis, baseline factors identified as strongly related to having persistent pain were maximum Lequesne score (odds ratio [OR] 1.09, P < 0.001), maximum hip/knee pain score (OR 1.61, P < 0.001), and number of painful hip and knee joints at baseline (OR 1.48, P = 0.004). Following adjustment for age, sex, and baseline score, differences in mean SF-36 change scores of the 2 groups were significant for all dimensions except for mental health. CONCLUSION: In older persons, a symptomatic hip or knee frequently progresses in terms of worsening symptoms and accrual of other symptomatic hip or knee joints. The impact of persistent symptoms on general health is substantial.

Zondervan KT, Cardon LR, Kennedy SH, Martin NG, Treloar SA. 2005. Multivariate genetic analysis of chronic pelvic pain and associated phenotypes. Behav Genet, 35 (2), pp. 177-188. | Show Abstract | Read more

Chronic pelvic pain (CPP) is a common condition in women that is difficult to diagnose. Although heritability estimates have been published for some conditions potentially underlying pelvic pain, the heritability of CPP itself has never been investigated. Using data from 623 MZ and 377 DZ female twin pairs aged 29-50 from an Australian twin cohort, we found an increased CPP concordance among MZs compared to DZs, with tetrachoric correlations of 0.43 (95% CI: 0.26-0.58) and 0.11 (95% CI: -0.16-0.38), respectively. This corresponded to a heritability of 0.41 (95% CI: 0.25-0.56). Lack of correlations with environmental indicators suggested that violation of the equal environments assumption was not responsible for this effect. Multivariate Cholesky decomposition models incorporating CPP and significantly correlated phenotypes showed that the entire CPP heritability could be explained by genetic variance underlying endometriosis (38%), dysmenorrhoea (23%), fibroids (24%), and somatic distress (15%), the latter a possible indicator of increased nociception. CPP itself is unlikely to be a useful independent phenotype to conduct genetic aetiological studies; contributing conditions such as endometriosis and variation in nociception are likely to provide more useful phenotypes.

Linsell L, Dawson J, Zondervan K, Rose P, Carr A, Randall T, Fitzpatrick R. 2005. Population survey comparing older adults with hip versus knee pain in primary care. Br J Gen Pract, 55 (512), pp. 192-198. | Show Abstract

BACKGROUND: Knee pain is nearly twice as prevalent as hip pain in elderly people, yet knee replacement is far less common than hip replacement. AIM: To investigate whether systematic differences in the primary care management of hip versus knee problems might explain the disparate rates of joint replacement. DESIGN OF STUDY: Cross-sectional, population-based postal survey. SETTING: Random sample of 5500 Oxfordshire residents aged 65 years and above. METHOD: Screening questions were used to identify symptomatic individuals: "During the past 12 months, have you had pain in or around either of your hips/knees on most days for 1 month or longer?". Standard (Lequesne) severity ratings were obtained for each hip and knee. Logistic regression was used to estimate odds ratios (ORs) for "knee cases" versus "hip cases" for selected healthcare services and attitudes toward replacement. RESULTS: Among 3341 responders, 212 hip cases and 612 knee cases were identified. Knee pain led to a GP consultation more often than hip pain (OR = 1.76, P = 0.04), but specialist referral was no more likely (OR = 0.85, P = 0.57). Similar percentages of hip and knee cases would agree to hip/knee replacement surgery if it was offered, but hip and knee cases differed in their views on the general success of joint replacement. CONCLUSIONS: Some variations in primary care management for hip versus knee pain were apparent. People with hip pain were mostly positive about replacement outcomes, whereas people with knee pain were more uncertain about replacement. Attitudes appeared to be influenced by knowing someone who had undergone such surgery.

Linsell L, Dawson J, Zondervan K, Randall T, Rose P, Carr A, Fitzpatrick R. 2005. Prospective study of elderly people comparing treatments following first primary care consultation for a symptomatic hip or knee. Fam Pract, 22 (1), pp. 118-125. | Show Abstract | Read more

BACKGROUND: Symptomatic knee problems in elderly people are considerably more common than hip problems, yet far more hips are replaced. OBJECTIVE: The purpose of this study was to investigate whether systematic differences occur in early primary care management of elderly patients who first consult with hip versus knee symptoms. METHODS: A prospective analysis was carried out of anonymized records in the MediPlus general practice database. This was a 3 year (1996-1998) prospective study of 310,843 patients aged 65+ regarding consultations about a new hip or knee problem. Survival analysis techniques were used to analyse time to and frequency of various interventions. RESULTS: A total of 1410 new hip and 3152 new knee consulters were identified. Baseline characteristics of the two groups were very similar. By 3 years following the first consultation, more of the hip cases had referral to a specialist (38.2% hips versus 31.5% knees, P <0.001) and joint replacement (9.6% hips versus 1.8% knees, P <0.001) recorded. Non-steroidal anti-inflammatory drug (NSAID) prescribing was high for both groups, with approximately 51% prescribed one of the safer forms and approximately 15% prescribed one with a lower safety record within 3 years. CONCLUSION: Rates of specialist referral and joint replacement in older people are much lower, or slower, for those with symptomatic knees relative to hips. In the absence of surgery, prescriptions for pain killers are similar for both groups. Elderly people with symptomatic knees are therefore at increased risk of drug side effects relative to those with symptomatic hips. This may represent another facet of unmet need for surgery in people with knee disease.

Babu KA, Rao KL, Reddy NG, Kanakavalli MK, Zondervan KT, Deenadayal M, Singh A, Shivaji S, Kennedy S. 2004. N-acetyl transferase 2 polymorphism and advanced stages of endometriosis in South Indian women. Reprod Biomed Online, 9 (5), pp. 533-540. | Show Abstract | Read more

Aylamine-N-acetyl transferase is a phase II detoxification enzyme encoded by the gene NAT2. Single nucleotide polymorphism (SNP) changes from the wild type NAT2 *4 allele result in allelic variants *5, *6 and *7. Homozygotes for the NAT2 *4 wild type are fast acetylators; heterozygotes with one wild-type allele and a variant NAT2 *5, *6 or *7 allele have reduced enzyme activity and individuals with two variant alleles are slow acetylators. Previous studies have implicated NAT2 as a susceptibility factor in endometriosis. This study investigated the NAT2 allele frequencies and genotype distributions in 252 unrelated women with endometriosis and 264 controls of South Indian origin. No differences were found between the frequencies of fast and slow acetylators in cases (34.9% and 65.1%) and controls (33.3% and 66.7%). Two NAT2 genotypes *7/*7 (1.2%) and *5/*6/*7 (1.6%) were detected in endometriosis cases only. Four new combinations, 6D (481 + 590 mutation), 7C (590 + 857), 7D (590 + 803 + 857) and 7E (481 + 590 + 803 + 857) were detected, which have not been reported earlier. Similar genotype and phenotype results were obtained in 33 affected sister-pairs. The case-control data from this study suggest there is no association between endometriosis and NAT2 in South Indian women; however, two new variant genotypes and seven SNP combinations were also identified in cases only, which suggests that the gene may still have some as yet undetermined role in the disease.

Grace VM, Zondervan KT. 2004. Chronic pelvic pain in New Zealand: prevalence, pain severity, diagnoses and use of the health services. Aust N Z J Public Health, 28 (4), pp. 369-375. | Show Abstract | Read more

UNLABELLED: Chronic pelvic pain (CPP) in women is often debilitating and isolating. Problems with diagnosis continue to make CPP one of the most perplexing conditions in gynaecology, and one of the most difficult to treat. OBJECTIVES: This paper reports the findings of a population-based study in New Zealand in 2001 designed to investigate the prevalence of chronic pelvic pain in women between the ages of 18 and 50. Chronic pelvic pain was defined as pain that is neither associated with the menstrual cycle nor sexual activity. The prevalence of dysmenorrhoea and dyspareunia was also sought. It further aimed to examine pain severity, diagnoses, and the use of the health services as these facets of CPP affect different groups of women within New Zealand. METHODS: A random sample of 2261 was generated from the New Zealand Electoral Roll, and a postal questionnaire was administered during 2001. The response rate was 66% (adjusted for non-receivers), giving a study group of 1,160 respondents. RESULTS: The three-month CPP prevalence rate was 25.4% (95% CI 22.8-27.9). Half of those women reporting CPP (47.7%) remained undiagnosed. The three-month prevalence of dysmenorrhoea was 55.2%, and dyspareunia 19.7%. Recent or past consulters of health services for CPP contained a higher proportion of women with a high pain burden than those not consulting health services. Only one-third of New Zealand women (34%) reported no form of chronic pelvic pain (i.e. no CPP, dysmenorrhoea or dyspareunia). These prevalence rates indicate that CPP should receive greater public education and clinical attention.

Durrant C, Zondervan KT, Cardon LR, Hunt S, Deloukas P, Morris AP. 2004. Linkage disequilibrium mapping via cladistic analysis of single-nucleotide polymorphism haplotypes. Am J Hum Genet, 75 (1), pp. 35-43. | Show Abstract | Read more

We present a novel approach to disease-gene mapping via cladistic analysis of single-nucleotide polymorphism (SNP) haplotypes obtained from large-scale, population-based association studies, applicable to whole-genome screens, candidate-gene studies, or fine-scale mapping. Clades of haplotypes are tested for association with disease, exploiting the expected similarity of chromosomes with recent shared ancestry in the region flanking the disease gene. The method is developed in a logistic-regression framework and can easily incorporate covariates such as environmental risk factors or additional unlinked loci to allow for population structure. To evaluate the power of this approach to detect disease-marker association, we have developed a simulation algorithm to generate high-density SNP data with short-range linkage disequilibrium based on empirical patterns of haplotype diversity. The results of the simulation study highlight substantial gains in power over single-locus tests for a wide range of disease models, despite overcorrection for multiple testing.

Dawson J, Linsell L, Zondervan K, Rose P, Randall T, Carr A, Fitzpatrick R. 2004. Epidemiology of hip and knee pain and its impact on overall health status in older adults. Rheumatology (Oxford), 43 (4), pp. 497-504. | Show Abstract | Read more

OBJECTIVES: To obtain prevalence rates of hip and knee pain in elderly people and compare combinations of symptoms with overall health status. METHODS: We performed a cross-sectional postal survey of a random sample of 5500 Oxfordshire residents aged 65 yr and older. Prevalence estimates were based on the screening question: 'During the past 12 months, have you had pain in or around either of your hip/knee joints on most days for one month or longer?' Overall health status was assessed with the SF-36 questionnaire. RESULTS: The response rate was 66.3% (3341/5039 eligible people), and was highest (approximately reverse similar 72%) for the 65-74 yr age-group. The percentage reporting hip pain was 19.2% [95% confidence interval (CI) 17.9-20.6], and 32.6% (95% CI 31.0-34.3) reported knee pain. The percentage reporting hip and knee pain was 11.3%, and 40.7% reported hip or knee pain. Less than half (48%) of the symptomatic respondents had unilateral problems affecting one hip or knee joint only. SF-36 scores worsened as the number of symptomatic hip and knee joints increased (P<0.001 for physical function, physical role limitation and bodily pain). CONCLUSIONS: Patterns of hip and knee symptoms are complex in older people. Amongst the symptomatic, most have more than one hip/knee affected. This has implications for treatment and health status measurement. In the absence of hip and knee symptoms, general health status scores of elderly people are similar to those of people aged under 65 yr.

Zondervan KT, Cardon LR. 2004. The complex interplay among factors that influence allelic association (vol 5, pg 89, 2004) NATURE REVIEWS GENETICS, 5 (3), pp. 238-238. | Read more

Zondervan KT, Weeks DE, Colman R, Cardon LR, Hadfield R, Schleffler J, Trainor AG, Coe CL, Kemnitz JW, Kennedy SH. 2004. Familial aggregation of endometriosis in a large pedigree of rhesus macaques. Hum Reprod, 19 (2), pp. 448-455. | Show Abstract | Read more

BACKGROUND: Endometriosis occurs in several non-human primate species that have menstrual cycles. This study investigated the prevalence and familial aggregation of endometriosis in one of those species, the rhesus macaque. METHODS: Between 1978 and 2001, 142 animals with endometriosis were identified from necropsy and surgical records and through the use of magnetic resonance imaging (MRI) at the Wisconsin National Primate Research Center, Madison, USA. All cases were used to build one large multigenerational pedigree and nine nuclear families comprising 1602 females in total. By 2002, the pedigrees contained 124 cases diagnosed at necropsy; 17 at surgery and three at MRI. Female animals that had died aged > or = 10 years without endometriosis, had both ovaries until at least 1 year prior to death, and had a full necropsy, were considered unaffected. RESULTS: The prevalence of endometriosis among necropsied animals aged > or = 10 years in the colony was 31.4% [95% confidence interval (CI) 26.9-35.9%]; prevalence increased with rising age and calendar age at death. Familial aggregation of endometriosis was strongly suggested by a significantly higher average kinship coefficient among affecteds compared with unaffecteds (P < 0.001) and a higher recurrence risk for full sibs (0.75; 95% CI 0.45-1.0) compared with maternal half sibs (0.26; 95% CI 0.10-0.41) and paternal half sibs (0.18; 95% CI 0.02-0.34). The segregation ratio among affected mothers (44.2%) was not significantly higher compared with unaffected mothers (36.6%). CONCLUSIONS: The results support familial aggregation of endometriosis in the rhesus macaque, and indicate that this is a promising animal model for the investigation of mode of inheritance, the location of potential genetic susceptibility loci and the influence of environmental factors.

Zondervan KT, Cardon LR. 2004. The complex interplay among factors that influence allelic association. Nat Rev Genet, 5 (2), pp. 89-100. | Show Abstract | Read more

Small effect sizes, common-disease/common-variant versus rare variant influences, biased single nucleotide polymorphism ascertainment and low linkage disequilibrium have recently been discussed as impediments to association studies. Such a focus on the individual factors that highlight their maximum potential effect (whether positive or deleterious) is often optimistic as, in practice, they do not operate in isolation. Instead, they work jointly to generate the disease gene architecture and to determine the ability of a study to discover it. Here, we consider how the effect size of the susceptibility locus, the frequency of the disease allele(s), the frequency of the marker allele(s) that are correlated with the disease allele(s) and the extent of linkage disequilibrium together influence genetic association studies.

Zondervan KT, Cardon LR. 2004. The complex interplay among factors that influence allelic association Nature Reviews Genetics, 5 (3), pp. 238-238. | Read more

Zondervan K, Cardon L, Kennedy S. 2002. Development of a Web site for the genetic epidemiology of endometriosis. Fertil Steril, 78 (4), pp. 777-781. | Show Abstract | Read more

OBJECTIVE: Endometriosis is a complex trait, in which genetic and environmental factors act together to produce the phenotype. So far, research into candidate genes has largely been based on biological and clinical hypotheses. Results of these studies and the wealth of gene and marker sequence information from the Human Genome Project could--when brought together--provide the researcher with new etiological avenues to explore. DESIGN: Online review. SETTING: The Web site being developed draws together evidence of genetic variants associated with endometriosis and new etiological hypotheses. It incorporates links to up-to-date genomic information relevant to the candidates from a range of bioinformatics databases. PATIENT(S): Endometriosis cases and controls in association studies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele and genotype frequencies. RESULT(S): The Web site summarizes the main hypotheses for endometriosis etiology that provide the basis for the search for genes involved, together with [1] the existing evidence of associations with candidate genes, with links to the relevant publications; [2] details of these candidate genes and the surrounding chromosomal area (location, function, polymorphisms, marker maps); [3] molecular biological findings, from studies of aberrant gene and protein expression in relevant tissues; and [4] chromosomal regions that have been implicated. CONCLUSION(S): This Web site should provide a useful information tool for the endometriosis researcher. We encourage researchers worldwide to use it, contribute to it, and share their knowledge about the condition.

Zondervan KT, Cardon LR, Kennedy SH. 2002. What makes a good case-control study? Design issues for complex traits such as endometriosis. Hum Reprod, 17 (6), pp. 1415-1423. | Show Abstract | Read more

The combined investigation of environmental and genetic risk-factors in complex traits will refocus attention on the case-control study. Endometriosis is an example of a complex trait for which most case-control studies have not followed the basic criteria of epidemiological study design. Appropriate control selection has been a particular problem. This article reviews the principles underlying the design of case-control studies, and their application to the study of endometriosis. Only if it is designed well is the case-control study a suitable alternative to the prospective cohort study. Use of newly diagnosed over prevalent cases is preferable, as the latter may alter risk estimates and complicate the interpretation of findings. Controls should be selected from the source population from which cases arose. Potential confounding should be addressed both in studies of environmental and genetic factors. For endometriosis, a possible design would be to: (i) use newly diagnosed cases with 'endometriotic' disease; (ii) collect information predating symptom onset; and (iii) use at least one population-based female control group matched on unadjustable confounders and screened for pelvic symptoms. In conclusion, future studies of complex traits such as endometriosis will have to incorporate both environmental and genetic factors. Only adequately designed studies will allow reliable results to be obtained and any true aetiologic heterogeneity expected to underlie a complex trait to be detected.

Zondervan K, Cardon L, Desrosiers R, Hyde D, Kemnitz J, Mansfield K, Roberts J, Scheffler J, Weeks DE, Kennedy S. 2002. The genetic epidemiology of spontaneous endometriosis in the rhesus monkey. Ann N Y Acad Sci, 955 (1), pp. 233-238. | Show Abstract | Read more

The etiology of endometriosis is uncertain, but there is increasing evidence that it is inherited as a complex genetic trait like diabetes or asthma. In such complex traits, multiple gene loci conferring susceptibility to the disease interact with each other and the environment to produce the phenotype. The study of such interactions in humans can be problematic. Thus, the availability of an animal model, which shares many aspects of anatomy and physiology with humans, is potentially a valuable tool for investigating the genetic epidemiology of the disease. Since endometriosis develops spontaneously in the rhesus monkey (Macaca mulatta) and the tissue is morphologically identical to its human counterpart, this population provides a unique opportunity to conduct such studies in this condition.

Zondervan K, Cardon L, Kennedy S. 2002. Designing studies into the genetic epidemiology of endometriosis. FERTILITY AND STERILITY, 77 (2), pp. S32-S32. | Read more

Zondervan K, Cardon L, Kennedy S. 2002. The genetic epidemiology of endometriosis - development of a website for the aggregation of global evidence. FERTILITY AND STERILITY, 77 (2), pp. S31-S31. | Read more

Nakago S, Hadfield RM, Zondervan KT, Mardon H, Manek S, Weeks DE, Barlow D, Kennedy S. 2001. Association between endometriosis and N-acetyl transferase 2 polymorphisms in a UK population. Mol Hum Reprod, 7 (11), pp. 1079-1083. | Show Abstract | Read more

The relationship between endometriosis and polymorphisms in the N-acetyl transferase 2 (NAT 2) gene was investigated in a UK population, as this gene has been previously implicated in the aetiology of the disease. Point mutations in the gene result in the variant alleles NAT 2 *5, *6 and *7 from the wild-type NAT 2 *4 allele. Homozygotes for the NAT 2 *4 wild type allele are fast NAT acetylators, while heterozygotes with one wild-type allele and a variant NAT 2 *5, *6 or *7 allele have reduced enzyme activity, and individuals with two variant alleles are slow acetylators. The NAT 2 *4/*6 genotype was significantly more common among affected women (35.2%) than population controls (8.1%; P = 0.0001) or unaffected women (4.2%; P = 0.02). Significantly more affected women (57.4%) were fast acetylators than were population controls (32.3%; P < 0.01) or unaffected women (33.3%; P < 0.05). These data suggest that altered NAT 2 enzyme activity may be a predisposition factor in endometriosis, or that NAT 2 alleles may be in linkage disequilibrium with a susceptibility allele in the same chromosomal region.

Zondervan KT, Yudkin PL, Vessey MP, Jenkinson CP, Dawes MG, Barlow DH, Kennedy SH. 2001. The community prevalence of chronic pelvic pain in women and associated illness behaviour. Br J Gen Pract, 51 (468), pp. 541-547. | Show Abstract

BACKGROUND: Chronic pelvic pain has often been described as a major women's health issue, but no information exists on the extent of the problem in the United Kingdom. AIM: To investigate the community prevalence of chronic pelvic pain and its effect on the lives of consulting and non-consulting women. DESIGN OF STUDY: Postal questionnaire survey. SETTING: Women aged 18 to 49 (n = 3916) randomly selected from the Oxfordshire Health Authority Register. METHOD: The questionnaire response rate (adjusted for non-deliveries) was 74% (2304/3106). Chronic pelvic pain was defined as recurrent or constant pelvic pain of at least six months' duration, unrelated to periods, intercourse, or pregnancy. Case subgroups comprised recent consulters, past consulters, and non-consulters. Women who reported dysmenorrhoea alone formed a comparison group. RESULTS: The three-month prevalence of chronic pelvic pain was 24.0% (95% CI = 22.1% to 25.8%). One-third of women reported pain that started more than five years ago. Recent consulters (32% of cases) were most affected by their symptoms in terms of pain severity, use of health care, physical and mental health scores, sleep quality, and pain-related absence from work. Non-consulters (41% of cases) did not differ from women with dysmenorrhoea in terms of symptom-related impairment. Irrespective of consulting behaviour, a high rate of symptom-related anxiety was found in women with chronic pelvic pain (31%) compared with women with dysmenorrhoea (7%). CONCLUSIONS: This study showed a high community prevalence of chronic pelvic pain in women of reproductive age. Cases varied substantially in the degree to which they were affected by their symptoms. The high symptom-related anxiety in these women emphasises the need for more information about chronic pelvic pain and its possible causes.

Zondervan KT, Cardon LR, Kennedy SH. 2001. The genetic basis of endometriosis. Curr Opin Obstet Gynecol, 13 (3), pp. 309-314. | Show Abstract | Read more

Family studies have long suggested a role for genetic factors in the aetiology of endometriosis. The influence of genes on disease development has mainly been researched independently of environmental factors, yet their interaction must play an important role. Greater exposure to retrograde menstruation and oestrogen is likely to increase the risk of endometriosis; toxic compounds such as dioxin may increase the risk, although the only direct evidence has come from primate studies. Previous association studies implicated GALT (a gene involved in galactose metabolism), and GSTM1 and NAT2 (genes encoding for the detoxification enzymes) as possible disease susceptibility genes. Recent findings have added to the evidence for the involvement of GSTM1 and NAT2, but have cast doubt on the role of GALT. However, the design of many genetic and epidemiological studies has been inadequate with respect to sample size, consistency in phenotype definition, and the choice of control populations. These features are likely to influence results, and could partly explain the lack of consistency in the findings. Future studies should use a consistent disease definition and be of appropriate epidemiological design.

Zondervan KT, Yudkin PL, Vessey MP, Jenkinson CP, Dawes MG, Barlow DH, Kennedy SH. 2001. Chronic pelvic pain in the community--symptoms, investigations, and diagnoses. Am J Obstet Gynecol, 184 (6), pp. 1149-1155. | Show Abstract | Read more

OBJECTIVES: This study was undertaken to investigate the overlap between chronic pelvic pain, dysmenorrhea, dyspareunia, irritable bowel syndrome, and genitourinary symptoms in the community and also to examine associated investigations and diagnoses. STUDY DESIGN: A postal questionnaire was used to survey 3916 women aged 18 through 49 randomly selected from the Oxfordshire Health Authority Register. The number of responders was 2304 (74% of 3106 questionnaire recipients). Chronic pelvic pain was described as recurrent or constant pelvic pain of > or =6 months' duration unrelated to periods, intercourse, or pregnancy. Case patients (n = 483) were subgrouped as follows: (1) chronic pelvic pain only, (2) chronic pelvic pain and irritable bowel syndrome, (3) chronic pelvic pain and genitourinary symptoms, and (4) chronic pelvic pain, genitourinary symptoms, and irritable bowel syndrome. RESULTS: Half the women with chronic pelvic pain also had either genitourinary symptoms or irritable bowel syndrome, or both. Prevalences of dysmenorrhea and dyspareunia were higher among women with chronic pelvic pain (81% and 41%, respectively) than among women without chronic pelvic pain (58% and 14%, respectively); rates did not differ among the chronic pelvic pain subgroups. Irritable bowel syndrome and stress were the most common diagnoses received by patients with chronic pelvic pain, but 50% had never received a diagnosis. CONCLUSIONS: There is substantial overlap between chronic pelvic pain and other abdominal symptoms in the community. Despite a high prevalence of chronic pelvic pain, many women have never had the condition diagnosed.

Zondervan K, Barlow DH. 2000. Epidemiology of chronic pelvic pain. Baillieres Best Pract Res Clin Obstet Gynaecol, 14 (3), pp. 403-414. | Show Abstract | Read more

An overview is given of the current knowledge of the epidemiology of chronic pelvic pain (CPP) in terms of prevalence, incidence, and associated risk factors. However, the lack of a consensus on the definition of CPP greatly hinders epidemiological studies. Although data are limited, the prevalence of CPP in the general population appears to be high. A single study found a 3-month prevalence (pelvic pain of at least 6 months' duration) of 15% in women aged 18-50 in the general US population. In the UK, an annual prevalence in primary care of 38/1000 was found in women aged 15-73, a rate comparable to that of asthma (37/1000) and back pain (41/1000). The monthly incidence in primary care was 1.6/1000. No incidence figures exist for the general population. Analysis of risk factors for CPP is highly complicated owing to its multifactorial aetiology. At present, it is only of some value using women with CPP identified at community level, since those in primary, secondary or tertiary care are likely to constitute highly selected sub-groups.

Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy SH. 1999. Patterns of diagnosis and referral in women consulting for chronic pelvic pain in UK primary care. Br J Obstet Gynaecol, 106 (11), pp. 1156-1161. | Show Abstract | Read more

OBJECTIVES: To describe duration of symptoms and patterns of diagnosis and referral in women with chronic pelvic pain. DESIGN: Retrospective cohort analysis of the MediPlus UK Primary Care Database. SETTING: One hundred and thirty-six general practices in the UK. STUDY GROUP: A cohort of 5051 incident cases of chronic pelvic pain. METHODS: The cohort was followed up from the start of their symptoms in 1992 until the end of the chronic pelvic pain episode or the end of 1995. MAIN OUTCOME MEASURES: Duration of symptoms, frequency of diagnoses and referral rates. RESULTS: A third of women had symptoms persisting for more than two years. Duration of symptoms increased significantly with age (P < 0.001) from a median of 13.7 months in 13-20 year olds to 20.2 months in women over the age of 60. Irritable bowel syndrome and cystitis were the most common diagnoses at all ages. Twenty-eight percent of women never received a diagnosis during three to four years of follow up after first consultation, and 60% of women had no evidence of a specialist referral. Women aged 21-50 and women whose final diagnosis was endometriosis received the largest number of diagnoses and had the highest referral rates. CONCLUSIONS: The numbers and types of diagnosis given to a woman with chronic pelvic pain and the likelihood of specialist referral depend on her age, as well as on the duration of symptoms. Women seen in secondary care for chronic pelvic pain are a highly selected group and are likely to represent only the tip of the iceberg.

Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy SH. 1999. Prevalence and incidence of chronic pelvic pain in primary care: evidence from a national general practice database. Br J Obstet Gynaecol, 106 (11), pp. 1149-1155. | Show Abstract | Read more

OBJECTIVES: To estimate the prevalence and incidence in primary care of chronic pelvic pain in women in the UK. DESIGN: Cross-sectional analysis of MediPlus UK Primary Care Database. SETTING: One hundred and thirty-six general practices in the UK. POPULATION: From 284,162 women aged 12-70 who were registered on the database and who had a general practice contact in 1991, 24,053 chronic pelvic pain cases were identified between 1991 and 1995. METHODS: Chronic pelvic pain was defined as pelvic pain lasting for at least six months, and cases were identified on the basis of contacts with general practice. Pain due to malignancy, chronic inflammatory bowel diseases or pregnancy, or which occurred only during menstruation or sexual intercourse, was excluded. MAIN OUTCOME MEASURES: Prevalence and incidence rates of chronic pelvic pain in primary care by age and region. RESULTS: Monthly prevalence and incidence rates of chronic pelvic pain were 21.5/1000 and 1.58/1000, respectively, with an annual prevalence of 38.3/1000. Monthly prevalence rates increased significantly with age (P < 0.001) from 18.2/1000 in 15-20 year olds to 27.6/1000 in women older than 60, as symptoms persisted longer in older women. Prevalence and incidence rates varied significantly between regions (P < 0.001), with the lowest monthly prevalence in Scotland (16.0/1000) and the highest in Wales (29.4/1000). CONCLUSIONS: Chronic pelvic pain is a common condition in the UK, with a prevalence in primary care comparable to migraine, back pain, and asthma. Its prevalence in the general population is likely to be considerably higher.

Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy SH. 1998. The prevalence of chronic pelvic pain in women in the United Kingdom: a systematic review. Br J Obstet Gynaecol, 105 (1), pp. 93-99. | Show Abstract | Read more

OBJECTIVE: To obtain a prevalence estimate for chronic pelvic pain in women in the United Kingdom by analysing published data. DESIGN: Systematic review of published papers. SETTING: The general population or hospitals in the United Kingdom. POPULATION: Women participating in relevant community surveys or control women participating in hospital-based studies. METHODS: Papers were retrieved by systematically searching the databases MEDLINE, EMBASE and PsycLit, and by hand searching. Studies were included if they 1. were community-based and reported prevalence rates of chronic pelvic pain, dyspareunia, dysmenorrhoea, or abdominal pain, or 2. referred to a clinical population but reported prevalence rates in a disease-free control group. MAIN OUTCOME MEASURES: Prevalence rates for chronic pelvic pain including any overlap with dyspareunia, dysmenorrhoea and abdominal pain. RESULTS: No community-based study has been performed that provides an estimate of the prevalence of chronic pelvic pain in the general UK population. A rate of 39% was reported in women undergoing laparoscopy for sterilisation or investigation of infertility in the single study from the United Kingdom investigating chronic pelvic pain unrelated to menstruation or intercourse. Prevalence rates for dyspareunia, dysmenorrhoea, and abdominal pain found in UK community-based studies were 8%, 45% to 97%, and 23% to 29%, respectively, but definitions used varied greatly. CONCLUSIONS: Because chronic pelvic pain can reduce the quality of life and general wellbeing, there is a need for a community-based study into the prevalence of chronic pelvic pain and its effect upon the lives of women in the UK.

Zondervan KT, Carpenter LM, Painter R, Vessey MP. 1996. Oral contraceptives and cervical cancer--further findings from the Oxford Family Planning Association contraceptive study. Br J Cancer, 73 (10), pp. 1291-1297. | Show Abstract | Read more

In 1983, we reported results from the Oxford Family Planning Association contraceptive study regarding the association between oral contraceptives (OCs) and cervical neoplasia, after a 10 year follow-up of a cohort of 17,000 women. Further findings from this study are reported here after an additional 12 years of follow-up. A nested case--control design was used in which cases were all women diagnosed under 45 years of age with invasive carcinoma (n = 33), carcinoma in situ (n = 121) or dysplasia (n = 159). Controls were randomly selected from among cohort members and matched to cases on exact year of birth and clinic attended at recruitment to study. Conditional logistic regression analysis was used to determine odds ratios (ORs) and 95% confidence intervals (CIs) associated with various aspects of OC use relative to never users adjusted for social class, smoking, age at first birth and ever use of diaphragm or condom. Ever users of OCs had a slightly elevated OR for all types of cervical neoplasia combined (OR = 1.40, 95% CI 1.00-1.96). Odds ratios were highest for invasive carcinoma (OR = 4.44, 95% CI 1.04-31.6), intermediate for carcinoma in situ (OR = 1.73, 95% CI 1.00-3.00) and lowest for dysplasia (OR = 1.07, 95% CI 0.69-1.66). The elevated risk associated with OC use appeared to be largely confined to current or recent (last use in the past 2 years) long-term users of OCs. Among current or recent users, ORs for all types of cervical neoplasia combined were 3.34 (95% CI 1.96-5.67) for 49-72 months of use, 1.69 (95% CI 0.97-2.95) for 73-96 months and 2.04 (95% CI 1.34-3.11) for 97 or more months. These results suggest a possible effect of OC use on later stages of cervical carcinogenesis, although residual confounding due to sexual factors or human papillomavirus (HPV) infection cannot be ruled out.

Zondervan KT, Ocké MC, Smit HA, Seidell JC. 1996. Do dietary and supplementary intakes of antioxidants differ with smoking status? Int J Epidemiol, 25 (1), pp. 70-79. | Show Abstract | Read more

BACKGROUND: Differences in dietary and supplementary intake of antioxidants were determine between different categories of smokers and never-smokers. METHODS: Data from a large, cross-sectional, population-based study were used. Subjects (n = 4244) were divided into five smoking categories according to the number of cigarettes smoked per day. Differences in intake of antioxidants or frequency of supplement use were assessed using multiple linear regression analysis and multiple logistic regression analysis, adjusting for potential confounders such as age, body mass index, education level, alcohol intake, and total energy intake. RESULTS: Men who smoked > 20 cigarettes/day had significantly lower intakes of beta-carotene and especially ascorbic acid compared to those who never smoked, resulting from an almost 60% lower fruit intake. Moderate and heavy smoking women also had lower ascorbic acid and fruit intake but differences were not as large as in men. A higher percentage of female heavy smokers compared with never-smokers consumed vitamin C (21.1% versus 14.1%), vitamin E (5.6% versus 1.8%), and multivitamin supplements (18.5% versus 12.2%). Among men only the moderate smokers differed significantly from never-smokers in supplement intake, in the sense that male moderate smokers had a higher percentage of multivitamin use (15.3% versus 12.2%) compared to never-smokers. CONCLUSIONS: Male heavy smokers not only have a lower dietary antioxidant intake than never-smokers, but additionally seem to use supplementation relatively infrequently.

Zondervan KT, Buitendijk SE, Anthony S, van Rijssel EJ, Verkerk PH. 1995. [Frequency and determinants of episiotomy in second-line obstetrics in The Netherlands]. Ned Tijdschr Geneeskd, 139 (9), pp. 449-452. | Show Abstract

OBJECTIVE: To study factors that influence the probability of episiotomy in Dutch gynaecologist-supervised deliveries. SETTING: Obstetric units of Dutch hospitals. DESIGN: Observational study. METHODS: Data of 65,313 gynaecologist-supervised, vaginal deliveries of live-born singletons registered in the Dutch National Obstetric Database of 1990, were used. Firstly, the effect of characteristics of the mother, the child, the pregnancy, and the delivery on the probability of episiotomy were assessed in univariate analyses. Subsequently logistic regression analysis was used to determine the effect of each variable, while adjusting for the other variables. RESULTS: The episiotomy rate in the total group of gynaecologist supervised deliveries was 39%. In the subgroup of vaginal deliveries of live born singletons, the rate was 46%. Besides the well-known risk factors such as parity, instrumental delivery and length of second stage of labour, ethnicity was also found to have an independent effect on the risk of an episiotomy. Mediterranean, Creole and Hindu women had a lower episiotomy risk than Dutch women (OR: 0.47 and 95% CI: 0.44-0.51). Gynaecologists more often performed episiotomy than midwives, after adjusting for possible confounding factors (OR: 1.54; 1.46-1.63). In University hospitals fewer episiotomies were performed than in large non-university hospitals (OR: 0.81; 0.76-0.87. CONCLUSION: The decision to perform episiotomy appears not to depend solely on factors related to perineal rupture or foetal complications. The probability of episiotomy is also influenced by attendant at delivery and type of hospital.

Zondervan KT, Buitendijk SE, Anthony S, Van Rijssel EJC, Verkerk PH. 1995. Frequency and determinants of episiotomy in Dutch gynaecologist-supervised deliveries Nederlands Tijdschrift voor Geneeskunde, 139 (9), pp. 449-452. | Show Abstract

Objective. To study factors that influence the probability of episiotomy in Dutch gynaecologist-supervised deliveries. Setting. Obstetric units of Dutch hospitals. Design. Observational study. Methods. Data of 65,313 gynaecologist-supervised, vaginal deliveries of live-born singletons registered in the Dutch National Obstetric Database of 1990, were used. Firstly, the effect of characteristics of the mother, the child, the pregnancy, and the delivery on the probability of episiotomy were assessed in univariate analyses. Subsequently logistic regression analysis was used to determine the effect of each variable, while adjusting for the other variables. Results. The episiotomy rate in the total group of gynaecologist supervised deliveries was 39%. In the subgroup of vaginal deliveries of live born singletons, the rate was 46%. Besides the well-known risk factors such as parity, instrumental delivery and length of second stage of labour, ethnicity was also found to have an independent effect on the risk of an episiotomy. Mediterranean, Creole and Hindu women had a lower episiotomy risk than Dutch women (OR: 0.47 and 95% CI: 0.44-0.51). Gynaecologists more often performed episiotomy than midwives, after adjusting for possible confounding factors (OR: 1.54; 1.46-1.63). In University hospitals fewer episiotomies were performed than in large non-university hospitals (OR: 0.81: 0.76-0.87). Conclusion. The decision to perform episiotomy appears not to depend solely on factors related to perineal rupture or foetal complications. The probability of episiotomy is also influenced by attendant at delivery and type of hospital.

Anthony S, Buitendijk SE, Zondervan KT, van Rijssel EJ, Verkerk PH. 1994. Episiotomies and the occurrence of severe perineal lacerations. Br J Obstet Gynaecol, 101 (12), pp. 1064-1067. | Show Abstract | Read more

OBJECTIVE: To investigate the relation between the use of mediolateral episiotomy and the occurrence of severe (third degree) perineal tears in hospital deliveries in the Netherlands. DESIGN: An observational study. SUBJECTS: Data were derived from the Dutch National Obstetric Database (LVR) of 1990, from which 43,309 spontaneous, occipito-anterior, vaginal deliveries of live, singleton infants were investigated. INTERVENTION: Medio-lateral episiotomy. MAIN OUTCOME MEASURE: The occurrence of severe perineal tears. RESULTS: The severe tear rate was 1.4% in the total study group. Using multiple logistic regression to control for possible confounding variables, the use of mediolateral episiotomy was found to be associated with a more than fourfold decrease in risk of severe lacerations (odds ratio 0.22, 95% CI 0.17 to 0.29). Further, in a logistic model deliveries in hospitals with restrictive use of episiotomy (< 11%) were compared with those in hospitals with liberal use of episiotomy (> 50%). Liberal use of episiotomy was not associated with a lower frequency of severe perineal tears. CONCLUSION: Although a protective effect of mediolateral episiotomy on the occurrence of severe lacerations was found, liberal use of mediolateral episiotomy should be discouraged on the basis of our findings.

BALLIEUX B, ZONDERVAN K, KIEVIT P, HAGEN E, VANDERWOUDE F, VANES L, DAHA M. 1994. DIFFERENTIAL BINDING OF PR3 AND MPO TO MONOLAYERS OF HUVEC - ANCA MEDIATED ADCC OF PR3/MPO INCUBATED HUVEC KIDNEY INTERNATIONAL, 46 (5), pp. 1462-1463.

Ballieux BE, Zondervan KT, Kievit P, Hagen EC, van Es LA, van der Woude FJ, Daha MR. 1994. Binding of proteinase 3 and myeloperoxidase to endothelial cells: ANCA-mediated endothelial damage through ADCC? Clin Exp Immunol, 97 (1), pp. 52-60. | Show Abstract | Read more

Binding of both proteinase 3 (PR3) and myeloperoxidase (MPO) to endothelial cells (EC) has been suggested to be involved in the vascular damage seen in patients with Wegener's granulomatosis or microscopic polyangiitis. In the present study we investigated in detail the interaction of MPO and PR3 with cultured human umbilical vein endothelial cells (HUVEC) and its matrix products. In addition, we investigated whether interaction of PR3 or MPO with HUVEC monolayers also resulted in antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-neutrophil cytoplasmic antibody (ANCA)-positive patient sera or rabbit IgG anti-PR3 or anti-MPO. Preincubation of HUVEC monolayers with PR3 or MPO resulted in a dose-dependent binding of both PR3 and MPO. However, HUVEC, preincubated with PR3 or MPO, followed by ANCA or by rabbit anti-PR3 or anti-MPO, were not susceptible to ADCC. Detailed analysis of the binding of PR3 to HUVEC monolayers showed that PR3 binds primarily to the extracellular matrix of endothelial cells, and to a very limited extent to the cells themselves. For MPO it was shown that it binds both to the extracellular matrix and to the endothelial cells themselves. However, after binding to HUVEC cultures, MPO was not detectable by polyclonal rabbit or human antibodies specific for MPO, probably because MPO is bound to sites not accessible for immunoglobulins. Binding of PR3 to HUVEC cultures (cells + matrix) was inhibited by fetal calf serum and by alpha 1-antitrypsin, but inactivation of enzymatic activity of PR3 by PMSF did not influence binding of PR3 to HUVEC cultures. Binding of MPO to HUVEC cultures was not influenced by fetal calf serum.

Sapkota Y, Steinthorsdottir V, Morris AP, Fassbender A, Rahmioglu N, De Vivo I, Buring JE, Zhang F, Edwards TL, Jones S et al. 2017. Meta-analysis identifies five novel loci associated with endometriosis highlighting key genes involved in hormone metabolism. Nat Commun, 8 pp. 15539. | Show Abstract | Read more

Endometriosis is a heritable hormone-dependent gynecological disorder, associated with severe pelvic pain and reduced fertility; however, its molecular mechanisms remain largely unknown. Here we perform a meta-analysis of 11 genome-wide association case-control data sets, totalling 17,045 endometriosis cases and 191,596 controls. In addition to replicating previously reported loci, we identify five novel loci significantly associated with endometriosis risk (P<5 × 10(-8)), implicating genes involved in sex steroid hormone pathways (FN1, CCDC170, ESR1, SYNE1 and FSHB). Conditional analysis identified five secondary association signals, including two at the ESR1 locus, resulting in 19 independent single nucleotide polymorphisms (SNPs) robustly associated with endometriosis, which together explain up to 5.19% of variance in endometriosis. These results highlight novel variants in or near specific genes with important roles in sex steroid hormone signalling and function, and offer unique opportunities for more targeted functional research efforts.

Uimari O, Rahmioglu N, Nyholt DR, Vincent K, Missmer SA, Becker C, Morris AP, Montgomery GW, Zondervan KT. 2017. Genome-wide genetic analyses highlight mitogen-activated protein kinase (MAPK) signaling in the pathogenesis of endometriosis. Hum Reprod, 32 (4), pp. 780-793. | Show Abstract | Read more

Study question: Do genome-wide association study (GWAS) data for endometriosis provide insight into novel biological pathways associated with its pathogenesis? Summary answer: GWAS analysis uncovered multiple pathways that are statistically enriched for genetic association signals, analysis of Stage A disease highlighted a novel variant in MAP3K4, while top pathways significantly associated with all endometriosis and Stage A disease included several mitogen-activated protein kinase (MAPK)-related pathways. What is known already: Endometriosis is a complex disease with an estimated heritability of 50%. To date, GWAS revealed 10 genomic regions associated with endometriosis, explaining <4% of heritability, while half of the heritability is estimated to be due to common risk variants. Pathway analyses combine the evidence of single variants into gene-based measures, leveraging the aggregate effect of variants in genes and uncovering biological pathways involved in disease pathogenesis. Study design size, duration: Pathway analysis was conducted utilizing the International Endogene Consortium GWAS data, comprising 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry with genotype data imputed up to 1000 Genomes Phase three reference panel. GWAS was performed for all endometriosis cases and for Stage A (revised American Fertility Society (rAFS) I/II, n = 1686) and B (rAFS III/IV, n = 1364) cases separately. The identified significant pathways were compared with pathways previously investigated in the literature through candidate association studies. Participants/materials, setting, methods: The most comprehensive biological pathway databases, MSigDB (including BioCarta, KEGG, PID, SA, SIG, ST and GO) and PANTHER were utilized to test for enrichment of genetic variants associated with endometriosis. Statistical enrichment analysis was performed using the MAGENTA (Meta-Analysis Gene-set Enrichment of variaNT Associations) software. Main results and the role of chance: The first genome-wide association analysis for Stage A endometriosis revealed a novel locus, rs144240142 (P = 6.45 × 10-8, OR = 1.71, 95% CI = 1.23-2.37), an intronic single-nucleotide polymorphism (SNP) within MAP3K4. This SNP was not associated with Stage B disease (P = 0.086). MAP3K4 was also shown to be differentially expressed in eutopic endometrium between Stage A endometriosis cases and controls (P = 3.8 × 10-4), but not with Stage B disease (P = 0.26). A total of 14 pathways enriched with genetic endometriosis associations were identified (false discovery rate (FDR)-P < 0.05). The pathways associated with any endometriosis were Grb2-Sos provides linkage to MAPK signaling for integrins pathway (P = 2.8 × 10-5, FDR-P = 3.0 × 10-3), Wnt signaling (P = 0.026, FDR-P = 0.026) and p130Cas linkage to MAPK signaling for integrins pathway (P = 6.0 × 10-4, FDR-P = 0.029); with Stage A endometriosis: extracellular signal-regulated kinase (ERK)1 ERK2 MAPK (P = 5.0 × 10-4, FDR-P = 5.0 × 10-4) and with Stage B endometriosis: two overlapping pathways that related to extracellular matrix biology-Core matrisome (P = 1.4 × 10-3, FDR-P = 0.013) and ECM glycoproteins (P = 1.8 × 10-3, FDR-P = 7.1 × 10-3). Genes arising from endometriosis candidate gene studies performed to date were enriched for Interleukin signaling pathway (P = 2.3 × 10-12), Apoptosis signaling pathway (P = 9.7 × 10-9) and Gonadotropin releasing hormone receptor pathway (P = 1.2 × 10-6); however, these pathways did not feature in the results based on GWAS data. Large scale data: Not applicable. Limitations, reasons for caution: The analysis is restricted to (i) variants in/near genes that can be assigned to pathways, excluding intergenic variants; (ii) the gene-based pathway definition as registered in the databases; (iii) women of European ancestry. Wider implications of the findings: The top ranked pathways associated with overall and Stage A endometriosis in particular involve integrin-mediated MAPK activation and intracellular ERK/MAPK acting downstream in the MAPK cascade, both acting in the control of cell division, gene expression, cell movement and survival. Other top enriched pathways in Stage B disease include ECM glycoprotein pathways important for extracellular structure and biochemical support. The results highlight the need for increased efforts to understand the functional role of these pathways in endometriosis pathogenesis, including the investigation of the biological effects of the genetic variants on downstream molecular processes in tissue relevant to endometriosis. Additionally, our results offer further support for the hypothesis of at least partially distinct causal pathophysiology for minimal/mild (rAFS I/II) vs. moderate/severe (rAFS III/IV) endometriosis. Study funding/competing interest(s): The genome-wide association data and Wellcome Trust Case Control Consortium (WTCCC) were generated through funding from the Wellcome Trust (WT084766/Z/08/Z, 076113 and 085475) and the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498). N.R. was funded by a grant from the Medical Research Council UK (MR/K011480/1). A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant WT098017). All authors declare there are no conflicts of interest.

Barban N, Jansen R, de Vlaming R, Vaez A, Mandemakers JJ, Tropf FC, Shen X, Wilson JF, Chasman DI, Nolte IM et al. 2016. Genome-wide analysis identifies 12 loci influencing human reproductive behavior. Nat Genet, 48 (12), pp. 1462-1472. | Show Abstract | Read more

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits.

Rahmioglu N, Nyholt DR, Morris AP, Missmer SA, Montgomery GW, Zondervan KT. 2014. Genetic variants underlying risk of endometriosis: insights from meta-analysis of eight genome-wide association and replication datasets. Hum Reprod Update, 20 (5), pp. 702-716. | Show Abstract | Read more

BACKGROUND: Endometriosis is a heritable common gynaecological condition influenced by multiple genetic and environmental factors. Genome-wide association studies (GWASs) have proved successful in identifying common genetic variants of moderate effects for various complex diseases. To date, eight GWAS and replication studies from multiple populations have been published on endometriosis. In this review, we investigate the consistency and heterogeneity of the results across all the studies and their implications for an improved understanding of the aetiology of the condition. METHODS: Meta-analyses were conducted on four GWASs and four replication studies including a total of 11 506 cases and 32 678 controls, and on the subset of studies that investigated associations for revised American Fertility Society (rAFS) Stage III/IV including 2859 cases. The datasets included 9039 cases and 27 343 controls of European (Australia, Belgium, Italy, UK, USA) and 2467 cases and 5335 controls of Japanese ancestry. Fixed and Han and Elkin random-effects models, and heterogeneity statistics (Cochran's Q test), were used to investigate the evidence of the nine reported genome-wide significant loci across datasets and populations. RESULTS: Meta-analysis showed that seven out of nine loci had consistent directions of effect across studies and populations, and six out of nine remained genome-wide significant (P < 5 × 10(-8)), including rs12700667 on 7p15.2 (P = 1.6 × 10(-9)), rs7521902 near WNT4 (P = 1.8 × 10(-15)), rs10859871 near VEZT (P = 4.7 × 10(-15)), rs1537377 near CDKN2B-AS1 (P = 1.5 × 10(-8)), rs7739264 near ID4 (P = 6.2 × 10(-10)) and rs13394619 in GREB1 (P = 4.5 × 10(-8)). In addition to the six loci, two showed borderline genome-wide significant associations with Stage III/IV endometriosis, including rs1250248 in FN1 (P = 8 × 10(-8)) and rs4141819 on 2p14 (P = 9.2 × 10(-8)). Two independent inter-genic loci, rs4141819 and rs6734792 on chromosome 2, showed significant evidence of heterogeneity across datasets (P < 0.005). Eight of the nine loci had stronger effect sizes among Stage III/IV cases, implying that they are likely to be implicated in the development of moderate to severe, or ovarian, disease. While three out of nine loci were inter-genic, the remaining were in or near genes with known functions of biological relevance to endometriosis, varying from roles in developmental pathways to cellular growth/carcinogenesis. CONCLUSIONS: Our meta-analysis shows remarkable consistency in endometriosis GWAS results across studies, with little evidence of population-based heterogeneity. They also show that the phenotypic classifications used in GWAS to date have been limited. Stronger associations with Stage III/IV disease observed for most loci emphasize the importance for future studies to include detailed sub-phenotype information. Functional studies in relevant tissues are needed to understand the effect of the variants on downstream biological pathways.

Nyholt DR, Low SK, Anderson CA, Painter JN, Uno S, Morris AP, MacGregor S, Gordon SD, Henders AK, Martin NG et al. 2012. Genome-wide association meta-analysis identifies new endometriosis risk loci. Nat Genet, 44 (12), pp. 1355-1359. | Show Abstract | Read more

We conducted a genome-wide association meta-analysis of 4,604 endometriosis cases and 9,393 controls of Japanese and European ancestry. We show that rs12700667 on chromosome 7p15.2, previously found to associate with disease in Europeans, replicates in Japanese (P = 3.6 × 10(-3)), and we confirm association of rs7521902 at 1p36.12 near WNT4. In addition, we establish an association of rs13394619 in GREB1 at 2p25.1 with endometriosis and identify a newly associated locus at 12q22 near VEZT (rs10859871). Excluding cases of European ancestry of minimal or unknown severity, we identified additional previously unknown loci at 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21.3 (rs1537377). All seven SNP effects were replicated in an independent cohort and associated at P <5 × 10(-8) in a combined analysis. Finally, we found a significant overlap in polygenic risk for endometriosis between the genome-wide association cohorts of European and Japanese descent (P = 8.8 × 10(-11)), indicating that many weakly associated SNPs represent true endometriosis risk loci and that risk prediction and future targeted disease therapy may be transferred across these populations.

Grundberg E, Small KS, Hedman ÅK, Nica AC, Buil A, Keildson S, Bell JT, Yang TP, Meduri E, Barrett A et al. 2012. Mapping cis- and trans-regulatory effects across multiple tissues in twins. Nat Genet, 44 (10), pp. 1084-1089. | Show Abstract | Read more

Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many expression quantitative trait locus (eQTL) studies, typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis effect on expression cannot be accounted for by common cis variants, a finding that reveals the contribution of low-frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene, and we identify several replicating trans variants that act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.

Min JL, Nicholson G, Halgrimsdottir I, Almstrup K, Petri A, Barrett A, Travers M, Rayner NW, Mägi R, Pettersson FH et al. 2012. Coexpression network analysis in abdominal and gluteal adipose tissue reveals regulatory genetic loci for metabolic syndrome and related phenotypes. PLoS Genet, 8 (2), pp. e1002505. | Show Abstract | Read more

Metabolic Syndrome (MetS) is highly prevalent and has considerable public health impact, but its underlying genetic factors remain elusive. To identify gene networks involved in MetS, we conducted whole-genome expression and genotype profiling on abdominal (ABD) and gluteal (GLU) adipose tissue, and whole blood (WB), from 29 MetS cases and 44 controls. Co-expression network analysis for each tissue independently identified nine, six, and zero MetS-associated modules of coexpressed genes in ABD, GLU, and WB, respectively. Of 8,992 probesets expressed in ABD or GLU, 685 (7.6%) were expressed in ABD and 51 (0.6%) in GLU only. Differential eigengene network analysis of 8,256 shared probesets detected 22 shared modules with high preservation across adipose depots (D(ABD-GLU) = 0.89), seven of which were associated with MetS (FDR P<0.01). The strongest associated module, significantly enriched for immune response-related processes, contained 94/620 (15%) genes with inter-depot differences. In an independent cohort of 145/141 twins with ABD and WB longitudinal expression data, median variability in ABD due to familiality was greater for MetS-associated versus un-associated modules (ABD: 0.48 versus 0.18, P = 0.08; GLU: 0.54 versus 0.20, P = 7.8×10(-4)). Cis-eQTL analysis of probesets associated with MetS (FDR P<0.01) and/or inter-depot differences (FDR P<0.01) provided evidence for 32 eQTLs. Corresponding eSNPs were tested for association with MetS-related phenotypes in two GWAS of >100,000 individuals; rs10282458, affecting expression of RARRES2 (encoding chemerin), was associated with body mass index (BMI) (P = 6.0×10(-4)); and rs2395185, affecting inter-depot differences of HLA-DRB1 expression, was associated with high-density lipoprotein (P = 8.7×10(-4)) and BMI-adjusted waist-to-hip ratio (P = 2.4×10(-4)). Since many genes and their interactions influence complex traits such as MetS, integrated analysis of genotypes and coexpression networks across multiple tissues relevant to clinical traits is an efficient strategy to identify novel associations.

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Nnoaham KE, Hummelshoj L, Kennedy SH, Jenkinson C, Zondervan KT. 2012. Developing symptom-based predictive models of endometriosis as a clinical screening tool: Results from a multicenter study Fertility and Sterility, 98 (3), | Show Abstract | Read more

Objective: To generate and validate symptom-based models to predict endometriosis among symptomatic women prior to undergoing their first laparoscopy. Design: Prospective, observational, two-phase study, in which women completed a 25-item questionnaire prior to surgery. Setting: Nineteen hospitals in 13 countries. Patient(s): Symptomatic women (n = 1,396) scheduled for laparoscopy without a previous surgical diagnosis of endometriosis. Intervention(s): None. Main Outcome Measure(s): Sensitivity and specificity of endometriosis diagnosis predicted by symptoms and patient characteristics from optimal models developed using multiple logistic regression analyses in one data set (phase I), and independently validated in a second data set (phase II) by receiver operating characteristic (ROC) curve analysis. Result(s): Three hundred sixty (46.7%) women in phase I and 364 (58.2%) in phase II were diagnosed with endometriosis at laparoscopy. Menstrual dyschezia (pain on opening bowels) and a history of benign ovarian cysts most strongly predicted both any and stage III and IV endometriosis in both phases. Prediction of any-stage endometriosis, although improved by ultrasound scan evidence of cyst/nodules, was relatively poor (area under the curve [AUC] = 68.3). Stage III and IV disease was predicted with good accuracy (AUC = 84.9, sensitivity of 82.3% and specificity 75.8% at an optimal cut-off of 0.24). Conclusion(s): Our symptom-based models predict any-stage endometriosis relatively poorly and stage III and IV disease with good accuracy. Predictive tools based on such models could help to prioritize women for surgical investigation in clinical practice and thus contribute to reducing time to diagnosis. We invite other researchers to validate the key models in additional populations. © 2012 by American Society for Reproductive Medicine.

Nnoaham KE, Hummelshoj L, Webster P, d'Hooghe T, de Cicco Nardone F, de Cicco Nardone C, Jenkinson C, Kennedy SH, Zondervan KT, World Endometriosis Research Foundation Global Study of Women's Health consortium. 2011. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril, 96 (2), pp. 366-373.e8. | Show Abstract | Read more

OBJECTIVE: To assess the impact of endometriosis on health-related quality of life (HRQoL) and work productivity. DESIGN: Multicenter cross-sectional study with prospective recruitment. SETTING: Sixteen clinical centers in ten countries. PATIENT(S): A total of 1,418 premenopausal women, aged 18-45 years, without a previous surgical diagnosis of endometriosis, having laparoscopy to investigate symptoms or to be sterilized. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Diagnostic delay, HRQoL, and work productivity. RESULT(S): There was a delay of 6.7 years, principally in primary care, between onset of symptoms and a surgical diagnosis of endometriosis, which was longer in centers where women received predominantly state-funded health care (8.3 vs. 5.5 years). Delay was positively associated with the number of pelvic symptoms (chronic pelvic pain, dysmenorrhoea, dyspareunia, and heavy periods) and a higher body mass index. Physical HRQoL was significantly reduced in affected women compared with those with similar symptoms and no endometriosis. Each affected woman lost on average 10.8 hours (SD 12.2) of work weekly, mainly owing to reduced effectiveness while working. Loss of work productivity translated into significant costs per woman/week, from US$4 in Nigeria to US$456 in Italy. CONCLUSION(S): Endometriosis impairs HRQoL and work productivity across countries and ethnicities, yet women continue to experience diagnostic delays in primary care. A higher index of suspicion is needed to expedite specialist assessment of symptomatic women. Future research should seek to clarify pain mechanisms in relation to endometriosis severity.

Painter JN, Nyholt DR, Morris A, Zhao ZZ, Henders AK, Lambert A, Wallace L, Martin NG, Kennedy SH, Treloar SA et al. 2011. High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19. Fertil Steril, 95 (7), pp. 2236-2240. | Show Abstract | Read more

OBJECTIVE: To refine a previously reported linkage peak for endometriosis on chromosome 10q26, and conduct follow-up analyses and a fine-mapping association study across the region to identify new candidate genes for endometriosis. DESIGN: Case-control study. SETTING: Academic research. PATIENT(S): Cases=3,223 women with surgically confirmed endometriosis; controls=1,190 women without endometriosis and 7,060 population samples. INTERVENTION(S): Analysis of 11,984 single nucleotide polymorphisms on chromosome 10. MAIN OUTCOME MEASURE(S): Allele frequency differences between cases and controls. RESULT(S): Linkage analyses on families grouped by endometriosis symptoms (primarily subfertility) provided increased evidence for linkage (logarithm of odds score=3.62) near a previously reported linkage peak. Three independent association signals were found at 96.59 Mb (rs11592737), 105.63 Mb (rs1253130), and 124.25 Mb (rs2250804). Analyses including only samples from linkage families supported the association at all three regions. However, only rs11592737 in the cytochrome P450 subfamily C (CYP2C19) gene was replicated in an independent sample of 2,079 cases and 7,060 population controls. CONCLUSION(S): The role of the CYP2C19 gene in conferring risk for endometriosis warrants further investigation.

Clarke GM, Anderson CA, Pettersson FH, Cardon LR, Morris AP, Zondervan KT. 2011. Basic statistical analysis in genetic case-control studies. Nat Protoc, 6 (2), pp. 121-133. | Show Abstract | Read more

This protocol describes how to perform basic statistical analysis in a population-based genetic association case-control study. The steps described involve the (i) appropriate selection of measures of association and relevance of disease models; (ii) appropriate selection of tests of association; (iii) visualization and interpretation of results; (iv) consideration of appropriate methods to control for multiple testing; and (v) replication strategies. Assuming no previous experience with software such as PLINK, R or Haploview, we describe how to use these popular tools for handling single-nucleotide polymorphism data in order to carry out tests of association and visualize and interpret results. This protocol assumes that data quality assessment and control has been performed, as described in a previous protocol, so that samples and markers deemed to have the potential to introduce bias to the study have been identified and removed. Study design, marker selection and quality control of case-control studies have also been discussed in earlier protocols. The protocol should take ~1 h to complete.

Min JL, Taylor JM, Richards JB, Watts T, Pettersson FH, Broxholme J, Ahmadi KR, Surdulescu GL, Lowy E, Gieger C et al. 2011. The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits. PLoS One, 6 (7), pp. e22070. | Show Abstract | Read more

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097-16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

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