register interest

Dr Samantha JL Knight

Research Area: Genetics and Genomics
Technology Exchange: Chromosome mapping and SNP typing
Scientific Themes: Genetics & Genomics and Cancer Biology
Keywords: translational research, Learning disability, B-cell chronic lymphocytic leukaemia, molecular diagnostics, genome imbalance, microarray technology, copy number variants and copy number abnormalities
Scanned image of a SNP microarray

Scanned image of a SNP microarray

Image of a peripheral blood film at x100 magnification showing multiple mature lymphocytes with condensed nuclear chromatin and basophilic cytoplasm consistent with chronic lymphocytic leukaemia. Some prolymphocytes are also present.

Image of a peripheral blood film at x100 magnification showing multiple mature lymphocytes with ...

TRANSLATIONAL GENETICS: BRINGING RESEARCH INTO CLINICAL PRACTICE

We aim to develop advanced technologies for use in the UK NHS and healthcare services globally and to translate research findings into clinical practice for a range of clinically important conditions.

Our laboratory is affiliated with the Genomic Medicine Theme of the Oxford NIHR Biomedical Research Centre (BRC), an initiative that helps to drive the translation of research from ‘bench to bedside’.

Much of our work has focused on  using microarray technology, an approach that has revolutionized our ability to perform high resolution genome-wide testing. Genetic changes identified include losses and gains of chromosomal material (copy number variants and copy number abnormalities) as well as copy neutral ones. Clinically, these changes may be benign, directly pathogenic or represent risk factors for disease. It is particularly important to identify those that contribute to disease not only to improve molecular diagnostic capability and counselling for families, but also to understand mechanisms of disease and biological pathways that, in the future, may lead to targeted therapies for appropriate patient groups.

Previously, our work resulted in the implementation of microarray testing for the molecular diagnosis of idiopathic learning disability (ILD) within the Oxford University Hospitals NHS Trust (OUHT). Now, microarray testing has been adopted nationally for ILD and has transformed clinical practice globally in this field.

Collaboratively, our work has contributed to the identification of novel syndromes, most notably a recurrent 17q21.31 microdeletion syndrome and 15q24 and 15q13.3 microdeletion syndromes.

To realize fully the potential of microarray testing in clinical practice, we are investigating now other clinical disorders, for example brain malformations and B-cell chronic lymphocytic leukaemia (CLL) and maximizing utility for other applications, for example supporting next generation sequencing (NGS) efforts, assessing the genetic integrity of induced pluripotent stem cell lines and for pre-implantation genetic diagnosis.

Our CLL microarray studies have shown already improved capability over cytogenetic approaches used currently in clinical practice to inform upon diagnosis, prognosis and

treatment selection. Now we are making further advances with the implementation of NGS, targeting specific genes of interest.

However, work is still needed to facilitate data analysis within a molecular diagnostic environment and to exploit fully the results to benefit patients. The goal of this continuing work is to enable individually tailored patient treatments, minimize side-effects and mortality and reduce NHS costs.

In the future, our focus will continue to complement the objectives of the Genomic Medicine Theme of the Oxford NIHR BRC.

Name Department Institution Country
Dr Anna Schuh (RDM) Nuffield Division of Clinical Laboratory Sciences Oxford University, John Radcliffe Hospital United Kingdom
Professor Jenny Taylor Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Dr Dagan Wells Nuffield Department of Obstetrics and Gynaecology University of Oxford United Kingdom
Dr Sarah Wordsworth Health Economics Research Centre University of Oxford United Kingdom
Dr Simon E Fisher Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Prof Andrew OM Wilkie FRS FMedSci FRCP (RDM) Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Lee Carpenter Stem Cell Research Laboratory, NHS Blood and Transplant Oxford University Hospitals NHS Trust, John Radcliffe Hospital United Kingdom
Professor Corrado Romano Oasi Institute for Research on Mental Retardation and Brain Aging Italy
Professor Evan Eichler Howard Hughes Medical Institute United States
Prof Fredrik Karpe (RDM) OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Dr Sabrina Tosi Leukaemia and Chromosome Research Laboratory Brunel University United Kingdom
Buchanan J, Wordsworth S, Clifford R, Robbe P, Taylor JC, Schuh A, Knight SJL. 2017. Using Genomic Information to Guide Ibrutinib Treatment Decisions in Chronic Lymphocytic Leukaemia: A Cost-Effectiveness Analysis. Pharmacoeconomics, 35 (8), pp. 845-858. | Show Abstract | Read more

BACKGROUND: Genomic tests may improve the stratification of patients to receive new therapies in several disease areas. However, the use of expensive targeted therapies can impact on the cost effectiveness of these tests. This study presents an economic evaluation of genomic testing in chronic lymphocytic leukaemia in the context of the UK National Health Service. METHODS: Cost-effectiveness and cost-utility analyses (using life-years and quality-adjusted life-years) were undertaken from a National Health Service and societal perspective. Five strategies were evaluated across several age groups using Markov modelling: three strategies that reflected varying current genetic testing practice and two configurations of genomic testing (including ibrutinib treatment). RESULTS: Genomic testing strategies yielded the most life-years/quality-adjusted life-years per patient, but were not cost effective compared with a threshold of £30,000 per life-year/quality-adjusted life-year gained. Cost-effectiveness acceptability curves indicated that there was some uncertainty surrounding this result. A genomic testing strategy becomes the most cost-effective option if a higher end-of-life cost-effectiveness threshold of £50,000 is applied, if a societal costing perspective is considered in 25-year-old patients or if the cost of ibrutinib treatment falls. CONCLUSION: Stratifying patients with chronic lymphocytic leukaemia to targeted treatment using genomic testing improves health outcomes, but will likely only represent a cost-effective use of limited National Health Service resources if a higher cost-effectiveness threshold or societal costing perspective is applied, or if the price of ibrutinib treatment is reduced. This result may be broadly indicative of the likely cost effectiveness of other genomic tests that inform the stratification of patients to high cost-targeted therapies.

Burns A, Alsolami R, Becq J, Timbs A, Bruce D, Robbe P, Vavoulis D, Cabes M, Dreau H, Taylor J et al. 2017. Whole-genome sequencing of chronic lymphocytic leukaemia reveals distinct differences in the mutational landscape between IgHV(mut) and IgHV(unmut) subgroups. Leukemia, | Show Abstract | Read more

Chronic lymphocytic leukaemia (CLL) consists of two biologically and clinically distinct subtypes defined by the abundance of somatic hypermutation (SHM) affecting the Ig variable heavy-chain locus (IgHV). The molecular mechanisms underlying these subtypes are incompletely understood. Here, we present a comprehensive whole-genome sequencing analysis of somatically acquired genetic events from 46 CLL patients, including a systematic comparison of coding and non-coding single-nucleotide variants, copy number variants and structural variants, regions of kataegis and mutation signatures between IgHV(mut) and IgHV(unmut) subtypes. We demonstrate that one-quarter of non-coding mutations in regions of kataegis outside the Ig loci are located in genes relevant to CLL. We show that non-coding mutations in ATM may negatively impact on ATM expression and find non-coding and regulatory region mutations in TCL1A, and in IgHV(unmut) CLL in IKZF3, SAMHD1,PAX5 and BIRC3. Finally, we show that IgHV(unmut) CLL is dominated by coding mutations in driver genes and an aging signature, whereas IgHV(mut) CLL has a high incidence of promoter and enhancer mutations caused by aberrant activation-induced cytidine deaminase activity. Taken together, our data support the hypothesis that differences in clinical outcome and biological characteristics between the two subgroups might reflect differences in mutation distribution, incidence and distinct underlying mutagenic mechanisms.Leukemia advance online publication, 27 June 2017; doi:10.1038/leu.2017.177.

Purshouse K, Schuh A, Fairfax BP, Knight S, Antoniou P, Dreau H, Popitsch N, Gatter K, Roberts I, Browning L et al. 2017. Whole-genome sequencing identifies homozygous BRCA2 deletion guiding treatment in dedifferentiated prostate cancer. Cold Spring Harb Mol Case Stud, 3 (3), pp. a001362. | Show Abstract | Read more

Whole-genome sequencing (WGS) has transformed the understanding of the genetic drivers of cancer and is increasingly being used in cancer medicine to identify personalized therapies. Here we describe a case in which the application of WGS identified a tumoral BRCA2 deletion in a patient with aggressive dedifferentiated prostate cancer that was repeat-biopsied after disease progression. This would not have been detected by standard BRCA testing, and it led to additional treatment with a maintenance poly ADP ribose polymerase (PARP) inhibitor following platinum-based chemotherapy. This case demonstrates that repeat biopsy upon disease progression and application of WGS to tumor samples has meaningful clinical utility and the potential to transform outcomes in patients with cancer.

Pagnamenta AT, Howard MF, Knight SJ, Keays DA, Quaghebeur G, Taylor JC, Kini U. 2016. Activation of an exonic splice-donor site in exon 30 of CDK5RAP2 in a patient with severe microcephaly and pigmentary abnormalities. Clin Case Rep, 4 (10), pp. 952-956. | Show Abstract | Read more

This report constitutes the first report of a cryptic exonic splice-donor site in CDK5RAP2, highlights the importance of evaluating novel splice mutations, and suggests that the phenotypic range associated with CDK5RAP2 mutations may include skin pigmentary abnormalities.

Hastings R, de Villiers CP, Hooper C, Ormondroyd L, Pagnamenta A, Lise S, Salatino S, Knight SJ, Taylor JC, Thomson KL et al. 2016. Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction. Circ Cardiovasc Genet, 9 (5), pp. 426-435. | Show Abstract | Read more

BACKGROUND: High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. METHODS AND RESULTS: Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin. CONCLUSIONS: Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin's roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.

Lange L, Pagnamenta AT, Lise S, Clasper S, Stewart H, Akha ES, Quaghebeur G, Knight SJ, Keays DA, Taylor JC, Kini U. 2016. A de novo frameshift in HNRNPK causing a Kabuki-like syndrome with nodular heterotopia. Clin Genet, 90 (3), pp. 258-262. | Show Abstract | Read more

Kabuki syndrome is a heterogeneous condition characterized by distinctive facial features, intellectual disability, growth retardation, skeletal abnormalities and a range of organ malformations. Although at least two major causative genes have been identified, these do not explain all cases. Here we describe a patient with a complex Kabuki-like syndrome that included nodular heterotopia, in whom testing for several single-gene disorders had proved negative. Exome sequencing uncovered a de novo c.931_932insTT variant in HNRNPK (heterogeneous nuclear ribonucleoprotein K). Although this variant was identified in March 2012, its clinical relevance could only be confirmed following the August 2015 publication of two cases with HNRNPK mutations and an overlapping phenotype that included intellectual disability, distinctive facial dysmorphism and skeletal/connective tissue abnormalities. Whilst we had attempted (unsuccessfully) to identify additional cases through existing collaborators, the two published cases were 'matched' using GeneMatcher, a web-based tool for connecting researchers and clinicians working on identical genes. Our report therefore exemplifies the importance of such online tools in clinical genetics research and the benefits of periodically reviewing cases with variants of unproven significance. Our study also suggests that loss of function variants in HNRNPK should be considered as a molecular basis for patients with Kabuki-like syndrome.

Goos JA, Fenwick AL, Swagemakers SM, McGowan SJ, Knight SJ, Twigg SR, Hoogeboom AJ, van Dooren MF, Magielsen FJ, Wall SA et al. 2016. Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis. Hum Mutat, 37 (8), pp. 732-736. | Show Abstract | Read more

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

Parker H, Rose-Zerilli MJ, Larrayoz M, Clifford R, Edelmann J, Blakemore S, Gibson J, Wang J, Ljungström V, Wojdacz TK et al. 2016. Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia. Leukemia, 30 (11), pp. 2179-2186. | Show Abstract | Read more

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Guièze R, Robbe P, Clifford R, de Guibert S, Pereira B, Timbs A, Dilhuydy MS, Cabes M, Ysebaert L, Burns A et al. 2015. Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL. Blood, 126 (18), pp. 2110-2117. | Show Abstract | Read more

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.

Ilkovski B, Pagnamenta AT, O'Grady GL, Kinoshita T, Howard MF, Lek M, Thomas B, Turner A, Christodoulou J, Sillence D et al. 2015. Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies. Hum Mol Genet, 24 (21), pp. 6146-6159. | Show Abstract | Read more

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

Pagnamenta AT, Howard MF, Wisniewski E, Popitsch N, Knight SJ, Keays DA, Quaghebeur G, Cox H, Cox P, Balla T et al. 2015. Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis. Hum Mol Genet, 24 (13), pp. 3732-3741. | Show Abstract | Read more

Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development.

Babbs C, Lloyd D, Pagnamenta AT, Twigg SR, Green J, McGowan SJ, Mirza G, Naples R, Sharma VP, Volpi EV et al. 2014. De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder. J Med Genet, 51 (11), pp. 737-747. | Show Abstract | Read more

BACKGROUND: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints. METHODS: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC). RESULTS: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD. CONCLUSIONS: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD.

Yang CT, French A, Goh PA, Pagnamenta A, Mettananda S, Taylor J, Knight S, Nathwani A, Roberts DJ, Watt SM, Carpenter L. 2014. Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins. Br J Haematol, 166 (3), pp. 435-448. | Show Abstract | Read more

Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium, CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.

Howard MF, Murakami Y, Pagnamenta AT, Daumer-Haas C, Fischer B, Hecht J, Keays DA, Knight SJ, Kölsch U, Krüger U et al. 2014. Mutations in PGAP3 impair GPI-anchor maturation, causing a subtype of hyperphosphatasia with mental retardation. Am J Hum Genet, 94 (2), pp. 278-287. | Show Abstract | Read more

Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome.

Yang CT, French A, Goh PA, Pagnamenta A, Mettananda S, Taylor J, Knight S, Nathwani A, Roberts DJ, Watt SM, Carpenter L. 2014. Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins British Journal of Haematology, 166 (3), pp. 435-448. | Show Abstract | Read more

Summary: Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144 + endothelium, CD235a + erythrocytes (myeloid lineage) and CD19 + B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31 + CD45 + erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. © 2014 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.

Clifford R, Louis T, Robbe P, Ackroyd S, Burns A, Timbs AT, Wright Colopy G, Dreau H, Sigaux F, Judde JG et al. 2014. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood, 123 (7), pp. 1021-1031. | Show Abstract | Read more

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

Laurell T, Lundin J, Anderlid BM, Gorski JL, Grigelioniene G, Knight SJ, Krepischi AC, Nordenskjöld A, Price SM, Rosenberg C et al. 2013. Molecular and clinical delineation of the 17q22 microdeletion phenotype. Eur J Hum Genet, 21 (10), pp. 1085-1092. | Show Abstract | Read more

Deletions involving 17q21-q24 have been identified previously to result in two clinically recognizable contiguous gene deletion syndromes: 17q21.31 and 17q23.1-q23.2 microdeletion syndromes. Although deletions involving 17q22 have been reported in the literature, only four of the eight patients reported were identified by array-comparative genomic hybridization (array-CGH) or flourescent in situ hybridization. Here, we describe five new patients with 1.8-2.5-Mb microdeletions involving 17q22 identified by array-CGH. We also present one patient with a large karyotypically visible deletion involving 17q22, fine-mapped to ~8.2 Mb using array-CGH. We show that the commonly deleted region in our patients spans 0.24 Mb and two genes; NOG and C17ORF67. The function of C17ORF67 is not known, whereas Noggin, the product of NOG, is essential for correct joint development. In common with the 17q22 patients reported previously, the disease phenotype of our patients includes intellectual disability, attention deficit hyperactivity disorder, conductive hearing loss, visual impairment, low set ears, facial dysmorphology and limb anomalies. All patients displayed NOG-related bone and joint features, including symphalangism and facial dysmorphology. We conclude that these common clinical features indicate a novel clinically recognizable, 17q22 contiguous microdeletion syndrome.

Alsolami R, Knight SJ, Schuh A. 2013. Clinical application of targeted and genome-wide technologies: can we predict treatment responses in chronic lymphocytic leukemia? Per Med, 10 (4), pp. 361-376. | Show Abstract | Read more

Chronic lymphocytic leukemia (CLL) is low-grade lymphoma of mature B cells and it is considered to be the most common type of hematological malignancy in the western world. CLL is characterized by a chronically relapsing course and clinical and biological heterogeneity. Many patients do not require any treatment for years. Although important progress has been made in the treatment of CLL, none of the conventional treatment options are curative. Recurrent chromosomal abnormalities have been identified and are associated with prognosis and pathogenesis of the disease. More recently, unbiased genome-wide technologies have identified multiple additional recurrent aberrations. The precise predictive value of these has not been established, but it is likely that the genetic heterogeneity observed at least partly reflects the clinical variability. The present article reviews our current knowledge of predictive markers in CLL using whole-genome technologies.

Bignell P, Knight S, Mackie P, Clifford R, Murrin R, Schuh A, Robson K. 2013. NOVEL GENE DELETIONS RESULT IN HAEMOCHROMATOSIS AMERICAN JOURNAL OF HEMATOLOGY, 88 (5), pp. E214-E214.

Twigg SR, Babbs C, van den Elzen ME, Goriely A, Taylor S, McGowan SJ, Giannoulatou E, Lonie L, Ragoussis J, Sadighi Akha E et al. 2013. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes. Hum Mol Genet, 22 (8), pp. 1654-1662. | Show Abstract | Read more

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Twigg SR, Vorgia E, McGowan SJ, Peraki I, Fenwick AL, Sharma VP, Allegra M, Zaragkoulias A, Sadighi Akha E, Knight SJ et al. 2013. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet, 45 (3), pp. 308-313. | Show Abstract | Read more

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.

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Twigg SRF, Vorgia E, Mcgowan SJ, Peraki I, Fenwick AL, Sharma VP, Allegra M, Zaragkoulias A, Akha ES, Knight SJL et al. 2013. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis Nature Genetics, 45 (3), pp. 308-313. | Show Abstract | Read more

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes. © 2013 Nature America, Inc. All rights reserved.

Laurell T, Lundin J, Anderlid B-M, Gorski JL, Grigelioniene G, Knight SJL, Krepischi ACV, Nordenskjöld A, Price SM, Rosenberg C et al. 2013. Molecular and clinical delineation of the 17q22 microdeletion phenotype European Journal of Human Genetics, 21 (10), pp. 1085-1092. | Show Abstract | Read more

Deletions involving 17q21-q24 have been identified previously to result in two clinically recognizable contiguous gene deletion syndromes: 17q21.31 and 17q23.1-q23.2 microdeletion syndromes. Although deletions involving 17q22 have been reported in the literature, only four of the eight patients reported were identified by array-comparative genomic hybridization (array-CGH) or flourescent in situ hybridization. Here, we describe five new patients with 1.8-2.5-Mb microdeletions involving 17q22 identified by array-CGH. We also present one patient with a large karyotypically visible deletion involving 17q22, fine-mapped to ∼8.2 Mb using array-CGH. We show that the commonly deleted region in our patients spans 0.24 Mb and two genes; NOG and C17ORF67. The function of C17ORF67 is not known, whereas Noggin, the product of NOG, is essential for correct joint development. In common with the 17q22 patients reported previously, the disease phenotype of our patients includes intellectual disability, attention deficit hyperactivity disorder, conductive hearing loss, visual impairment, low set ears, facial dysmorphology and limb anomalies. All patients displayed NOG-related bone and joint features, including symphalangism and facial dysmorphology. We conclude that these common clinical features indicate a novel clinically recognizable, 17q22 contiguous microdeletion syndrome.European Journal of Human Genetics advance online publication, 30 January 2013; doi:10.1038/ejhg.2012.306.

Lise S, Clarkson Y, Perkins E, Kwasniewska A, Sadighi Akha E, Schnekenberg RP, Suminaite D, Hope J, Baker I, Gregory L et al. 2012. Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development. PLoS Genet, 8 (12), pp. e1003074. | Show Abstract | Read more

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

Twigg SR, Lloyd D, Jenkins D, Elçioglu NE, Cooper CD, Al-Sannaa N, Annagür A, Gillessen-Kaesbach G, Hüning I, Knight SJ et al. 2012. Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization. Am J Hum Genet, 91 (5), pp. 897-905. | Show Abstract | Read more

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.

Newbury DF, Mari F, Sadighi Akha E, Macdermot KD, Canitano R, Monaco AP, Taylor JC, Renieri A, Fisher SE, Knight SJ. 2013. Dual copy number variants involving 16p11 and 6q22 in a case of childhood apraxia of speech and pervasive developmental disorder. Eur J Hum Genet, 21 (4), pp. 361-365. | Read more

Pagnamenta AT, Murray JE, Yoon G, Sadighi Akha E, Harrison V, Bicknell LS, Ajilogba K, Stewart H, Kini U, Taylor JC et al. 2012. A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss. Am J Med Genet A, 158A (10), pp. 2577-2582. | Show Abstract | Read more

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible.

Twigg SRF, Lloyd D, Jenkins D, Elçioglu NE, Cooper CDO, Al-Sannaa N, Annagür A, Gillessen-Kaesbach G, Hüning I, Knight SJL et al. 2012. Mutations in multidomain protein MEGF8 identify a carpenter syndrome subtype associated with defective lateralization American Journal of Human Genetics, 91 (5), pp. 897-905. | Show Abstract | Read more

Carpenter syndrome is an autosomal-recessive multiple-congenital- malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor- like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. © 2012 The American Society of Human Genetics.

Knight SJ, Yau C, Clifford R, Timbs AT, Sadighi Akha E, Dréau HM, Burns A, Ciria C, Oscier DG, Pettitt AR et al. 2012. Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia. Leukemia, 26 (7), pp. 1564-1575. | Show Abstract | Read more

Genome-wide array approaches and sequencing analyses are powerful tools for identifying genetic aberrations in cancers, including leukemias and lymphomas. However, the clinical and biological significance of such aberrations and their subclonal distribution are poorly understood. Here, we present the first genome-wide array based study of pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) that uses the computational statistical tool OncoSNP. We show that quantification of the proportion of copy number alterations (CNAs) and copy neutral loss of heterozygosity regions (cnLOHs) in each sample is feasible. Furthermore, we (i) reveal complex changes in the subclonal architecture of paired samples at relapse compared with pre-treatment, (ii) provide evidence supporting an association between increased genomic complexity and poor clinical outcome (iii) report previously undefined, recurrent CNA/cnLOH regions that expand or newly occur at relapse and therefore might harbor candidate driver genes of relapse and/or chemotherapy resistance. Our findings are likely to impact on future therapeutic strategies aimed towards selecting effective and individually tailored targeted therapies.

Pagnamenta AT, Murray JE, Yoon G, Akha ES, Harrison V, Bicknell LS, Ajilogba K, Stewart H, Kini U, Taylor JC et al. 2012. A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss American Journal of Medical Genetics, Part A, 158 A (10), pp. 2577-2582. | Show Abstract | Read more

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible. © 2012 Wiley Periodicals, Inc.

Pagnamenta AT, Lise S, Harrison V, Stewart H, Jayawant S, Quaghebeur G, Deng AT, Murphy VE, Sadighi Akha E, Rimmer A et al. 2012. Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies. J Hum Genet, 57 (1), pp. 70-72. | Show Abstract | Read more

The development of next generation sequencing (NGS) has radically transformed the scientific landscape, making it possible to sequence the exome of any given individual in a cost-effective way. The power of this approach has been demonstrated by a number of groups who have identified pathogenic mutations in small pedigrees that have been resistant to traditional genetic mapping. Recently it has become clear that exome sequencing has great potential with respect to sporadic disease and the identification of de novo mutations. This is highlighted by studies reporting whole-exome sequencing of patient-parental trios affected by learning disability, autism and schizophrenia. It is widely anticipated that the introduction of this technique into a clinical setting will revolutionise genetic diagnosis. However, the sensitivity of NGS exome sequencing is currently unclear. Here, we describe the exome sequencing of DNA samples from a patient with double cortex syndrome and her parents, resulting in the detection of a mosaic splicing mutation in LIS1. This variant was found at an allele frequency of just 18%, demonstrating that NGS methods have the capacity to identify pathogenic mosaic mutations present at a low level.

Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M, Sadighi-Akha E, Stranks AJ, Glanville J, Knight S, Jacobsen SE et al. 2011. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med, 208 (3), pp. 455-467. | Show Abstract | Read more

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.

Ballabio E, Regan R, Garimberti E, Harbott J, Bradtke J, Teigler-Schlegel A, Biondi A, Cazzaniga G, Giudici G, Wainscoat JS et al. 2011. Genomic imbalances are confined to non-proliferating cells in paediatric patients with acute myeloid leukaemia and a normal or incomplete karyotype. PLoS One, 6 (6), pp. e20607. | Show Abstract | Read more

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.

Wincent J, Bruno DL, van Bon BW, Bremer A, Stewart H, Bongers EM, Ockeloen CW, Willemsen MH, Keays DD, Baird G et al. 2010. Sixteen New Cases Contributing to the Characterization of Patients with Distal 22q11.2 Microduplications. Mol Syndromol, 1 (5), pp. 246-254. | Show Abstract | Read more

The chromosome region 22q11.2 has long been recognized to be susceptible to genomic rearrangement. More recently, this genomic instability has been shown to extend distally (involving LCR22E-H) to the commonly deleted/duplicated region. To date, 21 index cases with 'distal' 22q11.2 duplications have been reported. We report on the clinical and molecular characterization of 16 individuals with distal 22q11.2 duplications identified by DNA microarray analysis. Two of the individuals have been partly described previously. The clinical phenotype varied among the patients in this study, although the majority displayed various degrees of developmental delay and speech disturbances. Other clinical features included behavioral problems, hypotonia, and dysmorphic facial features. Notably, none of the patients was diagnosed with a congenital heart defect. We found a high degree of inherited duplications. Additional copy number changes of unclear clinical significance were identified in 5 of our patients, and it is possible that these may contribute to the phenotypic expression in these patients as has been suggested recently in a 2-hit 'digenic' model for 16p12.1 deletions. The varied phenotypic expression and incomplete penetrance observed for distal 22q11.2 duplications makes it exceedingly difficult to ascribe pathogenicity for these duplications. Given the observed enrichment of the duplication in patient samples versus healthy controls, it is likely that distal 22q11.2 duplications represent a susceptibility/risk locus for speech and mild developmental delay.

Wilkie AO, Byren JC, Hurst JA, Jayamohan J, Johnson D, Knight SJ, Lester T, Richards PG, Twigg SR, Wall SA. 2010. Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis. Pediatrics, 126 (2), pp. e391-e400. | Show Abstract | Read more

OBJECTIVES: We describe the first cohort-based analysis of the impact of genetic disorders in craniosynostosis. We aimed to refine the understanding of prognoses and pathogenesis and to provide rational criteria for clinical genetic testing. METHODS: We undertook targeted molecular genetic and cytogenetic testing for 326 children who required surgery because of craniosynostosis, were born in 1993-2002, presented to a single craniofacial unit, and were monitored until the end of 2007. RESULTS: Eighty-four children (and 64 relatives) had pathologic genetic alterations (86% single-gene mutations and 14% chromosomal abnormalities). The FGFR3 P250R mutation was the single largest contributor (24%) to the genetic group. Genetic diagnoses accounted for 21% of all craniosynostosis cases and were associated with increased rates of many complications. Children with an initial clinical diagnosis of nonsyndromic craniosynostosis were more likely to have a causative mutation if the synostoses were unicoronal or bicoronal (10 of 48 cases) than if they were sagittal or metopic (0 of 55 cases; P = .0003). Repeat craniofacial surgery was required for 58% of children with single-gene mutations but only 17% of those with chromosomal abnormalities (P = .01). CONCLUSIONS: Clinical genetic assessment is critical for the treatment of children with craniosynostosis. Genetic testing of nonsyndromic cases (at least for FGFR3 P250R and FGFR2 exons IIIa/c) should be targeted to patients with coronal or multisuture synostoses. Single-gene disorders that disrupt physiologic signaling in the cranial sutures often require reoperation, whereas chromosomal abnormalities follow a more-indolent course, which suggests a different, secondary origin of the associated craniosynostosis.

Spurlock G, Knight SJL, Thomas N, Kiehl T-R, Guha A, Upadhyaya M. 2010. Molecular evolution of a neurofibroma to malignant peripheral nerve sheath tumor (MPNST) in an NF1 patient: correlation between histopathological, clinical and molecular findings Journal of Cancer Research and Clinical Oncology, pp. 1-12.

Shaw AC, van Balkom ID, Bauer M, Cole TR, Delrue MA, Van Haeringen A, Holmberg E, Knight SJ, Mortier G, Nampoothiri S et al. 2010. Phenotype and natural history in Marshall-Smith syndrome. Am J Med Genet A, 152A (11), pp. 2714-2726. | Show Abstract | Read more

Marshall-Smith syndrome (MSS) is a distinctive entity of unknown etiology with fewer than 50 patients described in the medical literature to date. Through an International collaboration and use of an online wiki to facilitate data collection and sharing, we further delineate the phenotype and natural history of this syndrome. We present 15 new patients, the oldest being 30 years, provide an update on four previously published cases, and compare all patients with other patients reported in literature. Main clinical features are moderate to severe developmental delay with absent or limited speech, unusual behavior, dysharmonic bone maturation, respiratory compromise secondary to upper airway obstruction, short stature, and kyphoscoliosis. Facial features are characteristic with high forehead, underdeveloped midface, proptosis, anteverted nares, and everted lips. Minor abnormalities of brain morphology such as hypoplasia of the corpus callosum are common. Mortality from respiratory complications is high, but airway support increasingly allows survival into adulthood. Array-CGH was performed on 12 of the cohort and no copy number variants of clear clinical relevance were identified. The present study is the first reported use of an online wiki to aid delineation of a genetic syndrome, and illustrates its value in collecting detailed data in rare conditions.

White SM, Morgan A, Da Costa A, Lacombe D, Knight SJ, Houlston R, Whiteford ML, Newbury-Ecob RA, Hurst JA. 2010. The phenotype of Floating-Harbor syndrome in 10 patients. Am J Med Genet A, 152A (4), pp. 821-829. | Show Abstract | Read more

Floating-Harbor syndrome (FHS) is a rare condition typified by short stature, speech impairment, delayed bone age, and characteristic facies. The diagnosis can be difficult as the facial changes are subtle in infancy, and the features of short stature, delayed speech, and delayed bone age are frequently encountered in clinical practice. We refine the phenotype in FHS by reporting clinical findings in 10 typically affected individuals ranging in age from 7 to 34 years and present a mother and daughter who display some features of FHS. Bone age measurements were delayed when measured from age 6 months to 6 years but in some patients were normal between 6 and 12 years. Dysmorphic features at different ages are characterized. The lateral profile of the face and the characteristic body habitus aided diagnosis. Significant behavioral problems of hyperactivity, short attention span and aggression during childhood were reported for most individuals. The children studied had a severe and incapacitating disorder of speech and language. Intellectual functioning ranged from borderline normal to moderate intellectual disability. Early puberty was noted. Adult heights were 140-155 cm. Microarray analysis in eight of the patients provided no evidence that FHS is caused by a large-scale copy-number genomic change.

Pagnamenta AT, Wing K, Sadighi Akha E, Knight SJ, Bölte S, Schmötzer G, Duketis E, Poustka F, Klauck SM, Poustka A et al. 2009. A 15q13.3 microdeletion segregating with autism. Eur J Hum Genet, 17 (5), pp. 687-692. | Show Abstract | Read more

Autism and mental retardation (MR) show high rates of comorbidity and potentially share genetic risk factors. In this study, a rare approximately 2 Mb microdeletion involving chromosome band 15q13.3 was detected in a multiplex autism family. This genomic loss lies between distal break points of the Prader-Willi/Angelman syndrome locus and was first described in association with MR and epilepsy. Together with recent studies that have also implicated this genomic imbalance in schizophrenia, our data indicate that this CNV shows considerable phenotypic variability. Further studies should aim to characterise the precise phenotypic range of this CNV and may lead to the discovery of genetic or environmental modifiers.

Koolen DA, Sharp AJ, Hurst JA, Firth HV, Knight SJL. 2009. Correction Journal of Medical Genetics, 46 (8), pp. 576-576. | Read more

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van Bon BWM, Mefford HC, Menten B, Koolen DA, Sharp AJ, Nillesen WM, Innis JW, de Ravel TJL, Mercer CL, Fichera M et al. 2009. Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome JOURNAL OF MEDICAL GENETICS, 46 (8), pp. 511-523. | Show Abstract | Read more

Background: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. Methods: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. Results: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. Conclusions: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

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Lake A, Shield LA, Cordano P, Chui DTY, Osborne J, Crae S, Wilson KS, Tosi S, Knight SJL, Gesk S et al. 2009. Mutations of NFKBIA , encoding IκBα, are a recurrent finding in classical Hodgkin lymphoma but are not a unifying feature of non-EBV-associated cases International Journal of Cancer, 125 (6), pp. 1334-1342. | Show Abstract | Read more

A consistent feature of the Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is the constitutive activation of NF-κB transcription factors. In Epstein-Barr virus (EBV)-associated cases of cHL, expression of viral antigens most probably leads to NF-κB activation but for non-EBV-associated cases, the mechanism is not clear. Previous small studies have demonstrated deleterious mutations of NFKBIA, the gene encoding IκBα, in HRS cells. In the present study, we aimed to establish the frequency of NFKBIA mutation in cHL by investigating a larger series of cases and to determine whether these mutations are a characteristic feature of non-EBV-associated cHL. Single HRS cells from 20 cases of cHL were analysed by PCRs covering all 6 exons of the gene. Clonal deleterious mutations were detected in 3 cases and in 1 case both alleles of the gene were shown to harbour mutations. NFKBIA mutations were detected only in non-EBV-associated cases but the majority of these cases had wild-type NFKBIA. It remains possible that defects in genes encoding other inhibitors of NF-κB, such as TNFAIP3 (A20) and CYLD, are involved in the latter cases, as described for one case in this series. © 2009 UICC.

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Hannes FD, Sharp AJ, Mefford HC, de Ravel T, Ruivenkamp CA, Breuning MH, Fryns J-P, Devriendt K, Van Buggenhout G, Vogels A et al. 2009. Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant JOURNAL OF MEDICAL GENETICS, 46 (4), pp. 223-232. | Show Abstract | Read more

Background: Genomic disorders are often caused by non-allelic homologous recombination between segmental duplications. Chromosome 16 is especially rich in a chromosome-specific low copy repeat, termed LCR16. Methods and Results: A bacterial artificial chromosome (BAC) array comparative genome hybridisation (CGH) screen of 1027 patients with mental retardation and/or multiple congenital anomalies (MR/MCA) was performed. The BAC array CGH screen identified five patients with deletions and five with apparently reciprocal duplications of 16p13 covering 1.65 Mb, including 15 RefSeq genes. In addition, three atypical rearrangements overlapping or flanking this region were found. Fine mapping by highresolution oligonucleotide arrays suggests that these deletions and duplications result from non-allelic homologous recombination (NAHR) between distinct LCR16 subunits with.99% sequence identity. Deletions and duplications were either de novo or inherited from unaffected parents. To determine whether these imbalances are associated with the MR/MCA phenotype or whether they might be benign variants, a population of 2014 normal controls was screened. The absence of deletions in the control population showed that 16p13.11 deletions are significantly associated with MR/MCA (p = 0.0048). Despite phenotypic variability, common features were identified: three patients with deletions presented with MR, microcephaly and epilepsy (two of these had also short stature), and two other deletion carriers ascertained prenatally presented with cleft lip and midline defects. In contrast to its previous association with autism, the duplication seems to be a common variant in the population (5/1682, 0.29%). Conclusion: These findings indicate that deletions inherited from clinically normal parents are likely to be causal for the patients' phenotype whereas the role of duplications (de novo or inherited) in the phenotype remains uncertain. This difference in knowledge regarding the clinical relevance of the deletion and the duplication causes a paradigm shift in (cyto)genetic counselling.

Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S, Maloney VK, Crolla JA, Baralle D et al. 2008. Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N Engl J Med, 359 (16), pp. 1685-1699. | Show Abstract | Read more

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Martin J, Knight SJ, Sharp AJ, Eichler EE, Hurst J, Kini U. 2008. Potocki-Lupski syndrome mimicking a connective tissue disorder. Clin Dysmorphol, 17 (3), pp. 211-213. | Show Abstract | Read more

We report the case of a 16-year-old boy with 17p11.2 duplication (Potocki-Lupski syndrome, PTLS) and thoracic kyphosis. He was first referred to Clinical Genetics at the age of 21 2 years following a diagnosis of global developmental delay and benign hypotonia. He was noted to be a tall boy (height on the 90th centile and head circumference on the 97th centile) with long toes. He is the second child of non-consanguineous parents. Pregnancy was unremarkable, and he was born at full term by emergency Caesarean section for a brow presentation. He was a large baby with a birth weight of 3.8 kg. He was noted to be floppy and slow to feed soon after birth. At 4 months of age, following normal electromyography and nerve conduction studies, he was diagnosed with benign hypotonia, which later resolved spontaneously. His development was delayed globally. He first walked at 18 months and at the age of 6 years, he was noted to have difficulties with coordination. His first spoken words came only at the age of 6 years. He was a hyperactive child and had episodes of breath holding until the age of 5 years. © 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Sharp AJ, Mefford HC, Li K, Baker C, Skinner C, Stevenson RE, Schroer RJ, Novara F, De Gregori M, Ciccone R et al. 2008. A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures. Nat Genet, 40 (3), pp. 322-328. | Show Abstract | Read more

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4-BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is approximately 0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

Cited:

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Koolen DA, Sharp AJ, Hurst JA, Firth HV, Knight SJL, Goldenberg A, Saugier-Veber P, Pfundt R, Vissers LELM, Destree A et al. 2008. Clinical and molecular delineation of the 17q21.31 microdeletion syndrome JOURNAL OF MEDICAL GENETICS, 45 (11), pp. 710-720. | Show Abstract | Read more

Background: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. Aim: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. Results: We estimate the prevalence of the syndrome to be 1 in 16 000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (007: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau IMAM Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a < 500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p < 10 -5 ). Conclusion: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Koolen DA, Sistermans EA, Nilessen W, Knight SJ, Regan R, Liu YT, Kooy RF, Rooms L, Romano C, Fichera M et al. 2008. Identification of non-recurrent submicroscopic genome imbalances: the advantage of genome-wide microarrays over targeted approaches. Eur J Hum Genet, 16 (3), pp. 395-400. | Show Abstract | Read more

Genome-wide analysis of DNA copy-number changes using microarray-based technologies has enabled the detection of de novo cryptic chromosome imbalances in approximately 10% of individuals with mental retardation. So far, the majority of these submicroscopic microdeletions/duplications appear to be unique, hampering clinical interpretation and genetic counselling. We hypothesised that the genomic regions involved in these de novo submicroscopic aberrations would be candidates for recurrent copy-number changes in individuals with mental retardation. To test this hypothesis, we used multiplex ligation-dependent probe amplification (MLPA) to screen for copy number changes at eight genomic candidate regions in a European cohort of 710 individuals with idiopathic mental retardation. By doing so, we failed to detect additional submicroscopic rearrangements, indicating that the anomalies tested are non-recurrent in this cohort of patients. The break points flanking the candidate regions did not contain low copy repeats and/or sequence similarities, thus providing an explanation for its non-recurrent nature. On the basis of these data, we propose that the use of genome-wide microarrays is indicated when testing for copy-number changes in individuals with idiopathic mental retardation.

Wordsworth S, Buchanan J, Regan R, Davison V, Smith K, Dyer S, Campbell C, Blair E, Maher E, Taylor J, Knight SJ. 2007. Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom. Genomic Med, 1 (1-2), pp. 35-45. | Show Abstract | Read more

Array based comparative genomic hybridisation (aCGH) is a powerful technique for detecting clinically relevant genome imbalance and can offer 40 to > 1000 times the resolution of karyotyping. Indeed, idiopathic learning disability (ILD) studies suggest that a genome-wide aCGH approach makes 10-15% more diagnoses involving genome imbalance than karyotyping. Despite this, aCGH has yet to be implemented as a routine NHS service. One significant obstacle is the perception that the technology is prohibitively expensive for most standard NHS clinical cytogenetics laboratories. To address this, we investigated the cost-effectiveness of aCGH versus standard cytogenetic analysis for diagnosing idiopathic learning disability (ILD) in the NHS. Cost data from four participating genetics centres were collected and analysed. In a single test comparison, the average cost of aCGH was pound442 and the average cost of karyotyping was pound117 with array costs contributing most to the cost difference. This difference was not a key barrier when the context of follow up diagnostic tests was considered. Indeed, in a hypothetical cohort of 100 ILD children, aCGH was found to cost less per diagnosis ( pound3,118) than a karyotyping and multi-telomere FISH approach ( pound4,957). We conclude that testing for genomic imbalances in ILD using microarray technology is likely to be cost-effective because long-term savings can be made regardless of a positive (diagnosis) or negative result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice. The use of aCGH in routine clinical practice warrants serious consideration by healthcare providers.

Sharp AJ, Selzer RR, Veltman JA, Gimelli S, Gimelli G, Striano P, Coppola A, Regan R, Price SM, Knoers NV et al. 2007. Characterization of a recurrent 15q24 microdeletion syndrome. Hum Mol Genet, 16 (5), pp. 567-572. | Show Abstract | Read more

We describe multiple individuals with mental retardation and overlapping de novo submicroscopic deletions of 15q24 (1.7-3.9 Mb in size). High-resolution analysis showed that in three patients both proximal and distal breakpoints co-localized to highly identical segmental duplications (>51 kb in length, > 94% identity), suggesting non-allelic homologous recombination as the likely mechanism of origin. Sequencing studies in a fourth individual provided base pair resolution and showed that both breakpoints in this case were located in unique sequence. Despite the differences in the size and location of the deletions, all four individuals share several major features (growth retardation, microcephaly, digital abnormalities, hypospadias and loose connective tissue) and resemble one another facially (high anterior hair line, broad medial eyebrows, hypertelorism, downslanted palpebral fissures, broad nasal base, long smooth philtrum and full lower lip), indicating that this represents a novel syndrome caused by haploinsufficiency of one or more dosage-sensitive genes in the minimal deletion region. Our results define microdeletion of 15q24 as a novel recurrent genomic disorder.

Koolen DA, Vissers LE, Pfundt R, de Leeuw N, Knight SJ, Regan R, Kooy RF, Reyniers E, Romano C, Fichera M et al. 2006. A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism. Nat Genet, 38 (9), pp. 999-1001. | Show Abstract | Read more

Submicroscopic genomic copy number changes have been identified only recently as an important cause of mental retardation. We describe the detection of three interstitial, overlapping 17q21.31 microdeletions in a cohort of 1,200 mentally retarded individuals associated with a clearly recognizable clinical phenotype of mental retardation, hypotonia and a characteristic face. The deletions encompass the MAPT and CRHR1 genes and are associated with a common inversion polymorphism.

Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, Hurst JA, Stewart H, Price SM, Blair E, Hennekam RC et al. 2006. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet, 38 (9), pp. 1038-1042. | Show Abstract | Read more

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

Knight SJ, Regan R. 2006. Idiopathic learning disability and genome imbalance. Cytogenet Genome Res, 115 (3-4), pp. 215-224. | Show Abstract | Read more

Learning disability (LD) is a very common, lifelong and disabling condition, affecting about 3% of the population. Despite this, it is only over the past 10-15 years that major progress has been made towards understanding the origins of LD. In particular, genetics driven advances in technology have led to the unequivocal demonstration of the importance of genome imbalance in the aetiology of idiopathic LD (ILD). In this review we provide an overview of these advances, discussing technologies such as multi-telomere FISH and array CGH that have already emerged as well as new approaches that show diagnostic potential for the future. The advances to date have highlighted new considerations such as copy number polymorphisms (CNPs) that can complicate the interpretation of genome imbalance and its relevance to ILD. More importantly though, they have provided a remarkable approximately 15-20% improvement in diagnostic capability as well as facilitating genotype/phenotype correlations and providing new avenues for the identification and understanding of genes involved in neurocognitive function.

Wilson K, Ballabio E, Regan R, Hedman A, Jarrett R, Knight S, Tosi S. 2005. DOP-PCR products amplified from genomic DNA can be used reliably in whole genome array CGH studie for the detection of delections and duplications in leukaemia CHROMOSOME RESEARCH, 13 pp. 205-206.

Price TS, Regan R, Mott R, Hedman A, Honey B, Daniels RJ, Smith L, Greenfield A, Tiganescu A, Buckle V et al. 2005. SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data. Nucleic Acids Res, 33 (11), pp. 3455-3464. | Show Abstract | Read more

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.

Knight SJ, Flint J. 2004. The use of subtelomeric probes to study mental retardation. Methods Cell Biol, 75 (75), pp. 799-831. | Show Abstract

In this chapter, we focus on the genetic basis of mental retardation (MR), specifically the use of subtelomeric probes to provide new diagnoses in idiopathic MR. We discuss both the background to the clinical demand for diagnoses and the technological advances that culminated in the development of subtelomeric testing strategies. We explain the theory behind these strategies and briefly outline the protocols involved, giving the advantages, limitations, and pitfalls of the analyses. Finally, we give an overview of the MR subtelomeric studies to date and how subtelomeric testing has become a widely used tool in clinical diagnostic laboratories, particularly in the diagnosis of unexplained MR, but also in other fields of clinical medicine. The conclusion addresses the overall impact that subtelomeric testing has had on the diagnosis of MR, the implications for patients and their families, and future research avenues for exploring the genetic causes of MR and improving our overall understanding of neurocognitive development.

Ross NL, Mavrogiannis LA, Sargent CA, Knight SJ, Wadekar R, DeLisi LE, Crow TJ. 2003. Quantitation of X-Y homologous genes in patients with schizophrenia by multiplex polymerase chain reaction. Psychiatr Genet, 13 (2), pp. 115-119. | Show Abstract | Read more

OBJECTIVES: The genetic basis of schizophrenia is obscure. In an XX male patient with schizophrenia we previously showed that one X;Y translocation breakpoint was in pseudoautosomal region 1 (PAR1) with the effect that the proximal segment of PAR1 from the PAR1 boundary to acetylserotonin N-methyl transferase (ASMT) distally was triplicated in this patient. This study determined whether dosage imbalances of X-Y homologous regions in general are associated with schizophrenia. METHODS: A multiplex semi-quantitative polymerase chain reaction assay was developed to quantify MIC2 gene as a representative of PAR1 and compare it with the SYBL1 gene which maps in pseudoautosomal region 2 (PAR2) and protocadherin XY (PCDHXY), located at Xq21.3. Each of these three loci was co-amplified with the autosomal gene MSX2 using Cy5-labelled primers and the products separated by electrophoresis in polyacrylamide gels. Results were expressed as ratios of peak area of the target gene to MSX2 which served as an internal dosage control. RESULTS: Using genomes with sex chromosome aneuploidies, the method was found sensitive enough to detect a two-fold difference in gene copy number. We confirmed the MIC2 triplication in the XX male patient but found no significant difference in gene dosage of MIC2, PCDHXY and SYBL1 in a panel of 17 patients with schizophrenia compared to controls. CONCLUSIONS: No evidence was obtained for gene dosage imbalances in MIC2, PCDHXY and SYBL1 in patients with schizophrenia.

Flint J, Knight S. 2003. The use of telomere probes to investigate submicroscopic rearrangements associated with mental retardation. Curr Opin Genet Dev, 13 (3), pp. 310-316. | Show Abstract | Read more

Idiopathic mental retardation is a common condition the origins of which are poorly understood. Following initial reports that small chromosomal rearrangements affecting telomeres could be an important aetiological contributor, several new methods for screening patients have been developed. Over the past few years, 22 studies have reported results from 2585 patients. The prevalence of abnormalities in the entire group is 5.1%; but the figure is higher (6.8%) in individuals with moderate to severe mental retardation. About half the cases are caused by a de novo deletion, and about half by a balanced translocation segregating in a patient's family. Despite the large sample size available, it is still not clear whether a combination of thorough clinical examination and assiduous cytogenetic investigation might not be as effective at detecting subtelomeric anomalies as molecular assays.

Knight SJ, Flint J. 2002. Multi-telomere FISH. Methods Mol Biol, 204 pp. 155-179. | Read more

de Vries BB, Lees M, Knight SJ, Regan R, Corney D, Flint J, Barnicoat A, Winter RM. 2001. Submicroscopic 8pter deletion, mild mental retardation, and behavioral problems caused by a familial t(8;20)(p23;p13). Am J Med Genet, 99 (4), pp. 314-319. | Show Abstract | Read more

Microscopically visible distal 8p deletions have been associated with growth and mental impairment, minor facial anomalies, congenital heart defects, and behavioral problems. We report two cousins with mild retardation and behavioral problems, including inappropriate sexual behavior and pyromania. Familial learning difficulties on the grandfather's side incompatible with Mendelian inheritance prompted telomere screening, which detected a submicroscopic terminal 8p deletion of < 5.1 Mb. The cousins' mothers both carried a t(8;20)(p23;p13) balanced translocation. The frequently observed microcephaly in patients with microscopically visible deletions of 8pter is lacking in both cousins, suggesting that the gene(s) causing the microcephaly is centromeric to the deleted region. The absence of cardiac defects in the cousins confirms the more proximal location of gene(s) causing these abnormalities in other reported cases with microscopically visible 8pter deletions and supports involvement of the GATA4 gene. Moreover, the current cases predict the presence of a putative gene(s) involved in behavior in the most telomeric 5.1 Mb of the p-arm of chromosome 8. This first clinical report of a submicroscopic subtelomeric 8p deletion gives more insight into the so-called 8p- syndrome and demonstrates the difficulty in making a clinical diagnosis for a submicroscopic 8pter deletion in an individual patient with mental retardation.

de Vries BB, White SM, Knight SJ, Regan R, Homfray T, Young ID, Super M, McKeown C, Splitt M, Quarrell OW et al. 2001. Clinical studies on submicroscopic subtelomeric rearrangements: a checklist. J Med Genet, 38 (3), pp. 145-150. | Show Abstract | Read more

BACKGROUND: Submicroscopic subtelomeric chromosome defects have been found in 7.4% of children with moderate to severe mental retardation and in 0.5% of children with mild retardation. Effective clinical preselection is essential because of the technical complexities and cost of screening for subtelomere deletions. METHODS: We studied 29 patients with a known subtelomeric defect and assessed clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history. Controls were 110 children with mental retardation of unknown aetiology with normal G banded karyotype and no detectable submicroscopic subtelomeric abnormalities. RESULTS: Prenatal onset of growth retardation was found in 37% compared to 9% of the controls (p<0.0005). A higher percentage of positive family history for mental retardation was reported in the study group than the controls (50% v 21%, p=0.002). Miscarriage(s) were observed in only 8% of the mothers of subtelomeric cases compared to 30% of controls (p=0.028) which was, however, not significant after a Bonferroni correction. Common features (>30%) among subtelomeric deletion cases were microcephaly, short stature, hypertelorism, nasal and ear anomalies, hand anomalies, and cryptorchidism. Two or more facial dysmorphic features were observed in 83% of the subtelomere patients. None of these features was significantly different from the controls. Using the results, a five item checklist was developed which allowed exclusion from further testing in 20% of the mentally retarded children (95% CI 13-28%) in our study without missing any subtelomere cases. As our control group was selected for the "chromosomal phenotype", the specificity of the checklist is likely to be higher in an unselected group of mentally retarded subjects. CONCLUSIONS: Our results suggest that good indicators for subtelomeric defects are prenatal onset of growth retardation and a positive family history for mental retardation. These clinical criteria, in addition to features suggestive of a chromosomal phenotype, resulted in the development of a five item checklist which will improve the diagnostic pick up rate of subtelomeric defects among mentally retarded subjects.

De Vries BB, Knight SJ, Homfray T, Smithson SF, Flint J, Winter RM. 2001. Submicroscopic subtelomeric 1qter deletions: a recognisable phenotype? J Med Genet, 38 (3), pp. 175-178. | Read more

de Vries BB, Bitner-Glindzicz M, Knight SJ, Tyson J, MacDermont KD, Flint J, Malcolm S, Winter RM. 2000. A boy with a submicroscopic 22qter deletion, general overgrowth and features suggestive of FG syndrome. Clin Genet, 58 (6), pp. 483-487. | Show Abstract | Read more

Over recent years, submicroscopic subtelomeric rearrangements have been shown to be a significant cause of mental retardation and, therefore, such abnormalities should be considered in every child with moderate to severe retardation with additional features suggestive of a chromosomal abnormality. The FG syndrome is an X-linked recessive mental retardation syndrome with congenital hypotonia, relative macrocephaly, a characteristic facies and constipation. We describe a severely mentally retarded boy with a history of severe constipation, truncal hypotonia, facial dysmorphism, fetal pads, and joint laxity, leading to an initial diagnosis of FG syndrome at the age of 3 years. Clinical re-evaluation at the age of 6 years, when he showed signs of general overgrowth, initiated a telomere screen, and a submicroscopic 22q13.3 telomere deletion was detected. The features suggestive of FG syndrome in this boy with a 22q13.3--> qter deletion may indicate testing for submicroscopic 22qter deletions in patients with atypical features of FG syndrome without a definite X-linked family history.

Knight SJ, Flint J. 2000. Screening chromosome ends for learning disability. BMJ, 321 (7271), pp. 1240. | Read more

Knight SJ, Lese CM, Precht KS, Kuc J, Ning Y, Lucas S, Regan R, Brenan M, Nicod A, Lawrie NM et al. 2000. An optimized set of human telomere clones for studying telomere integrity and architecture. Am J Hum Genet, 67 (2), pp. 320-332. | Show Abstract | Read more

Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions.

Knight SJ, Flint J. 2000. Perfect endings: a review of subtelomeric probes and their use in clinical diagnosis. J Med Genet, 37 (6), pp. 401-409. | Show Abstract | Read more

Chromosomal rearrangements involving the ends of chromosomes (telomeres) are emerging as an important cause of human genetic diseases. This review describes the development of first and second generation sets of telomere specific clones, together with advances in fluorescence in situ hybridisation (FISH) technology, which have made the prospect of screening for telomeric rearrangements a realistic goal. Initial FISH studies using the telomere specific clones indicate that they will be a valuable diagnostic tool for the investigation of mental retardation, the characterisation of known abnormalities detected by conventional cytogenetic analysis, spontaneous recurrent miscarriages, infertility, haematological malignancies, and preimplantation diagnosis, as well as other fields of clinical interest. In addition, they may help investigate telomere structure and function and can be used in the identification of dosage sensitive genes involved in human genetic disease.

Knight-Jones E, Knight S, Heussler H, Regan R, Flint J, Martin K. 2000. Neurodevelopmental profile of a new dysmorphic syndrome associated with submicroscopic partial deletion of 1p36.3. Dev Med Child Neurol, 42 (3), pp. 201-206. | Show Abstract | Read more

We describe four children with dysmorphic syndrome with severe learning disability (SLD). Their chromosomes had been normal on conventional cytogenetic examination. However, screening using a multiprobe fluorescence in situ hybridisation (FISH) technique for subtelomeric abnormalities revealed a deletion of the p arm of chromosome 1. The physical features include body asymmetry, microcephaly, distinctive facies with deep-set eyes, sharply defined eye sockets, and mid-face hypoplasia; the neurodevelopmental profile was characterised by SLD, motor delay with hypotonia, markedly delayed visual maturation, and postural asymmetry together with epilepsy. This phenotype is consistent with that described for partial monosomy for 1p36.3.

Knight SJ, Regan R, Nicod A, Horsley SW, Kearney L, Homfray T, Winter RM, Bolton P, Flint J. 1999. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet, 354 (9191), pp. 1676-1681. | Show Abstract | Read more

BACKGROUND: No explanation for moderate to severe mental retardation is apparent in about 40% of cases. Although small chromosomal rearrangements may account for some undiagnosed cases, a lack of genome-wide screening methods has made it impossible to ascertain the frequency of such abnormalities. METHODS: A fluorescence in-situ hybridisation (FISH) test was used to examine the integrity of chromosome ends in 284 children with unexplained moderate to severe retardation, and in 182 children with unexplained mild retardation. 75 normal men were also tested. When a chromosomal rearrangement was found, its size was estimated, and members of the child's family were investigated. FINDINGS: Subtle chromosomal abnormalities occurred with a frequency of 7.4% in the children with moderate to severe mental retardation, and of 0.5% in the children with mild retardation. The abnormalities had an estimated population prevalence of 2.1 per 10,000, and were familial in almost half of cases. INTERPRETATION: Once recognisable syndromes have been excluded, abnormalities that include the ends of chromosomes are the commonest cause of mental retardation in children with undiagnosed moderate to severe mental retardation. Owing to the high prevalence of familial cases, screening for subtle chromosomal rearrangements is warranted in children with unexplained moderate to severe mental retardation.

Brackley KJ, Kilby MD, Morton J, Whittle MJ, Knight SJ, Flint J. 1999. A case of recurrent congenital fetal anomalies associated with a familial subtelomeric translocation. Prenat Diagn, 19 (6), pp. 570-574. | Show Abstract | Read more

The potential of a new fluorescent in situ hybridization technique is discussed, which uses a complete set of telomeric probes to reveal cryptic chromosome rearrangements that remain undetected by standard cytogenetic analysis. We report the obstetric history of a patient who had a termination of pregnancy at 20 weeks for a fetus with multiple congenital anomalies but a normal male karyotype using conventional G-banding analysis on a mid-trimester placental biopsy. In a subsequent pregnancy, a diaphragmatic hernia and intra-uterine growth restriction were detected at 34 weeks' gestation and a fetal blood sample showed a normal female karotype. However, her child was born with dysmorphic features and additional severe abnormalities including microcephaly, anophthalmos and left fixed talipes. The child has shown marked developmental delay. In view of a strong family history of congenital abnormalities and recurrent miscarriage suggestive of a familial translocation, a fluorescent in situ hybridization technique using specific telomeric probes was performed on blood from the affected child and her parents. An unbalanced subtelomeric translocation was detected involving the long arms of chromosomes 2 and 7 in the child and a balanced translocation was detected in her father. Accurate genetic counselling and the opportunity for early prenatal diagnosis can now be offered to this family.

Slavotinek A, Rosenberg M, Knight S, Gaunt L, Fergusson W, Killoran C, Clayton-Smith J, Kingston H, Campbell RH, Flint J et al. 1999. Screening for submicroscopic chromosome rearrangements in children with idiopathic mental retardation using microsatellite markers for the chromosome telomeres. J Med Genet, 36 (5), pp. 405-411. | Show Abstract

Recently much attention has been given to the detection of submicroscopic chromosome rearrangements in patients with idiopathic mental retardation. We have screened 27 subjects with mental retardation and dysmorphic features for such rearrangements using a genetic marker panel screening. The screening was a pilot project using markers from the subtelomeric regions of all 41 chromosome arms. The markers were informative for monosomy in both parents at 3661902 loci (40.6%, 95% confidence interval 37.0-44.2%) in the 22 families where DNA was available from both parents. In two of the 27 subjects, submicroscopic chromosomal aberrations were detected. The first patient had a 5-6 Mb deletion of chromosome 18q and the second patient had a 4 Mb deletion of chromosome 1p. The identification of two deletions in 27 cases gave an aberration frequency of 7.5% without adjustment for marker informativeness (95% confidence interval 1-24%) and an estimated frequency of 18% if marker informativeness for monosomy was taken into account. This frequency is higher than previous estimates of the number of subtelomeric chromosome abnormalities in children with idiopathic mental retardation (5-10%) although the confidence interval is overlapping. Our study suggests that in spite of the low informativeness of this pilot screening, submicroscopic chromosome aberrations may be a common cause of dysmorphic features and mental retardation.

Horsley SW, Knight SJ, Nixon J, Huson S, Fitchett M, Boone RA, Hilton-Jones D, Flint J, Kearney L. 1998. Del(18p) shown to be a cryptic translocation using a multiprobe FISH assay for subtelomeric chromosome rearrangements. J Med Genet, 35 (9), pp. 722-726. | Show Abstract | Read more

We have previously described a fluorescence in situ hybridisation (FISH) assay for the simultaneous analysis of all human subtelomeric regions using a single microscope slide. Here we report the use of this multiprobe FISH assay in the study of a patient whose karyotype was reported by G banding analysis as 46,XX,del(18)(p11.2). Although the proband had some features suggestive of a chromosomal abnormality, relatively few of the specific features of del(18p) were present. She was a 37 year old female with mild distal spinal muscular atrophy (SMA), arthritis of the hands, an abnormal chest shape (pectus excavatum), and an unusual skin condition (keratosis pilaris). Reverse chromosome painting with degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplified del(18p) chromosomes as a probe confirmed the abnormality as del(18p), with no evidence of any other chromosome involvement. Subsequently, the multiprobe FISH assay confirmed deletion of 18p subtelomeric sequence. However, the assay also showed that sequences corresponding to the 2p subtelomeric probe were present on the tip of the shortened 18p. The patient is therefore monosomic for 18p11.2-pter and trisomic for 2p25-pter, and the revised karyotype is 46,XX,der(18)t(2;18)(p25; p11.2). We believe that a proportion of all cases reported as telomeric deletions may be cryptic translocations involving other chromosome subtelomeric regions. Further studies such as this are necessary to define accurately the clinical characteristics associated with pure monosomy in chromosomal deletion syndromes.

Slavotinek A, Rosenberg M, Knight S, Fergusson W, Gaunt L, Clayton-Smith J, Kingston H, Flint J, Biesecker L, Donnai D. 1998. The detection of submicroscopic chromosome rearrangements in children with idiopathic mental retardation and physical differences EUROPEAN JOURNAL OF HUMAN GENETICS, 6 pp. 91-91.

Ritchie RJ, Chakrabarti L, Knight SJ, Harding RM, Davies KE. 1997. Population genetics of the FRAXE and FRAXF GCC repeats, and a novel CGG repeat, in Xq28. Am J Med Genet, 73 (4), pp. 463-469. | Show Abstract | Read more

Most of the rare folate sensitive fragile sites cloned to date arise from expansion of a CGG:CCG trinucleotide repeat array. Analysis of the CAG repeat at the Huntington Disease (HD) locus showed a positively skewed repeat distribution leading to the proposal that microsatellites are subject to a mutational bias toward expansion. Such a mutational bias predicts an increase in mean repeat size at all microsatellite loci. We present an analysis of repeats at two fragile site loci, FRAXE and FRAXF, and a novel CGG repeat in Xq28, in five different human populations, which suggests that these loci may also be subject to the same mutation process. The novel repeat array may represent the first evidence for the existence of a fourth fragile site in Xq27.3-28.

Giraudeau F, Aubert D, Young I, Horsley S, Knight S, Kearney L, Vergnaud G, Flint J. 1997. Molecular-cytogenetic detection of a deletion of 1p36.3. J Med Genet, 34 (4), pp. 314-317. | Show Abstract | Read more

We report a deletion of 1p36.3 in a child with microcephaly, mental retardation, broad forehead, deep set eyes, depressed nasal bridge, flat midface, relative prognathism, and abnormal ears. The phenotype is consistent with that described for partial monosomy for 1p36.3. Reverse chromosome painting and microsatellite and Southern blot analyses were used to map the extent of the deletion. Fluorescence in situ hybridisation (FISH) analysis using probes from every telomere indicates that the rearrangement is likely to be a chromosomal truncation or rearrangement involving subtelomeric repetitive DNA. The deletion was identified by screening a sample of children and adults with idiopathic mental retardation. In conjunction with previous work on this sample, we estimate that 7.4% of the group have subtelomeric rearrangements.

Abrams MT, Doheny KF, Mazzocco MM, Knight SJ, Baumgardner TL, Freund LS, Davies KE, Reiss AL. 1997. Cognitive, behavioral, and neuroanatomical assessment of two unrelated male children expressing FRAXE. Am J Med Genet, 74 (1), pp. 73-81. | Show Abstract | Read more

Standardized cognitive, behavioral, and neuroanatomical data are presented on 2 unrelated boys with the FRAXE (FMR2) GCC expansion mutation. In the context of normal IQ, both boys had a history of developmental delay, including significant problems with communication, attention, and overactivity. Additionally, one child was diagnosed with autistic disorder. Data from these 2 cases are compared to analogous information from previous reports about individuals with the FRAXE or FRAXA (FMR1) mutation. These comparisons support the idea that FRAXE is associated with nonspecific developmental delay and possibly high-functioning autism.

Horsley S, Knight SJ, Regan R, Cardy DLN, Lawrie NM, Flint J, Kearney L. 1997. A fluorescence in situ hybridisation (FISH) assay for submicroscopic chromosome rearrangements involving telomers. CYTOGENETICS AND CELL GENETICS, 77 (1-2), pp. W2-W2.

Knight SJ, Horsley SW, Regan R, Lawrie NM, Maher EJ, Cardy DL, Flint J, Kearney L. 1997. Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur J Hum Genet, 5 (1), pp. 1-8. | Show Abstract

We report an innovative fluorescence in situ hybridization technique which exploits a unique resource of 41 telomere-specific probes and allows the simultaneous analysis of the subtelomeric region of every chromosome for deletion, triplication and balanced translocation events. This technique requires only a single microscope slide per patient and is expected to be a useful diagnostic tool with applications in the fields of idiopathic mental retardation, the detection of congenital abnormalities and in some forms of cancer. This will lead to more accurate genetic counselling of patients and their families and will provide the basis for future diagnostic, therapeutic and preventative measures.

Biancalana V, Taine L, Bouix JC, Finck S, Chauvin A, De Verneuil H, Knight SJ, Stoll C, Lacombe D, Mandel JL. 1996. Expansion and methylation status at FRAXE can be detected on EcoRI blots used for FRAXA diagnosis: analysis of four FRAXE families with mild mental retardation in males. Am J Hum Genet, 59 (4), pp. 847-854. | Show Abstract

The original test for the analysis of the CCG expansion at the FRAXE locus involves Southern blot analysis of HindIII digests. We show that, by using a different probe, the FRAXE mutation can be detected easily on the same EcoRI or EagI+EcoRI blots as are used for detection of FRAXA. Unexpectedly, we found that both the expansion and methylation status can be determined on a single EcoRI digest, because of the presence of a methylation-sensitive EcoRI site very close to the CCG repeat. We thus detected in a series of mentally retarded individuals previously tested for FRAXA expansion a FRAXE proband who led to the identification of a large sibship (7 of 10 children carrying a mutation). We also show that two fragile X families without FRAXA mutation that previously have been described by Oberlé et al. have the FRAXE expansion. In another family also ascertained initially by cytogenetic finding of a fragile X site, we performed the combined cytogenetic and molecular prenatal diagnosis of a mutated male fetus. All nine males (>3 years old) in whom we found a methylated mutation had mild mental retardation. Our results suggest that the threshold of repeat length for abnormal methylation and fragile-site expression may be smaller at FRAXE than at FRAXA.

Ning Y, Roschke A, Smith ACM, Macha M, Precht K, Riethman H, Ledbetter DH, Flint J, Horsley S, Regan R et al. 1996. A complete set of human telomeric probes and their clinical application. National Institutes of Health and Institute of Molecular Medicine collaboration. Nat Genet, 14 (1), pp. 86-89. | Show Abstract | Read more

Human chromosomes terminate with specialized telomeric structures including the simple tandem repeat (TTAGGG)n and additional complex subtelomeric repeats. Unique sequence DNA for each telomere is located 100-300 kilobases (kb) from the end of most chromosomes. A high concentration of genes and a number of candidate genes for recognizable syndromes are known to be present in telomeric regions. The human telomeric regions represent a major diagnostic challenge in clinical cytogenetics, because most of the terminal bands are G negative, and cryptic deletions and translocations in the telomeric regions are therefore difficult to detect by conventional cytogenetic methods. In fact, several submicroscopic chromosomal abnormalities in patients with undiagnosed mental retardation or multiple congenital anomalies have been identified by other molecular methods such as DNA polymorphism analysis. To improve the sensitivity for deletion detection and to determine whether such cryptic rearrangements represent a significant source of human pathology that has not been previously appreciated, it would be valuable to have specific FISH probes for all human telomeres. We report here the isolation and characterization of a complete set of specific FISH probes representing each human telomere. As most of these clones are at a known distance of within 100-300 kb from the end of the chromosome arm, this provides a 10-fold improvement in deletion detection sensitivity compared with high-resolution cytogenetics (2-3 Mb resolution). While testing these probes, we serendipitously identified a family with multiple members carrying a cryptic 1q;11p rearrangement in the balanced or unbalanced state.

Holden JJ, Julien-Inalsingh C, Chalifoux M, Wing M, Scott E, Fidler K, Swift I, Maidment B, Knight SJ, Davies KE, White BN. 1996. Trinucleotide repeat expansion in the FRAXE locus is not common among institutionalized individuals with non-specific developmental disabilities. Am J Med Genet, 64 (2), pp. 420-423. | Show Abstract | Read more

Expansion of a polymorphic GCC-repeat at the FRAXE locus has been associated with expression of chromosome fragility at this site and cognitive impairment in some individuals previously testing negative for CGG-repeat expansion in the fragile X mental retardation-1 (FMR1) gene. To determine the frequency of FRAXE triplet repeat expansion among persons with developmental disability, 396 individuals from two institutions were studied, all of whom were negative for FMR1 repeat expansion. Clinically, there was a wide range of mental impairment, with the majority (61.1%) being severely to profoundly affected. The distribution of FRAXE GCC-repeat numbers in the study population was 5-38: 28 (5.6%) with 10-14 repeats; 366 (73.8%) with 15-19 repeats; 74 (14.9%) with 20-24 repeats; 20 (4.0%) with 25-29 repeats; and 5 (1.0%) with 30-38 repeats, with no individuals demonstrating repeat expansion. One profoundly retarded male was found to have a deletion of about 40 bp. Southern blots of HindIII-digested DNAs from individuals with > or = 26 repeats all showed normal patterns. These results suggest that FRAXE GCC-repeat expansion is not a common cause of developmental disability in institutionalized persons with mild to profound mental retardation.

Lindsay S, Splitt M, Edney S, Berney TP, Knight SJ, Davies KE, O'Brien O, Gale M, Burn J. 1996. PPM-X: a new X-linked mental retardation syndrome with psychosis, pyramidal signs, and macroorchidism maps to Xq28. Am J Hum Genet, 58 (6), pp. 1120-1126. | Show Abstract

We report a three-generation family manifesting a previously undescribed X-linked mental retardation syndrome. Four of the six moderately retarded males have had episodes of manic-depressive psychosis. The phenotype also includes pyramidal signs, Parkinsonian features, and macroorchidism, but there are no characteristic dysmorphic facial features. Affected males do not show fragile sites at distal Xq on cytogenetic analysis, nor do they have expansions of the CGG repeats at the FRAXA, FRAXE, or FRAXF loci. Linkage analyses were undertaken, and a maximal LOD score of 3.311 at theta = .0 was observed with the microsatellite marker DXS1123 in Xq28. A recombination was detected in one of the affected males with DXS1691 (Xq28), which gives the proximal boundary of the localization. No distal recombination has been detected at any of the loci tested.

Knight SJ, Ritchie RJ, Chakrabarti L, Cross G, Taylor GR, Mueller RF, Hurst J, Paterson J, Yates JR, Dow DJ, Davies KE. 1996. A study of FRAXE in mentally retarded individuals referred for fragile X syndrome (FRAXA) testing in the United Kingdom. Am J Hum Genet, 58 (5), pp. 906-913. | Show Abstract

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.

Chakrabarti L, Knight SJ, Flannery AV, Davies KE. 1996. A candidate gene for mild mental handicap at the FRAXE fragile site. Hum Mol Genet, 5 (2), pp. 275-282. | Show Abstract | Read more

The cytogenetic expression of the folate sensitive fragile site, FRAXE, is due to the expansion of a GCC repeat in proximal Xq28 of the human X chromosome and is associated with a mild form of mental handicap. Normal individuals have 6-35 copies of the repeat whereas cytogenetically positive, developmentally delayed males have > 200 copies and show methylation of the associated CpG island. Through the use of conserved sequences adjacent to the FRAXE GCC repeat, we have isolated a 1495 bp cDNA which begins 331 bp distal to the FRAXE site and extends to a region > 170 kb distal in Xq28. The cDNA sequence possesses both a putative start of translation and a poly-A tail. The predicted protein has amino acid motifs which share significant homologies with the human AF-4 gene which encodes a putative transcription factor. On northern analysis, the cDNA detects a 9.5 kb transcript in human brain, placenta and lung. This transcript is present in multiple human brain tissues, but is more abundant in the hippocampus and the amygdala, thus providing possible functional insights. RT-PCR of normal adult brain RNA provides evidence for the existence of the 1495 bp transcript represented by the isolated cDNA.

Flannery AV, Hirst MC, Knight SJ, Ritchie RJ, Davies KE. 1995. The fragile X syndrome. Biochim Biophys Acta, 1271 (2-3), pp. 293-303. | Read more

Mulley JC, Yu S, Loesch DZ, Hay DA, Donnelly A, Gedeon AK, Carbonell P, López I, Glover G, Gabarrón I. 1995. FRAXE and mental retardation. J Med Genet, 32 (3), pp. 162-169. | Show Abstract | Read more

Mental impairment and instability of the CCG repeat at FRAXE is described in six kindreds. Cosegregation of FRAXA and FRAXE was found within one of these kindreds. Cytogenetic expression of FRAXE was shown to skip a generation when associated with a reduction in size of the CCG expansion when transmitted through a male; however, in general, transmission occurred through females and a copy number increased from one generation to the next. In these respects the behaviour of FRAXE paralleled that of FRAXA. A relationship between FRAXE and non-specific mental impairment is strongly suggested by the occurrence in these families of more mentally impaired male and female carriers, after removal of index cases, than could reasonably be expected by chance.

Ritchie RJ, Knight SJ, Hirst MC, Grewal PK, Bobrow M, Cross GS, Davies KE. 1994. The cloning of FRAXF: trinucleotide repeat expansion and methylation at a third fragile site in distal Xqter. Hum Mol Genet, 3 (12), pp. 2115-2121. | Show Abstract | Read more

Three fragile sites, FRAXA, FRAXE and FRAXF lie in the Xq27-28 region of the human X chromosome. The expression of FRAXA is associated with the fragile X syndrome, the most prevalent form of inherited mental retardation whilst the expression of FRAXE is associated with a rarer and comparatively milder form of mental handicap. Both the FRAXA and FRAXE sites have been cloned and the fragile site expression found to be due to the expansion of analogous CGG/GCC trinucleotide repeat arrays. We describe here the cloning of the third fragile site, FRAXF, and demonstrate that it involves the expansion of a (GCCGTC)n(GCC)n compound array. PCR analyses across the repeat of normal individuals show that the number of triplets in the array ranges from 12-26 and the most common allele consists of 14 triplet units. Sequencing analyses show that 95% of normal individuals have three copies of the GCCGTC motif and in these individuals, the size variation observed by PCR is due to copy number alterations in the GCC array. In a cytogenetically positive male with developmental delay, the array is expanded by > 900 triplets and the adjacent CpG-rich region is methylated. The array is also expanded in cytogenetically positive carrier females from the family originally used to define the FRAXF site. We conclude that the expanded array corresponds to the FRAXF fragile site.

Hamel BC, Smits AP, de Graaff E, Smeets DF, Schoute F, Eussen BH, Knight SJ, Davies KE, Assman-Hulsmans CF, Oostra BA. 1994. Segregation of FRAXE in a large family: clinical, psychometric, cytogenetic, and molecular data. Am J Hum Genet, 55 (5), pp. 923-931. | Show Abstract

During an ongoing study on X-linked mental retardation, we ascertained a large family in which mild mental retardation was cosegregating with a fragile site at Xq27-28. Clinical, psychometric, cytogenetic, and molecular studies were performed. Apart from mild mental retardation, affected males and females did not show a specific clinical phenotype. Psychometric assessment of four representative affected individuals revealed low academic achievements, with verbal and performance IQs of 61-75 and 70-82, respectively. Cytogenetically the fragile site was always present in affected males and was not always present in affected females. With FISH the fragile site was located within the FRAXE region. The expanded GCC repeat of FRAXE was seen in affected males and females either as a discrete band or as a broad smear. No expansion was seen in unaffected males, whereas three unaffected females did have an enlarged GCC repeat. Maternal transmission of FRAXE may lead to expansion or contraction of the GCC repeat length, whereas in all cases of paternal transmission contraction was seen. In striking contrast to the situation in fragile X syndrome, affected males may have affected daughters. In addition, there appears to be no premutation of the FRAXE GCC repeat, since in the family studied here all males lacking the normal allele were found to be affected.

Knight SJ, Voelckel MA, Hirst MC, Flannery AV, Moncla A, Davies KE. 1994. Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap. Am J Hum Genet, 55 (1), pp. 81-86. | Show Abstract

We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.

Hirst MC, Knight SJ, Christodoulou Z, Grewal PK, Fryns JP, Davies KE. 1993. Origins of the fragile X syndrome mutation. J Med Genet, 30 (8), pp. 647-650. | Show Abstract | Read more

The fragile X syndrome is a common cause of mental impairment. In view of the low reproductive fitness of affected males, the high incidence of the syndrome has been suggested to be the result of a high rate of new mutations occurring exclusively in the male germline. Extensive family studies, however, have failed to identify any cases of a new mutation. Alternatively, it has been suggested that a selective advantage of unaffected heterozygotes may, in part, explain the high incidence of the syndrome. Molecular investigations have shown that the syndrome is caused by the amplification of a CGG trinucleotide repeat in the FMR-1 gene which leads to the loss of gene expression. Further to this, genetic studies have suggested that there is evidence of linkage disequilibrium between the fragile X disease locus and flanking polymorphic markers. More recently, this analysis has been extended and has led to the observation that a large number of fragile X chromosomes appear to be lineage descendants of founder mutation events. Here, we present a study of the FRAXAC1 polymorphic marker in our patient cohort. We find that its allele distribution is strikingly different on fragile X chromosomes, confirming the earlier observations and giving further support to the suggestions of a fragile X founder effect.

Knight SJ, Flannery AV, Hirst MC, Campbell L, Christodoulou Z, Phelps SR, Pointon J, Middleton-Price HR, Barnicoat A, Pembrey ME. 1993. Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation. Cell, 74 (1), pp. 127-134. | Show Abstract | Read more

We have cloned the fragile site FRAXE and demonstrate that individuals with this fragile site possess amplifications of a GCC repeat adjacent to a CpG island in Xq28 of the human X chromosome. Normal individuals have 6-25 copies of the GCC repeat, whereas mentally retarded, FRAXE-positive individuals have > 200 copies and also have methylation at the CpG island. This situation is similar to that seen at the FRAXA locus and is another example in which a trinucleotide repeat expansion is associated with a human genetic disorder. In contrast with the fragile X syndrome, the GCC repeat can expand or contract and is equally unstable when passed through the male or female line. These results also have implications for the understanding of chromosome fragility.

Flynn GA, Hirst MC, Knight SJ, Macpherson JN, Barber JC, Flannery AV, Davies KE, Buckle VJ. 1993. Identification of the FRAXE fragile site in two families ascertained for X linked mental retardation. J Med Genet, 30 (2), pp. 97-100. | Show Abstract | Read more

Chromosome fragility in two families not exhibiting amplification of the CGG trinucleotide associated with the fragile X site has been examined. Fluorescence in situ hybridisation with cosmid DNA from loci immediately flanking FRAXA and other distal loci have confirmed that cytogenetic fragility in these subjects is the result of expression of a new folate sensitive fragile X site, FRAXE.

Hirst MC, Knight SJ, Bell MV, Super M, Davies KE. 1992. The fragile X syndrome. Clin Sci (Lond), 83 (3), pp. 255-264. | Show Abstract | Read more

An amplification of a highly unstable DNA element has been identified at the fragile X locus in Xq27.3. This sequence appears to be both the source of the primary mutation causing the fragile X syndrome, apparently having its causative effect through the methylation of the FMR-1 HTF island and the region of cytogenetic fragility. The direct analysis of the genotype of carrier and affected individuals can be used as a direct diagnosis tool which will improve both the accuracy and speed of diagnosis. The identification of hereditary unstable DNA in a disease with such a wide level of non-penetrance and variable phenotype may give clues as to the basis of non-penetrance in other human genetic disorders.

Knight SJ, Hirst MC, Roche A, Christodoulou Z, Huson SM, Winter R, Fitchett M, McKinley MJ, Lindenbaum RH, Nakahori Y. 1992. Molecular studies of the fragile X syndrome. Am J Med Genet, 43 (1-2), pp. 217-223. | Show Abstract | Read more

We have studied families segregating for the fragile X syndrome for the presence of amplification of the CGG repeat sequence adjacent to the HpaII Tiny Fragment (HTF) island in the FMR-1 gene. We demonstrate that 138/143 fragile X positive, mentally retarded males show a characteristic smear of fragments corresponding to somatic variation in the amplification of the CGG sequence. In 7/8 normal transmitting males (NTM's), we show that there is a small amplification of sequence but no evidence for somatic variation. Defined mutated fragments in the size range found in NTM's are seen in daughters of NTM's. The daughters of these female carriers show either a defined fragment in the NTM size range, a defined larger fragment or a heterogeneous pattern of fragments. In the latter 2 cases the clinical phenotype of the females cannot easily be predicted, presumably because of variable X inactivation. In some families, the observed DNA genotype does not correlate with the phenotype; in others we demonstrate the occurrence of individuals with a mosaic DNA genotype. The implications of these data for diagnosis of the disease are discussed.

Knight SJ, Hirst MC, Davies KE. 1992. Molecular analysis of the fragile X syndrome. Dis Markers, 10 (1), pp. 1-5. | Show Abstract

Carriers of the fragile X mutation possess more than the normal number of copies of a trinucleotide repeat (CGG) within the coding region of a gene designated as FMR-1 in Xq27. The clinical phenotype is determined by the number of copies of the CGG repeat. DNA-based methods for the detection of the fragile X mutation are now available which greatly assist in the genetic diagnosis of this disorder. Direct detection of the mutation enables the identification of fragile X negative normal transmitting males and fragile X negative carrier females.

Hirst MC, Nakahori Y, Knight SJ, Schwartz C, Thibodeau SN, Roche A, Flint TJ, Connor JM, Fryns JP, Davies KE. 1991. Genotype prediction in the fragile X syndrome. J Med Genet, 28 (12), pp. 824-829. | Show Abstract | Read more

Fragile X positive, mentally retarded males have been shown to have an insertion or amplification of DNA sequences at, or close to, the site of expression of the fragile site. We show here the application of the detection of such changes to the diagnosis of affected males and female carriers and the identification of normal transmitting males. One fragile X negative male with the clinical features of the Martin-Bell syndrome also possesses an inserted/amplified DNA sequence. The implications of these results for screening for the fragile X syndrome are discussed.

Nakahori Y, Knight SJ, Holland J, Schwartz C, Roche A, Tarleton J, Wong S, Flint TJ, Froster-Iskenius U, Bentley D. 1991. Molecular heterogeneity of the fragile X syndrome. Nucleic Acids Res, 19 (16), pp. 4355-4359. | Show Abstract | Read more

The fragile X syndrome is an X-linked disorder which has been shown to be associated with the length variation of a DNA fragment containing a CGG trinucleotide repeat element at or close to the fragile site. Phenotypically normal carriers of the disorder generally have a smaller length variation than affected individuals. We have cloned the region in cosmids and defined the area containing the amplified sequence. We have used probes from the region to analyse the mutation in families. We show that the mutation evolves in different ways in different individuals of the same family. In addition we show that not all fragile X positive individuals show this amplification of DNA sequence even though they show expression of the fragile site at levels greater than 25%. One patient has alterations in the region adjacent to the CGG repeat elements. Three patients in fragile X families have the normal fragment with amplification in a small population of their cells. These observations indicate that there is molecular heterogeneity in the fragile X syndrome and that the DNA fragment length variation is not the only sequence responsible for the expression of the fragile site or the disease phenotype.

Brook JD, Knight SJ, Roberts SH, Harley HG, Walsh KV, Rundle SA, Freyne K, Koch MC, Epstein ND, Wieringa B. 1991. The physical map of chromosome arm 19q: some new assignments, confirmations and re-assessments. Hum Genet, 87 (1), pp. 65-72. | Show Abstract | Read more

We have constructed and analysed somatic cell hybrids from cell lines containing balanced reciprocal translocations involving chromosome 19 and providing two new breakpoints on 19q. These and other hybrids have been tested with a series of markers from 19q to enhance the existing map. Several new cloned DNA sequences that map to 19q13.3-19qter are reported; the locus D19Z1 has been analysed by CHEF gel electrophoresis.

Consalez GG, Thomas NS, Stayton CL, Knight SJ, Johnson M, Hopkins LC, Harper PS, Elsas LJ, Warren ST. 1991. Assignment of Emery-Dreifuss muscular dystrophy to the distal region of Xq28: the results of a collaborative study. Am J Hum Genet, 48 (3), pp. 468-480. | Show Abstract

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked humeroperoneal dystrophy associated with cardiomyopathy that is distinct from the Duchenne and Becker forms of X-linked muscular dystrophy. Linkage analysis has assigned EDMD to the terminal region of the human X chromosome long arm. We report here further linkage analysis in two multigenerational EDMD families using seven Xq28 marker loci. Cumulative lod scores suggest that EDMD is approximately 2 cM from DXS52 (lod = 15.67) and very close to the factor VIII (F8C) and the red/green color pigment (R/GCP) loci, with respective lod scores of 9.62 and 10.77, without a single recombinant. Several recombinations between EDMD and three proximal Xq28 markers suggest that the EDMD gene is located in distal Xq28. Multipoint linkage analysis indicates that the odds are 2,000:1 that EDMD lies distal to DXS305. These data substantially refine the ability to perform accurate carrier detection, prenatal diagnosis, and the presymptomatic diagnosis of at-risk males for EDMD by linkage analysis. The positioning of the EDMD locus close to the loci for F8C and R/GCP will assist in future efforts to identify and isolate the disease gene.

HIRST M, NAKAHORI Y, KNIGHT S, ROCHE A, RAUT C, RACK K, FLYNN G, BUCKLE V, BELL M, CLAUSSEN U et al. 1991. A YAC CONTIG ACROSS THE FRAGILE-X REGION DERIVED FROM CHROMOSOME MICRODISSECTION MARKERS CYTOGENETICS AND CELL GENETICS, 58 (3-4), pp. 2067-2067.

Hirst MC, Roche A, Flint TJ, MacKinnon RN, Bassett JH, Nakahori Y, Watson JE, Bell MV, Patterson MN, Boyd Y. 1991. Linear order of new and established DNA markers around the fragile site at Xq27.3. Genomics, 10 (1), pp. 243-249. | Show Abstract | Read more

We have used recombinant clones derived from microdissection of the fragile X region to characterize breakpoints around the fragile site at Xq27.3. So far, no microdissection markers derived from Xq28 material have been found, thus allowing a rapid screening for clones surrounding the fragile site by their presence in a somatic cell hybrid containing Xq27.2-Xqter. A total of 43 new DNA markers from Xq27 have been sublocalized within this chromosome band. Of these new DNA markers, 5 lie in an interval defined as containing the fragile X region. The saturation of Xq27 with DNA markers by microdissection demonstrates the power of this technique and provides the resources for generating a complete physical map of the region.

HIRST M, KNIGHT S, DAVIES K, CROSS G, OCRAFT K, RAEBURN S, HEEGER S, EUNPU D, JENKINS E, LINDENBAUM R. 1991. Prenatal diagnosis of fragile X syndrome. Lancet, 338 (8772), pp. 956-958. | Read more

Warren ST, Knight SJ, Peters JF, Stayton CL, Consalez GG, Zhang FP. 1990. Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage. Proc Natl Acad Sci U S A, 87 (10), pp. 3856-3860. | Show Abstract | Read more

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.

Twigg SR, Babbs C, van den Elzen ME, Goriely A, Taylor S, McGowan SJ, Giannoulatou E, Lonie L, Ragoussis J, Sadighi Akha E et al. 2013. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes. Hum Mol Genet, 22 (8), pp. 1654-1662. | Show Abstract | Read more

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Twigg SR, Vorgia E, McGowan SJ, Peraki I, Fenwick AL, Sharma VP, Allegra M, Zaragkoulias A, Sadighi Akha E, Knight SJ et al. 2013. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet, 45 (3), pp. 308-313. | Show Abstract | Read more

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.

Cited:

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Scopus

Twigg SRF, Vorgia E, Mcgowan SJ, Peraki I, Fenwick AL, Sharma VP, Allegra M, Zaragkoulias A, Akha ES, Knight SJL et al. 2013. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis Nature Genetics, 45 (3), pp. 308-313. | Show Abstract | Read more

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes. © 2013 Nature America, Inc. All rights reserved.

Newbury DF, Mari F, Sadighi Akha E, Macdermot KD, Canitano R, Monaco AP, Taylor JC, Renieri A, Fisher SE, Knight SJ. 2013. Dual copy number variants involving 16p11 and 6q22 in a case of childhood apraxia of speech and pervasive developmental disorder. Eur J Hum Genet, 21 (4), pp. 361-365. | Read more

Pagnamenta AT, Murray JE, Yoon G, Sadighi Akha E, Harrison V, Bicknell LS, Ajilogba K, Stewart H, Kini U, Taylor JC et al. 2012. A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss. Am J Med Genet A, 158A (10), pp. 2577-2582. | Show Abstract | Read more

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7 Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible.

Knight SJ, Yau C, Clifford R, Timbs AT, Sadighi Akha E, Dréau HM, Burns A, Ciria C, Oscier DG, Pettitt AR et al. 2012. Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia. Leukemia, 26 (7), pp. 1564-1575. | Show Abstract | Read more

Genome-wide array approaches and sequencing analyses are powerful tools for identifying genetic aberrations in cancers, including leukemias and lymphomas. However, the clinical and biological significance of such aberrations and their subclonal distribution are poorly understood. Here, we present the first genome-wide array based study of pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) that uses the computational statistical tool OncoSNP. We show that quantification of the proportion of copy number alterations (CNAs) and copy neutral loss of heterozygosity regions (cnLOHs) in each sample is feasible. Furthermore, we (i) reveal complex changes in the subclonal architecture of paired samples at relapse compared with pre-treatment, (ii) provide evidence supporting an association between increased genomic complexity and poor clinical outcome (iii) report previously undefined, recurrent CNA/cnLOH regions that expand or newly occur at relapse and therefore might harbor candidate driver genes of relapse and/or chemotherapy resistance. Our findings are likely to impact on future therapeutic strategies aimed towards selecting effective and individually tailored targeted therapies.

Pagnamenta AT, Murray JE, Yoon G, Akha ES, Harrison V, Bicknell LS, Ajilogba K, Stewart H, Kini U, Taylor JC et al. 2012. A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss American Journal of Medical Genetics, Part A, 158 A (10), pp. 2577-2582. | Show Abstract | Read more

Primary microcephaly is a genetically heterogeneous condition characterized by reduced head circumference (-3 SDS or more) and mild-to-moderate learning disability. Here, we describe clinical and molecular investigations of a microcephalic child with sensorineural hearing loss. Although consanguinity was unreported initially, detection of 13.7Mb of copy neutral loss of heterozygosity (cnLOH) on chromosome 9 implicated the CDK5RAP2 gene. Targeted sequencing identified a homozygous E234X mutation, only the third mutation to be described in CDK5RAP2, the first in an individual of non-Pakistani descent. Sensorineural hearing loss is not generally considered to be consistent with autosomal recessive microcephaly and therefore it seems likely that the deafness in this individual is caused by the co-occurrence of a further gene mutation, independent of CDK5RAP2. Nevertheless, further detailed clinical descriptions of rare CDK5RAP2 patients, including hearing assessments will be needed to resolve fully the phenotypic range associated with mutations in this gene. This study also highlights the utility of SNP-array testing to guide disease gene identification where an autosomal recessive condition is plausible. © 2012 Wiley Periodicals, Inc.

Pagnamenta AT, Wing K, Sadighi Akha E, Knight SJ, Bölte S, Schmötzer G, Duketis E, Poustka F, Klauck SM, Poustka A et al. 2009. A 15q13.3 microdeletion segregating with autism. Eur J Hum Genet, 17 (5), pp. 687-692. | Show Abstract | Read more

Autism and mental retardation (MR) show high rates of comorbidity and potentially share genetic risk factors. In this study, a rare approximately 2 Mb microdeletion involving chromosome band 15q13.3 was detected in a multiplex autism family. This genomic loss lies between distal break points of the Prader-Willi/Angelman syndrome locus and was first described in association with MR and epilepsy. Together with recent studies that have also implicated this genomic imbalance in schizophrenia, our data indicate that this CNV shows considerable phenotypic variability. Further studies should aim to characterise the precise phenotypic range of this CNV and may lead to the discovery of genetic or environmental modifiers.

Sharp AJ, Mefford HC, Li K, Baker C, Skinner C, Stevenson RE, Schroer RJ, Novara F, De Gregori M, Ciccone R et al. 2008. A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures. Nat Genet, 40 (3), pp. 322-328. | Show Abstract | Read more

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4-BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is approximately 0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

Wordsworth S, Buchanan J, Regan R, Davison V, Smith K, Dyer S, Campbell C, Blair E, Maher E, Taylor J, Knight SJ. 2007. Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom. Genomic Med, 1 (1-2), pp. 35-45. | Show Abstract | Read more

Array based comparative genomic hybridisation (aCGH) is a powerful technique for detecting clinically relevant genome imbalance and can offer 40 to > 1000 times the resolution of karyotyping. Indeed, idiopathic learning disability (ILD) studies suggest that a genome-wide aCGH approach makes 10-15% more diagnoses involving genome imbalance than karyotyping. Despite this, aCGH has yet to be implemented as a routine NHS service. One significant obstacle is the perception that the technology is prohibitively expensive for most standard NHS clinical cytogenetics laboratories. To address this, we investigated the cost-effectiveness of aCGH versus standard cytogenetic analysis for diagnosing idiopathic learning disability (ILD) in the NHS. Cost data from four participating genetics centres were collected and analysed. In a single test comparison, the average cost of aCGH was pound442 and the average cost of karyotyping was pound117 with array costs contributing most to the cost difference. This difference was not a key barrier when the context of follow up diagnostic tests was considered. Indeed, in a hypothetical cohort of 100 ILD children, aCGH was found to cost less per diagnosis ( pound3,118) than a karyotyping and multi-telomere FISH approach ( pound4,957). We conclude that testing for genomic imbalances in ILD using microarray technology is likely to be cost-effective because long-term savings can be made regardless of a positive (diagnosis) or negative result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice. The use of aCGH in routine clinical practice warrants serious consideration by healthcare providers.

Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, Hurst JA, Stewart H, Price SM, Blair E, Hennekam RC et al. 2006. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet, 38 (9), pp. 1038-1042. | Show Abstract | Read more

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

Price TS, Regan R, Mott R, Hedman A, Honey B, Daniels RJ, Smith L, Greenfield A, Tiganescu A, Buckle V et al. 2005. SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data. Nucleic Acids Res, 33 (11), pp. 3455-3464. | Show Abstract | Read more

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.

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