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1.  Genes implicated in thiopurine-induced toxicity: Comparing TPMT enzyme activity with clinical phenotype and exome data in a paediatric IBD cohort 
Scientific Reports  2016;6:34658.
The aim of our study was to assess the utility of next generation sequencing (NGS) for predicting toxicity and clinical response to thiopurine drugs in paediatric patients with inflammatory bowel disease. Exome data for 100 patients were assessed against biochemically measured TPMT enzyme activity, clinical response and adverse effects. The TPMT gene and a panel of 15 other genes implicated in thiopurine toxicity were analysed using a gene based statistical test (SKAT-O test). Nine patients out of 100 (Crohn’s disease- 67, ulcerative colitis- 23 and IBDU-10) had known TPMT mutations associated with deficient enzyme activity. A novel and a highly pathogenic TPMT variant not detectable through standard genotyping, was identified through NGS in an individual intolerant to thiopurines. Of the 14 patients intolerant to thiopurines, NGS identified deleterious TPMT variants in 5 individuals whereas the biochemical test identified 8 individuals as intolerant (sensitivity 35.7% and 57.14%; specificity 93.75% and 50% respectively). SKAT-O test identified a significant association between MOCOS gene and TPMT activity (p = 0.0015), not previously reported. Although NGS has the ability to detect rare or novel variants not otherwise identified through standard genotyping, it demonstrates no clear advantage over the biochemical test in predicting toxicity in our modest cohort.
PMCID: PMC5050412  PMID: 27703193
2.  Deleterious coding variants in multi-case families with non-syndromic cleft lip and/or palate phenotypes 
Scientific Reports  2016;6:30457.
Nonsyndromic Cleft Lip and/or Palate (NSCLP) is regarded as a multifactorial condition in which clefting is an isolated phenotype, distinguished from the largely monogenic, syndromic forms which include clefts among a spectrum of phenotypes. Nonsyndromic clefting has been shown to arise through complex interactions between genetic and environmental factors. However, there is increasing evidence that the broad NSCLP classification may include a proportion of cases showing familial patterns of inheritance and contain highly penetrant deleterious variation in specific genes. Through exome sequencing of multi-case families ascertained in Bogota, Colombia, we identify 28 non-synonymous single nucleotide variants that are considered damaging by at least one predictive score. We discuss the functional impact of candidate variants identified. In one family we find a coding variant in the MSX1 gene which is predicted damaging by multiple scores. This variant is in exon 2, a highly conserved region of the gene. Previous sequencing has suggested that mutations in MSX1 may account for ~2% of NSCLP. Our analysis further supports evidence that a proportion of NSCLP cases arise through monogenic coding mutations, though further work is required to unravel the complex interplay of genetics and environment involved in facial clefting.
PMCID: PMC4960602  PMID: 27456059
3.  Longitudinal copy number, whole exome and targeted deep sequencing of ‘good risk’ IGHV-mutated CLL patients with progressive disease 
Leukemia  2016;30(6):1301-1310.
The biological features of IGHV-M CLL responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in thirteen patients presenting with cMBL or Stage A disease and good risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom ten have required therapy. Using cytogenetics, FISH, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing, at diagnosis we identified mutations in established CLL driver genes in nine (69%), non-coding mutations (PAX5 enhancer region) in three, and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition prior to therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good risk disease.
PMCID: PMC4861248  PMID: 26847028
4.  Progressive myoclonic epilepsy with Fanconi syndrome 
JRSM Open  2016;7(6):2054270415623145.
This report illustrates the difficulties in diagnosing complex cases and demonstrates how whole exome sequencing can resolve complex phenotypes.
PMCID: PMC4900196  PMID: 27293772
Progressive myoclonic epilepsy; valproate; Fanconi syndrome; next generation sequencing; exome; KCTD7
5.  Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion 
PLoS ONE  2016;11(2):e0149162.
In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.
PMCID: PMC4752330  PMID: 26871591
6.  Whole genome sequences are required to fully resolve the linkage disequilibrium structure of human populations 
BMC Genomics  2015;16(1):666.
An understanding of linkage disequilibrium (LD) structures in the human genome underpins much of medical genetics and provides a basis for disease gene mapping and investigating biological mechanisms such as recombination and selection. Whole genome sequencing (WGS) provides the opportunity to determine LD structures at maximal resolution.
We compare LD maps constructed from WGS data with LD maps produced from the array-based HapMap dataset, for representative European and African populations. WGS provides up to 5.7-fold greater SNP density than array-based data and achieves much greater resolution of LD structure, allowing for identification of up to 2.8-fold more regions of intense recombination. The absence of ascertainment bias in variant genotyping improves the population representativeness of the WGS maps, and highlights the extent of uncaptured variation using array genotyping methodologies. The complete capture of LD patterns using WGS allows for higher genome-wide association study (GWAS) power compared to array-based GWAS, with WGS also allowing for the analysis of rare variation. The impact of marker ascertainment issues in arrays has been greatest for Sub-Saharan African populations where larger sample sizes and substantially higher marker densities are required to fully resolve the LD structure.
WGS provides the best possible resource for LD mapping due to the maximal marker density and lack of ascertainment bias. WGS LD maps provide a rich resource for medical and population genetics studies. The increasing availability of WGS data for large populations will allow for improved research utilising LD, such as GWAS and recombination biology studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1854-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4558963  PMID: 26335686
Linkage disequilibrium map; Population structure; Whole-genome sequencing; Recombination; Next-generation sequencing
7.  Exome Analysis of Patients with Concurrent Pediatric Inflammatory Bowel Disease and Autoimmune Disease 
Inflammatory Bowel Diseases  2015;21(6):1229-1236.
Article first published online 17 April 2015.
Supplemental Digital Content is Available in the Text.
Pediatric Inflammatory Bowel Disease (PIBD) is a chronic condition seen in genetically predisposed individuals. Genome-wide association studies have implicated >160 genomic loci in IBD with many genes coding for proteins in key immune pathways. This study looks at autoimmune disease burden in patients diagnosed with PIBD and interrogates exome data of a subset of patients.
Patients were recruited from the Southampton Genetics of PIBD cohort. Clinical diagnosis of autoimmune disease in these individuals was ascertained from medical records. For a subset of patients with PIBD and concurrent asthma, exome data was interrogated to ascertain the burden of pathogenic variants within genes implicated in asthma. Association testing was conducted between cases and population controls using the SKAT-O test.
Forty-nine (28.3%) PIBD children (18.49% CD, 8.6% UC, and 21.15% IBDU patients) had a concurrent clinical diagnosis of at least one other autoimmune disorder; asthma was the most prevalent, affecting 16.2% of the PIBD cohort. Rare and common variant association testing revealed 6 significant genes (P < 0.05) before Bonferroni adjustment. Three of these genes were previously implicated in both asthma and IBD (ZPBP2 IL1R1, and IL18R1) and 3 in asthma only (PYHIN1, IL2RB, and GSTP1).
One-third of our cohort had a concurrent autoimmune condition. We observed higher incidence of asthma compared with the overall pediatric prevalence. Despite a small sample size, SKAT-O evaluated a significant burden of rare and common mutations in 6 genes. Variant burden suggests that a systemic immune dysregulation rather than organ-specific could underpin immune dysfunction for a subset of patients.
PMCID: PMC4450895  PMID: 25895113
pediatric inflammatory bowel disease; comorbidity; exome sequencing; autoimmune disorders; asthma; genetics
8.  Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia Caused by a Novel R782G Mutation in CSF1R 
Scientific Reports  2015;5:10042.
We report a new family with autosomal dominant inheritance of a late onset rapidly progressive leukodystrophy in which exome sequencing has revealed a novel mutation p.R782G in the Colony-Stimulating Factor 1 Receptor gene (CSF1R). Neuropathology of two affected family members showed cerebral white matter degeneration with axonal swellings and pigmented macrophages. The few recently reported families with CSF1R mutations had been previously labelled “hereditary diffuse leukencephalopathy with axonal spheroids” (HDLS) and “pigmentary orthochromatic leukodystrophy” (POLD), disorders which now appear to form a disease continuum. The term “adult-onset leukoencephalopathy with axonal spheroids and pigmented glia” (ALSP) has been proposed to encompass this spectrum. As CSF1R regulates microglia this mutation implies that dysregulation of microglia is the primary cause of the disease.
PMCID: PMC4432561  PMID: 25975230
9.  Erratum to: a SNP profiling panel for sample tracking in whole-exome sequencing studies 
Genome Medicine  2015;7(1):44.
This is an Erratum to Genome Medicine 2013, 5:89, highlighting an error in Table 1 of the original article. Please see related article:
PMCID: PMC4422541  PMID: 25949530
10.  Genome-wide analysis of multiethnic cohorts identifies new loci influencing intraocular pressure and susceptibility to glaucoma 
Hysi, Pirro G | Cheng, Ching-Yu | Springelkamp, Henriët | Macgregor, Stuart | Bailey, Jessica N Cooke | Wojciechowski, Robert | Vitart, Veronique | Nag, Abhishek | Hewitt, Alex W | Höhn, René | Venturini, Cristina | Mirshahi, Alireza | Ramdas, Wishal D. | Thorleifsson, Gudmar | Vithana, Eranga | Khor, Chiea-Chuen | Stefansson, Arni B | Liao, Jiemin | Haines, Jonathan L | Amin, Najaf | Wang, Ya Xing | Wild, Philipp S | Ozel, Ayse B | Li, Jun Z | Fleck, Brian W | Zeller, Tanja | Staffieri, Sandra E | Teo, Yik-Ying | Cuellar-Partida, Gabriel | Luo, Xiaoyan | Allingham, R Rand | Richards, Julia E | Senft, Andrea | Karssen, Lennart C | Zheng, Yingfeng | Bellenguez, Céline | Xu, Liang | Iglesias, Adriana I | Wilson, James F | Kang, Jae H | van Leeuwen, Elisabeth M | Jonsson, Vesteinn | Thorsteinsdottir, Unnur | Despriet, Dominiek D.G. | Ennis, Sarah | Moroi, Sayoko E | Martin, Nicholas G | Jansonius, Nomdo M | Yazar, Seyhan | Tai, E-Shyong | Amouyel, Philippe | Kirwan, James | van Koolwijk, Leonieke M.E. | Hauser, Michael A | Jonasson, Fridbert | Leo, Paul | Loomis, Stephanie J | Fogarty, Rhys | Rivadeneira, Fernando | Kearns, Lisa | Lackner, Karl J | de Jong, Paulus T.V.M. | Simpson, Claire L | Pennell, Craig E | Oostra, Ben A | Uitterlinden, André G | Saw, Seang-Mei | Lotery, Andrew J | Bailey-Wilson, Joan E | Hofman, Albert | Vingerling, Johannes R | Maubaret, Cécilia | Pfeiffer, Norbert | Wolfs, Roger C.W. | Lemij, Hans G | Young, Terri L | Pasquale, Louis R | Delcourt, Cécile | Spector, Timothy D | Klaver, Caroline C.W. | Small, Kerrin S | Burdon, Kathryn P | Stefansson, Kari | Wong, Tien-Yin | Viswanathan, Ananth | Mackey, David A | Craig, Jamie E | Wiggs, Janey L | van Duijn, Cornelia M | Hammond, Christopher J | Aung, Tin
Nature genetics  2014;46(10):1126-1130.
Elevated intraocular pressure (IOP) is an important risk factor in developing glaucoma and IOP variability may herald glaucomatous development or progression. We report the results of a genome-wide association study meta-analysis of 18 population cohorts from the International Glaucoma Genetics Consortium (IGGC), comprising 35,296 multiethnic participants for IOP. We confirm genetic association of known loci for IOP and primary open angle glaucoma (POAG) and identify four new IOP loci located on chromosome 3q25.31 within the FNDC3B gene (p=4.19×10−08 for rs6445055), two on chromosome 9 (p=2.80×10−11 for rs2472493 near ABCA1 and p=6.39×10−11 for rs8176693 within ABO) and one on chromosome 11p11.2 (best p=1.04×10−11 for rs747782). Separate meta-analyses of four independent POAG cohorts, totaling 4,284 cases and 95,560 controls, show that three of these IOP loci are also associated with POAG.
PMCID: PMC4177225  PMID: 25173106
11.  Age-Related Macular Degeneration: A Disease of Systemic or Local Complement Dysregulation? 
Journal of Clinical Medicine  2014;3(4):1234-1257.
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. The role of complement in the development of AMD is now well-established. While some studies show evidence of complement dysregulation within the eye, others have demonstrated elevated systemic complement activation in association with AMD. It is unclear which one is the primary driver of disease. This has important implications for designing novel complement-based AMD therapies. We present a summary of the current literature and suggest that intraocular rather than systemic modulation of complement may prove more effective.
PMCID: PMC4470180  PMID: 26237601
age-related macular degeneration; complement pathway; complement system proteins/genetics; pathway analysis; proteomics
12.  Meta-analysis of genome-wide association studies identifies novel loci that influence cupping and the glaucomatous process 
Springelkamp, Henriët. | Höhn, René | Mishra, Aniket | Hysi, Pirro G. | Khor, Chiea-Chuen | Loomis, Stephanie J. | Bailey, Jessica N. Cooke | Gibson, Jane | Thorleifsson, Gudmar | Janssen, Sarah F. | Luo, Xiaoyan | Ramdas, Wishal D. | Vithana, Eranga | Nongpiur, Monisha E. | Montgomery, Grant W. | Xu, Liang | Mountain, Jenny E. | Gharahkhani, Puya | Lu, Yi | Amin, Najaf | Karssen, Lennart C. | Sim, Kar-Seng | van Leeuwen, Elisabeth M. | Iglesias, Adriana I. | Verhoeven, Virginie J. M. | Hauser, Michael A. | Loon, Seng-Chee | Despriet, Dominiek D. G. | Nag, Abhishek | Venturini, Cristina | Sanfilippo, Paul G. | Schillert, Arne | Kang, Jae H. | Landers, John | Jonasson, Fridbert | Cree, Angela J. | van Koolwijk, Leonieke M. E. | Rivadeneira, Fernando | Souzeau, Emmanuelle | Jonsson, Vesteinn | Menon, Geeta | Weinreb, Robert N. | de Jong, Paulus T. V. M. | Oostra, Ben A. | Uitterlinden, André G. | Hofman, Albert | Ennis, Sarah | Thorsteinsdottir, Unnur | Burdon, Kathryn P. | Spector, Timothy D. | Mirshahi, Alireza | Saw, Seang-Mei | Vingerling, Johannes R. | Teo, Yik-Ying | Haines, Jonathan L. | Wolfs, Roger C. W. | Lemij, Hans G. | Tai, E-Shyong | Jansonius, Nomdo M. | Jonas, Jost B. | Cheng, Ching-Yu | Aung, Tin | Viswanathan, Ananth C. | Klaver, Caroline C. W. | Craig, Jamie E. | Macgregor, Stuart | Mackey, David A. | Lotery, Andrew J. | Stefansson, Kari | Bergen, Arthur A. B. | Young, Terri L. | Wiggs, Janey L. | Pfeiffer, Norbert | Wong, Tien-Yin | Pasquale, Louis R. | Hewitt, Alex W. | van Duijn, Cornelia M. | Hammond, Christopher J.
Nature Communications  2014;5:4883.
Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.
Glaucoma is the most common cause of irreversible blindness worldwide. Here, the authors carry out a large meta-analysis of genetic data from individuals of European and Asian ancestry and identify 10 new loci associated with vertical cup-disc ratio, a key factor in the clinical assessment of patients with glaucoma.
PMCID: PMC4199103  PMID: 25241763
13.  Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus 
Lu, Yi | Vitart, Veronique | Burdon, Kathryn P | Khor, Chiea Chuen | Bykhovskaya, Yelena | Mirshahi, Alireza | Hewitt, Alex W | Koehn, Demelza | Hysi, Pirro G | Ramdas, Wishal D | Zeller, Tanja | Vithana, Eranga N | Cornes, Belinda K | Tay, Wan-Ting | Tai, E Shyong | Cheng, Ching-Yu | Liu, Jianjun | Foo, Jia-Nee | Saw, Seang Mei | Thorleifsson, Gudmar | Stefansson, Kari | Dimasi, David P | Mills, Richard A | Mountain, Jenny | Ang, Wei | Hoehn, René | Verhoeven, Virginie J M | Grus, Franz | Wolfs, Roger | Castagne, Raphaële | Lackner, Karl J | Springelkamp, Henriët | Yang, Jian | Jonasson, Fridbert | Leung, Dexter Y L | Chen, Li J | Tham, Clement C Y | Rudan, Igor | Vatavuk, Zoran | Hayward, Caroline | Gibson, Jane | Cree, Angela J | MacLeod, Alex | Ennis, Sarah | Polasek, Ozren | Campbell, Harry | Wilson, James F | Viswanathan, Ananth C | Fleck, Brian | Li, Xiaohui | Siscovick, David | Taylor, Kent D | Rotter, Jerome I | Yazar, Seyhan | Ulmer, Megan | Li, Jun | Yaspan, Brian L | Ozel, Ayse B | Richards, Julia E | Moroi, Sayoko E | Haines, Jonathan L | Kang, Jae H | Pasquale, Louis R | Allingham, R Rand | Ashley-Koch, Allison | Mitchell, Paul | Wang, Jie Jin | Wright, Alan F | Pennell, Craig | Spector, Timothy D | Young, Terri L | Klaver, Caroline C W | Martin, Nicholas G | Montgomery, Grant W | Anderson, Michael G | Aung, Tin | Willoughby, Colin E | Wiggs, Janey L | Pang, Chi P | Thorsteinsdottir, Unnur | Lotery, Andrew J | Hammond, Christopher J | van Duijn, Cornelia M | Hauser, Michael A | Rabinowitz, Yaron S | Pfeiffer, Norbert | Mackey, David A | Craig, Jamie E | Macgregor, Stuart | Wong, Tien Y
Nature genetics  2013;45(2):155-163.
Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10−8). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4–1.88, P = 2.7 × 10−10, and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29–1.68, P = 4.9 × 10−9). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10−4; tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.
PMCID: PMC3720123  PMID: 23291589
14.  Megalencephaly Syndromes: Exome Pipeline Strategies for Detecting Low-Level Mosaic Mutations 
PLoS ONE  2014;9(1):e86940.
Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation.
PMCID: PMC3908952  PMID: 24497998
15.  Complement factor I and age-related macular degeneration 
Molecular Vision  2014;20:1253-1257.
The complement system has been implicated in the pathogenesis of age-related macular degeneration (AMD). Complement factor I (CFI) is a serum protease that inhibits all complement pathways. A previous multicenter study identified a single missense CFI mutation (p.Gly119Arg) in 20/3,567 (0.56%) of AMD cases versus 1/3,937 (0.025%) of controls, thus suggesting that this mutation confers a high risk of AMD. A second CFI mutation, p.Gly188Ala, was identified in one patient with AMD.
We screened 521 unrelated AMD cases and 627 controls for the p.Gly119Arg and p.Gly188Ala variants. All participants were Caucasian and >55 years, and recruited through Southampton Eye Unit or research clinics in Guernsey. All participants underwent dilated fundal examination by an experienced retinal specialist. SNP assays were performed using KASP™ biochemistry.
The p.Gly119Arg mutation was identified in 7/521 AMD cases compared to 1/627 age-matched controls (odds ratio [OR] = 8.47, confidence interval [CI] = 1.04–69.00, p = 0.027). There was a varied phenotype among the seven cases with the mutation, which was present in 4/254 (1.6%) cases with active or end-stage wet AMD and 3/267 dry AMD cases (1.1%). The p.Gly188Ala substitution was identified in 1/521 cases and 1/627 controls.
Our results identified a much higher frequency of heterozygosity for p.Gly119Arg in both cases and controls than in previous studies. Of note is that our sub-cohort from Guernsey had a particularly high frequency of p.Gly119Arg heterozygosity in affected individuals (4%) compared to our sub-cohort from the mainland (0.71%). Although these data support the conclusions of van de Ven et al. that the p.Gly119Arg substitution confers a high risk of AMD, our data suggest that this missense mutation is not as rare or as highly penetrant as previously reported. There was no difference in frequency for a second CFI variant, p.Gly188Ala, between the cases and the controls.
PMCID: PMC4165324  PMID: 25352734
16.  Whole Exome Sequencing Identifies Novel Recurrently Mutated Genes in Patients with Splenic Marginal Zone Lymphoma 
PLoS ONE  2013;8(12):e83244.
The pathogenesis of splenic marginal zone lymphoma (SMZL) remains largely unknown. Recent high-throughput sequencing studies have identified recurrent mutations in key pathways, most notably NOTCH2 mutations in >25% of patients. These studies are based on small, heterogeneous discovery cohorts, and therefore only captured a fraction of the lesions present in the SMZL genome. To identify further novel pathogenic mutations within related biochemical pathways, we applied whole exome sequencing (WES) and copy number (CN) analysis to a biologically and clinically homogeneous cohort of seven SMZL patients with 7q abnormalities and IGHV1-2*04 gene usage. We identified 173 somatic non-silent variants, affecting 160 distinct genes. In additional to providing independent validation of the presence of mutation in several previously reported genes (NOTCH2, TNFAIP3, MAP3K14, MLL2 and SPEN), our study defined eight additional recurrently mutated genes in SMZL; these genes are CREBBP, CBFA2T3, AMOTL1, FAT4, FBXO11, PLA2G4D, TRRAP and USH2A. By integrating our WES and CN data we identified three mutated putative candidate genes targeted by 7q deletions (CUL1, EZH2 and FLNC), with FLNC positioned within the well-characterized 7q minimally deleted region. Taken together, this work expands the reported directory of recurrently mutated cancer genes in this disease, thereby expanding our understanding of SMZL pathogenesis. Ultimately, this work will help to establish a stratified approach to care including the possibility of targeted therapy.
PMCID: PMC3862727  PMID: 24349473
17.  A SNP profiling panel for sample tracking in whole-exome sequencing studies 
Genome Medicine  2013;5(9):89.
Whole-exome sequencing provides a cost-effective means to sequence protein coding regions within the genome, which are significantly enriched for etiological variants. We describe a panel of single nucleotide polymorphisms (SNPs) to facilitate the validation of data provenance in whole-exome sequencing studies. This is particularly significant where multiple processing steps necessitate transfer of sample custody between clinical, laboratory and bioinformatics facilities. SNPs captured by all commonly used exome enrichment kits were identified, and filtered for possible confounding properties. The optimised panel provides a simple, yet powerful, method for the assignment of intrinsic, highly discriminatory identifiers to genetic samples.
PMCID: PMC3978886  PMID: 24070238
18.  Genome-wide association study of age-related macular degeneration identifies associated variants in the TNXB–FKBPL–NOTCH4 region of chromosome 6p21.3 
Human Molecular Genetics  2012;21(18):4138-4150.
Age-related macular degeneration (AMD) is a leading cause of visual loss in Western populations. Susceptibility is influenced by age, environmental and genetic factors. Known genetic risk loci do not account for all the heritability. We therefore carried out a genome-wide association study of AMD in the UK population with 893 cases of advanced AMD and 2199 controls. This showed an association with the well-established AMD risk loci ARMS2 (age-related maculopathy susceptibility 2)–HTRA1 (HtrA serine peptidase 1) (P =2.7 × 10−72), CFH (complement factor H) (P =2.3 × 10−47), C2 (complement component 2)–CFB (complement factor B) (P =5.2 × 10−9), C3 (complement component 3) (P =2.2 × 10−3) and CFI (P =3.6 × 10−3) and with more recently reported risk loci at VEGFA (P =1.2 × 10−3) and LIPC (hepatic lipase) (P =0.04). Using a replication sample of 1411 advanced AMD cases and 1431 examined controls, we confirmed a novel association between AMD and single-nucleotide polymorphisms on chromosome 6p21.3 at TNXB (tenascin XB)–FKBPL (FK506 binding protein like) [rs12153855/rs9391734; discovery P =4.3 × 10−7, replication P =3.0 × 10−4, combined P =1.3 × 10−9, odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.3–1.6] and the neighbouring gene NOTCH4 (Notch 4) (rs2071277; discovery P =3.2 × 10−8, replication P =3.8 × 10−5, combined P =2.0 × 10−11, OR = 1.3, 95% CI = 1.2–1.4). These associations remained significant in conditional analyses which included the adjacent C2–CFB locus. TNXB, FKBPL and NOTCH4 are all plausible AMD susceptibility genes, but further research will be needed to identify the causal variants and determine whether any of these genes are involved in the pathogenesis of AMD.
PMCID: PMC3428154  PMID: 22694956
19.  Nonlinear association between CGG repeat number and age of menopause in FMR1 premutation carriers 
FMR1 premutations are known to be associated with premature ovarian failure (POF), but the underlying mechanism is unknown. We present evidence for a nonlinear association between menopause age and premutation size suggesting that premutations in the mid-size range are at greatest risk for POF, while larger premutations are at lower risk.
PMCID: PMC3725422  PMID: 16251893
FMR1; permutation; premature ovarian failure
20.  Reproductive and menstrual history of females with fragile X expansions 
FRAXA premutations have been associated with premature ovarian failure (POF) or menopause before the age of 40. We have studied women in families ascertained because of a mentally retarded full mutation relative and determined their age of menopause, serum hormone levels in premenopausal individuals and the outcome of any pregnancies. Survival analysis was used as a measure of menopause and demonstrated a significant decrease in age of menopause in premutation carriers compared with their full mutation carrier and normal relatives. Serum FSH was also raised in premutation carriers, although oestradiol, inhibin A and inhibin B were not significantly different. However, we did not find an excess of dizygous twins or pregnancy loss/trisomies, both of which are associated with aging ovaries. Thus premutation carriers as a group have an earlier menopause and raised serum FSH but do not appear to manifest other features of an aging ovary.
PMCID: PMC3725540  PMID: 10854106
Fragile X; premutation; premature ovarian failure
21.  Support Vector Machine Classifier for Estrogen Receptor Positive and Negative Early-Onset Breast Cancer 
PLoS ONE  2013;8(7):e68606.
Two major breast cancer sub-types are defined by the expression of estrogen receptors on tumour cells. Cancers with large numbers of receptors are termed estrogen receptor positive and those with few are estrogen receptor negative. Using genome-wide single nucleotide polymorphism genotype data for a sample of early-onset breast cancer patients we developed a Support Vector Machine (SVM) classifier from 200 germline variants associated with estrogen receptor status (p<0.0005). Using a linear kernel Support Vector Machine, we achieved classification accuracy exceeding 93%. The model indicates that polygenic variation in more than 100 genes is likely to underlie the estrogen receptor phenotype in early-onset breast cancer. Functional classification of the genes involved identifies enrichment of functions linked to the immune system, which is consistent with the current understanding of the biological role of estrogen receptors in breast cancer.
PMCID: PMC3716652  PMID: 23894323
22.  Evidence of association of APOE with age-related macular degeneration - a pooled analysis of 15 studies 
Human mutation  2011;32(12):1407-1416.
Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment in high-income countries. Previous studies report inconsistent associations between AMD and apolipoprotein E (APOE), a lipid transport protein involved in low-density cholesterol modulation. Potential interaction between APOE and sex, and smoking status, has been reported. We present a pooled analysis (n=21,160) demonstrating associations between late AMD and APOε4 (OR=0.72 per haplotype; CI: 0.65–0.74; P=4.41×10−11) and APOε2 (OR=1.83 for homozygote carriers; CI: 1.04–3.23; P=0.04), following adjustment for age-group and sex within each study and smoking status. No evidence of interaction between APOE and sex or smoking was found. Ever smokers had significant increased risk relative to never smokers for both neovascular (OR=1.54; CI: 1.38–1.72; P=2.8×10−15) and atrophic (OR=1.38; CI: 1.18–1.61; P=3.37×10−5) AMD but not early AMD (OR=0.94; CI: 0.86–1.03; P=0.16), implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyondε2 and ε4 haplotypes. Our expanded analysis substantially improves our understanding of the association between the APOE locus and AMD. It further provides evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology.
PMCID: PMC3217135  PMID: 21882290
age-related macular degeneration; AMD; apolipoprotein E; APOE; case-control association study
23.  Variations in Apolipoprotein E Frequency With Age in a Pooled Analysis of a Large Group of Older People 
American Journal of Epidemiology  2011;173(12):1357-1364.
Variation in the apolipoprotein E gene (APOE) has been reported to be associated with longevity in humans. The authors assessed the allelic distribution of APOE isoforms ε2, ε3, and ε4 among 10,623 participants from 15 case-control and cohort studies of age-related macular degeneration (AMD) in populations of European ancestry (study dates ranged from 1990 to 2009). The authors included only the 10,623 control subjects from these studies who were classified as having no evidence of AMD, since variation within the APOE gene has previously been associated with AMD. In an analysis stratified by study center, gender, and smoking status, there was a decreasing frequency of the APOE ε4 isoform with increasing age (χ2 for trend = 14.9 (1 df); P = 0.0001), with a concomitant increase in the ε3 isoform (χ2 for trend = 11.3 (1 df); P = 0.001). The association with age was strongest in ε4 homozygotes; the frequency of ε4 homozygosity decreased from 2.7% for participants aged 60 years or less to 0.8% for those over age 85 years, while the proportion of participants with the ε3/ε4 genotype decreased from 26.8% to 17.5% across the same age range. Gender had no significant effect on the isoform frequencies. This study provides strong support for an association of the APOE gene with human longevity.
PMCID: PMC3145394  PMID: 21498624
aged; apolipoprotein E2; apolipoprotein E3; apolipoprotein E4; apolipoproteins E; longevity; meta-analysis; multicenter study
24.  Genome-wide association of breast cancer: composite likelihood with imputed genotypes 
We describe composite likelihood-based analysis of a genome-wide breast cancer case–control sample from the Cancer Genetic Markers of Susceptibility project. We determine 14 380 genome regions of fixed size on a linkage disequilibrium (LD) map, which delimit comparable levels of LD. Although the numbers of single-nucleotide polymorphisms (SNPs) are highly variable, each region contains an average of ∼35 SNPs and an average of ∼69 after imputation of missing genotypes. Composite likelihood association mapping yields a single P-value for each region, established by a permutation test, along with a maximum likelihood disease location, SE and information weight. For single SNP analysis, the nominal P-value for the most significant SNP (msSNP) requires substantial correction given the number of SNPs in the region. Therefore, imputing genotypes may not always be advantageous for the msSNP test, in contrast to composite likelihood. For the region containing FGFR2 (a known breast cancer gene) the largest χ2 is obtained under composite likelihood with imputed genotypes (χ22 increases from 20.6 to 22.7), and compares with a single SNP-based χ22 of 19.9 after correction. Imputation of additional genotypes in this region reduces the size of the 95% confidence interval for location of the disease gene by ∼40%. Among the highest ranked regions, SNPs in the NTSR1 gene would be worthy of examination in additional samples. Meta-analysis, which combines weighted evidence from composite likelihood in different samples, and refines putative disease locations, is facilitated through defining fixed regions on an underlying LD map.
PMCID: PMC3025787  PMID: 20959865
composite likelihood; association mapping; breast cancer; imputed genotypes; FGFR2 gene
25.  Genome-wide association study of primary open angle glaucoma risk and quantitative traits 
Molecular Vision  2012;18:1083-1092.
Primary open angle glaucoma (POAG) is a characteristic optic neuropathy which progresses to irreversible vision loss. Few genes have been detected that influence POAG susceptibility and other genes are therefore likely to be involved. We analyzed carefully characterized POAG cases in a genome-wide association study (GWAS).
We performed a GWAS in 387 POAG cases using public control data (WTCCC2). We also investigated the quantitative phenotypes, cup:disc ratio (CDR), central corneal thickness (CCT), and intra-ocular pressure (IOP). Promising single nucleotide polymorphisms (SNPs), based on various prioritisation criteria, were genotyped in a cohort of 294 further POAG cases and controls.
We found 2 GWAS significant results in the discovery stage for association, one of which which had multiple evidence in the gene ‘neural precursor cell expressed, developmentally down-regulated 9’ (NEDD9; rs11961171, p=8.55E-13) and the second on chromosome 16 with no supporting evidence. Taking into account all the evidence from risk and quantitative trait ocular phenotypes we chose 86 SNPs for replication in an independent sample. Our most significant SNP was not replicated (p=0.59). We found 4 nominally significant results in the replication cohort, but none passed correction for multiple testing. Two of these, for phenotypes CDR (rs4385494, discovery p=4.51x10–5, replication p=0.029) and CCT (rs17128941, discovery p=5.52x10–6, replication=0.027), show the consistent direction of effects between the discovery and replication data. We also assess evidence for previously associated known genes and find evidence for the genes ‘transmembrane and coiled-coil domains 1’ (TMCO1) and ‘cyclin-dependent kinase inhibitor 2B’ (CDKN2B).
Although we were unable to replicate any novel results for POAG risk, we did replicate two SNPs with consistent effects for CDR and CCT, though they do not withstand correction for multiple testing. There has been a range of publications in the last couple of years identifying POAG risk genes and genes involved in POAG related ocular traits. We found evidence for 3 known genes (TMCO1, CDKN2B, and S1 RNA binding domain 1 [SRBD1]) in this study. Novel rare variants, not detectable by GWAS, but by new methods such as exome sequencing may hold the key to unravelling the remaining contribution of genetics to complex diseases such as POAG.
PMCID: PMC3351427  PMID: 22605921

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