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1.  Association of a rare NOTCH4 coding variant with systemic sclerosis: a family-based whole exome sequencing study 
Background
Systemic sclerosis (SSc) is a rheumatologic disease with a multifactorial etiology. Genome-wide association studies imply a polygenic, complex mode of inheritance with contributions from variation at the human leukocyte antigen locus and non-coding variation at a locus on chromosome 6p21, among other modestly impactful loci. Here we describe an 8-year-old female proband presenting with diffuse cutaneous SSc/scleroderma and a family history of SSc in a grandfather and maternal aunt.
Methods
We employed whole exome sequencing (WES) of three members of this family. We examined rare missense, nonsense, splice-altering, and coding indels matching an autosomal dominant inheritance model. We selected one missense variant for Sanger sequencing confirmation based on its predicted impact on gene function and location in a known SSc genetic locus.
Results
Bioinformatic analysis found eight candidate variants meeting our criteria. We identified a very rare missense variant in the regulatory NODP domain of NOTCH4 located at the 6p21 locus, c.4245G > A:p.Met1415Ile, segregating with the phenotype. This allele has a frequency of 1.83 × 10−5 by the data of the Exome Aggregation Consortium.
Conclusion
This family suggests a novel mechanism of SSc pathogenesis in which a rare and penetrant coding variation can substantially elevate disease risk in contrast to the more modest non-coding variation typically found at this locus. These results suggest that modulation of the NOTCH4 gene might be responsible for the association signal at chromosome 6p21 in SSc.
Electronic supplementary material
The online version of this article (doi:10.1186/s12891-016-1320-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12891-016-1320-4
PMCID: PMC5103422  PMID: 27829420
Whole exome sequencing; Systemic sclerosis; Scleroderma; NOTCH4; Mendelian genetics
2.  Genomic copy number variation association study in Caucasian patients with nonsyndromic cryptorchidism 
BMC Urology  2016;16:62.
Background
Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population.
Methods
Genome wide genotyping were performed in 559 cases and 1772 controls (Group 1) using Illumina HumanHap550 v1, HumanHap550 v3 or Human610-Quad platforms and in 353 cases and 1149 controls (Group 2) using the Illumina Human OmniExpress 12v1 or Human OmniExpress 12v1-1. Signal intensity data including log R ratio (LRR) and B allele frequency (BAF) for each single nucleotide polymorphism (SNP) were used for CNV detection using PennCNV software. After sample quality control, gene- and CNV-based association tests were performed using cleaned data from Group 1 (493 cases and 1586 controls) and Group 2 (307 cases and 1102 controls) using ParseCNV software. Meta-analysis was performed using gene-based test results as input to identify significant genes, and CNVs in or around significant genes were identified in CNV-based association test results. Called CNVs passing quality control and signal intensity visualization examination were considered for validation using TaqMan CNV assays and QuantStudio® 3D Digital PCR System.
Results
The meta-analysis identified 373 genome wide significant (p < 5X10−4) genes/loci including 49 genes/loci with deletions and 324 with duplications. Among them, 17 genes with deletion and 1 gene with duplication were identified in CNV-based association results in both Group 1 and Group 2. Only 2 genes (NUCB2 and UPF2) containing deletions passed CNV quality control in both groups and signal intensity visualization examination, but laboratory validation failed to verify these deletions.
Conclusions
Our data do not support that structural variation is a major cause of nonsyndromic cryptorchidism.
Electronic supplementary material
The online version of this article (doi:10.1186/s12894-016-0180-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12894-016-0180-4
PMCID: PMC5073740  PMID: 27769252
Cryptorchidism; Genetics; CNV
3.  Pathway analysis supports association of nonsyndromic cryptorchidism with genetic loci linked to cytoskeleton-dependent functions 
Human Reproduction (Oxford, England)  2015;30(10):2439-2451.
STUDY QUESTION
What are the genetic loci that increase susceptibility to nonsyndromic cryptorchidism, or undescended testis?
SUMMARY ANSWER
A genome-wide association study (GWAS) suggests that susceptibility to cryptorchidism is heterogeneous, with a subset of suggestive signals linked to cytoskeleton-dependent functions and syndromic forms of the disease.
WHAT IS KNOWN ALREADY
Population studies suggest moderate genetic risk of cryptorchidism and possible maternal and environmental contributions to risk. Previous candidate gene analyses have failed to identify a major associated locus, although variants in insulin-like 3 (INSL3), relaxin/insulin-like family peptide receptor 2 (RXFP2) and other hormonal pathway genes may increase risk in a small percentage of patients.
STUDY DESIGN, SIZE, DURATION
This is a case–control GWAS of 844 boys with nonsyndromic cryptorchidism and 2718 control subjects without syndromes or genital anomalies, all of European ancestry.
PARTICIPANTS/MATERIALS, SETTING, METHODS
All boys with cryptorchidism were diagnosed and treated by a pediatric specialist. In the discovery phase, DNA was extracted from tissue or blood samples and genotyping performed using the Illumina HumanHap550 and Human610-Quad (Group 1) or OmniExpress (Group 2) platform. We imputed genotypes genome-wide, and combined single marker association results in meta-analyses for all cases and for secondary subphenotype analyses based on testis position, laterality and age, and defined genome-wide significance as P = 7 × 10−9 to correct for multiple testing. Selected markers were genotyped in an independent replication group of European cases (n = 298) and controls (n = 324). We used several bioinformatics tools to analyze top (P < 10−5) and suggestive (P < 10−3) signals for significant enrichment of signaling pathways, cellular functions and custom gene lists after multiple testing correction.
MAIN RESULTS AND THE ROLE OF CHANCE
In the full analysis, we identified 20 top loci, none reaching genome-wide significance, but one passing this threshold in a subphenotype analysis of proximal testis position (rs55867206, near SH3PXD2B, odds ratio = 2.2 (95% confidence interval 1.7, 2.9), P = 2 × 10−9). An additional 127 top loci emerged in at least one secondary analysis, particularly of more severe phenotypes. Cytoskeleton-dependent molecular and cellular functions were prevalent in pathway analysis of suggestive signals, and may implicate loci encoding cytoskeletal proteins that participate in androgen receptor signaling. Genes linked to human syndromic cryptorchidism, including hypogonadotropic hypogonadism, and to hormone-responsive and/or differentially expressed genes in normal and cryptorchid rat gubernaculum, were also significantly overrepresented. No tested marker showed significant replication in an independent population. The results suggest heterogeneous, multilocus and potentially multifactorial susceptibility to nonsyndromic cryptorchidism.
LIMITATIONS, REASONS FOR CAUTION
The present study failed to identify genome-wide significant markers associated with cryptorchidism that could be replicated in an independent population, so further studies are required to define true positive signals among suggestive loci.
WIDER IMPLICATIONS OF THE FINDINGS
As the only GWAS to date of nonsyndromic cryptorchidism, these data will provide a basis for future efforts to understand genetic susceptibility to this common reproductive anomaly and the potential for additive risk from environmental exposures.
STUDY FUNDING/COMPETING INTERESTS
This work was supported by R01HD060769 (the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD)), P20RR20173 (the National Center for Research Resources (NCRR), currently P20GM103464 from the National Institute of General Medical Sciences (NIGMS)), an Institute Development Fund to the Center for Applied Genomics at The Children's Hospital of Philadelphia, and Nemours Biomedical Research. The authors have no competing interests to declare.
doi:10.1093/humrep/dev180
PMCID: PMC4573451  PMID: 26209787
cryptorchidism; genetics; cytoskeleton; testis; pathways
4.  Rare variants at 16p11.2 are associated with common variable immunodeficiency 
Background
Common variable immunodeficiency (CVID) is characterized clinically by inadequate quantity and quality of serum immunoglobulins with increased susceptibility to infections resulting in significant morbidity and mortality. Only a few genes have been uncovered and the genetic background of CVID remains elusive to date for the majority of patients.
Objective
To seek novel associations of genes and genetic variants with CVID.
Methods
We performed association analyses in a discovery cohort of 164 CVID cases and 19,542 healthy control subjects genotyped on the Immuno BeadChip from Illumina (iCHIP); replication of findings were examined in an independent cohort of 135 CVID cases and 2,066 healthy control subjects, followed by meta-analysis.
Results
We identified 11 SNPs at the 16p11.2 locus associated with CVID at genome-wide significant level in the discovery cohort. The most significant SNP, rs929867 (p = 6.21×10−9) is in the gene FUS (fused-in-sarcoma) with four other SNPs mapping to ITGAM (integrin CD11b). Results were confirmed in our replication cohort. Conditional association analysis suggests a single association signal at the 16p11.2 locus. A strong trend of association was also seen by 38 SNPs (p < 5×10−5) in the MHC region, supporting that this is a genuine CVID locus. Interestingly we found that 80% of patients with the rare ITGAM variants have reduced counts of switched-memory B-cells.
Conclusion
We report a novel association of CVID with rare variants at the FUS/ITGAM (CD11b) locus on 16p11.2. The association signal is enriched for promoter/enhancer markers in the ITGAM gene. ITGAM encodes the integrin CD11b, a part of complement receptor 3 (CR3/Mac-1), a novel candidate gene implicated here for the first time in the pathogenesis of CVID.
doi:10.1016/j.jaci.2014.12.1939
PMCID: PMC4461447  PMID: 25678086
immunodeficiency; immunogenetics; genome-wide association study; ITGAM; rare variants
5.  Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases 
Nature medicine  2015;21(9):1018-1027.
Genome-wide association studies (GWASs) have identified hundreds of susceptibility genes, including shared associations across clinically distinct autoimmune diseases. We performed an inverse χ2 meta-analysis across ten pediatric-age-of-onset autoimmune diseases (pAIDs) in a case-control study including more than 6,035 cases and 10,718 shared population-based controls. We identified 27 genome-wide significant loci associated with one or more pAIDs, mapping to in silico–replicated autoimmune-associated genes (including IL2RA) and new candidate loci with established immunoregulatory functions such as ADGRL2, TENM3, ANKRD30A, ADCY7 and CD40LG. The pAID-associated single-nucleotide polymorphisms (SNPs) were functionally enriched for deoxyribonuclease (DNase)-hypersensitivity sites, expression quantitative trait loci (eQTLs), microRNA (miRNA)-binding sites and coding variants. We also identified biologically correlated, pAID-associated candidate gene sets on the basis of immune cell expression profiling and found evidence of genetic sharing. Network and protein-interaction analyses demonstrated converging roles for the signaling pathways of type 1, 2 and 17 helper T cells (TH1, TH2 and TH17), JAK-STAT, interferon and interleukin in multiple autoimmune diseases.
doi:10.1038/nm.3933
PMCID: PMC4863040  PMID: 26301688
6.  AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N-Acetylaspartate 
JIMD Reports  2014;14:77-85.
Background: Whole exome sequencing (WES) offers a powerful diagnostic tool to rapidly and efficiently sequence all coding genes in individuals presenting for consideration of phenotypically and genetically heterogeneous disorders such as suspected mitochondrial disease. Here, we report results of WES and functional validation in a consanguineous Indian kindred where two siblings presented with profound developmental delay, congenital hypotonia, refractory epilepsy, abnormal myelination, fluctuating basal ganglia changes, cerebral atrophy, and reduced N-acetylaspartate (NAA).
Methods: Whole blood DNA from one affected and one unaffected sibling was captured by Agilent SureSelect Human All Exon kit and sequenced on the Illumina HiSeq2000. Mutations were validated by Sanger sequencing in all family members. Protein from wild-type and mutant fibroblasts was isolated to assess mutation effects on protein expression and enzyme activity.
Results: A novel SLC25A12 homozygous missense mutation, c.1058G>A; p.Arg353Gln, segregated with disease in this kindred. SLC25A12 encodes the neuronal aspartate-glutamate carrier 1 (AGC1) protein, an essential component of the neuronal malate/aspartate shuttle that transfers NADH and H+ reducing equivalents from the cytosol to mitochondria. AGC1 activity enables neuronal export of aspartate, the glial substrate necessary for proper neuronal myelination. Recombinant mutant p.Arg353Gln AGC1 activity was reduced to 15% of wild type. One prior reported SLC25A12 mutation caused complete loss of AGC1 activity in a child with epilepsy, hypotonia, hypomyelination, and reduced brain NAA.
Conclusions: These data strongly suggest that SLC25A12 disease impairs neuronal AGC1 activity. SLC25A12 sequencing should be considered in children with infantile epilepsy, congenital hypotonia, global delay, abnormal myelination, and reduced brain NAA.
Electronic supplementary material
The online version of this chapter (doi:10.1007/8904_2013_287) contains supplementary material, which is available to authorized users.
doi:10.1007/8904_2013_287
PMCID: PMC4213337  PMID: 24515575
7.  Variants in CXCR4 associate with juvenile idiopathic arthritis susceptibility 
BMC Medical Genetics  2016;17:24.
Background
Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children, the etiology of which involves a strong genetic component, but much of the underlying genetic determinants still remain unknown. Our aim was to identify novel genetic variants that predispose to JIA.
Methods
We performed a genome-wide association study (GWAS) and replication in a total of 1166 JIA cases and 9500 unrelated controls of European ancestry. Correlation of SNP genotype and gene expression was investigated. Then we conducted targeted resequencing of a candidate locus, among a subset of 480 cases and 480 controls. SUM test was performed to evaluate the association of the identified rare functional variants.
Results
The CXCR4 locus on 2q22.1 was found to be significantly associated with JIA, peaking at SNP rs953387. However, this result is subjected to subpopulation stratification within the subjects of European ancestry. After adjusting for principal components, nominal significant association remained (p < 10−4). Because of its interesting known function in immune regulation, we carried out further analyses to assess its relationship with JIA. Expression of CXCR4 was correlated with CXCR4 rs953387 genotypes in lymphoblastoid cell lines (p = 0.014) and T-cells (p = 0.0054). In addition, rare non-synonymous and stop-gain sequence variants in CXCR4, putatively damaging for CXCR4 function, were significantly enriched in JIA cases (p = 0.015).
Conclusion
Our results suggest the association of CXCR4 variants with JIA, implicating that this gene may be involved in the pathogenesis of autoimmune disease. However, because this locus is subjected to population stratification within the subjects of European ancestry, additional replication is still necessary for this locus to be considered a true risk locus for JIA. This cell-surface chemokine receptor has already been targeted in other diseases and may serve as a tractable therapeutic target for a specific subset of pediatric arthritis patients with additional replication and functional validation of the locus.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-016-0285-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12881-016-0285-3
PMCID: PMC4804485  PMID: 27005825
Juvenile idiopathic arthritis; Genome-wide association study; CXCR4; Targeted resequencing
8.  Genome-wide association study of maternal and inherited effects on left-sided cardiac malformations 
Human Molecular Genetics  2014;24(1):265-273.
Congenital left-sided lesions (LSLs) are serious, heritable malformations of the heart. However, little is known about the genetic causes of LSLs. This study was undertaken to identify common variants acting through the genotype of the affected individual (i.e. case) or the mother (e.g. via an in utero effect) that influence the risk of LSLs. A genome-wide association study (GWAS) was performed using data from 377 LSL case-parent triads, with follow-up studies in an independent sample of 224 triads and analysis of the combined data. Associations with both the case and maternal genotypes were assessed using log-linear analyses under an additive model. An association between LSLs and the case genotype for one intergenic SNP on chromosome 16 achieved genome-wide significance in the combined data (rs8061121, combined P = 4.0 × 10−9; relative risk to heterozygote: 2.6, 95% CI: 1.9–3.7). In the combined data, there was also suggestive evidence of association between LSLs and the case genotype for a variant in the synaptoporin gene (rs1975649, combined P = 3.4 × 10−7; relative risk to heterozygote: 1.6, 95% CI: 1.4–2.0) and between LSLs and the maternal genotype for an intergenic SNP on chromosome 10 (rs11008222, combined P = 6.3 × 10−7; relative risk to heterozygote: 1.6, 95% CI: 1.4–2.0). This is the first GWAS of LSLs to evaluate associations with both the case and maternal genotypes. The results of this study identify three candidate LSL susceptibility loci, including one that appears to be associated with the risk of LSLs via the maternal genotype.
doi:10.1093/hmg/ddu420
PMCID: PMC4326325  PMID: 25138779
9.  Genetic sharing and heritability of paediatric age of onset autoimmune diseases 
Nature Communications  2015;6:8442.
Autoimmune diseases (AIDs) are polygenic diseases affecting 7–10% of the population in the Western Hemisphere with few effective therapies. Here, we quantify the heritability of paediatric AIDs (pAIDs), including JIA, SLE, CEL, T1D, UC, CD, PS, SPA and CVID, attributable to common genomic variations (SNP-h2). SNP-h2 estimates are most significant for T1D (0.863±s.e. 0.07) and JIA (0.727±s.e. 0.037), more modest for UC (0.386±s.e. 0.04) and CD (0.454±0.025), largely consistent with population estimates and are generally greater than that previously reported by adult GWAS. On pairwise analysis, we observed that the diseases UC-CD (0.69±s.e. 0.07) and JIA-CVID (0.343±s.e. 0.13) are the most strongly correlated. Variations across the MHC strongly contribute to SNP-h2 in T1D and JIA, but does not significantly contribute to the pairwise rG. Together, our results partition contributions of shared versus disease-specific genomic variations to pAID heritability, identifying pAIDs with unexpected risk sharing, while recapitulating known associations between autoimmune diseases previously reported in adult cohorts.
Autoimmune diseases are genetically complex disorders that affect up to 10% of the Western population. Here Li et al. quantify the heritability of a range of autoimmune diseases in the largest paediatric cohort examined to date, illustrating that genetic and non-genetic components variably contribute to the susceptibility of each disease.
doi:10.1038/ncomms9442
PMCID: PMC4633631  PMID: 26450413
11.  Phenotype-specific association of the TGFBR3 locus with nonsyndromic cryptorchidism 
The Journal of urology  2014;193(5):1637-1645.
Purpose
Based on a genome-wide association study (GWAS) of testicular dysgenesis syndrome (TDS) reporting possible association with TGFBR3, we analyzed GWAS data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal.
Materials and Methods
We excluded samples based on strict quality control criteria, leaving 844 cases and 2718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform. Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of Group 1 and 2 data and selective genotyping of independent cases (n=330) and controls (n=324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum.
Results
We identified suggestive (p≤1×10−4) association of markers in/near TGFBR3 including rs9661103 (OR 1.40, 95% CI 1.20,1.64, p=2.71×10−5) and rs10782968 (OR 1.58, CI 1.26,1.98, p=9.36×10−5) in Groups 1 and 2, respectively. In subgroup analyses, we observed strongest association of rs17576372 (OR 1.42, CI 1.24,1.60; p=1.67×10−4) with proximal and rs11165059 (OR 1.32, CI 1.15,1.38; p=9.42×10−4) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior GWAS signal (rs12082710) was marginal (OR 1.13, CI 0.99,1.28, p=0.09 for Group 1) and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild type and cryptorchid rat fetal gubernaculum.
Conclusions
These data suggest complex or phenotype-specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFβ signaling.
doi:10.1016/j.juro.2014.10.097
PMCID: PMC4406821  PMID: 25390077
Cryptorchidism; genetic association studies; TGFRB3; gubernaculum
12.  Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder 
Nature genetics  2011;44(1):78-84.
Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.
doi:10.1038/ng.1013
PMCID: PMC4310555  PMID: 22138692
13.  Association of variants of the interleukin-23 receptor (IL23R) gene with susceptibility to pediatric Crohn’s disease 
Background & Aims
Recently an association was demonstrated between the single nucleotide polymorphism (SNP), rs11209026, within the interleukin-23 receptor (IL23R) locus and Crohn’s disease (CD) as a consequence of a genome wide association study of this disease in adults. We examined the effects of this and other previously reported SNPs at this locus with respect to CD in children.
Methods
Utilizing data from our ongoing genome-wide association study in our cohort of 142 pediatric CD cases and 281 matched controls, we investigated the association of the previously reported SNPs at the IL23R locus with the childhood form of this disease.
Results
Using a Fisher’s exact test, the minor allele frequency (MAF) of rs1120902 in the cases was 1.75% while it was 6.61% in controls, yielding a protective odds ratio (OR) of 0.25 (95% CI 0.10 – 0.65; one-sided P = 9.2×10−4). Furthermore, of all the SNPs previously reported, rs11209026 was the most strongly associated. A subsequent family-based association test (which is more resistant to population stratification) with 65 sets of trios derived from our initial patient cohort yielded significant association with rs11209026 in a transmission disequilibrium test (one-sided P=0.0017). In contrast, no association was detected to the CARD15 gene for the IBD phenotype.
Conclusions
The OR of the IL23R variant in our pediatric study is highly comparable with that reported previously in a non-Jewish adult IBD case-control cohort (OR=0.26). As such, variants in IL23R gene confer a similar magnitude of risk of CD to children as for their adult counterparts.
doi:10.1016/j.cgh.2007.04.024
PMCID: PMC4287202  PMID: 17618837
IL23R; gene; association; Crohn’s Disease
15.  Common variants at 12q15 and 12q24 are associated with infant head circumference 
Taal, H Rob | Pourcain, Beate St | Thiering, Elisabeth | Das, Shikta | Mook-Kanamori, Dennis O | Warrington, Nicole M | Kaakinen, Marika | Kreiner-Møller, Eskil | Bradfield, Jonathan P | Freathy, Rachel M | Geller, Frank | Guxens, Mònica | Cousminer, Diana L | Kerkhof, Marjan | Timpson, Nicholas J | Ikram, M Arfan | Beilin, Lawrence J | Bønnelykke, Klaus | Buxton, Jessica L | Charoen, Pimphen | Chawes, Bo Lund Krogsgaard | Eriksson, Johan | Evans, David M | Hofman, Albert | Kemp, John P | Kim, Cecilia E | Klopp, Norman | Lahti, Jari | Lye, Stephen J | McMahon, George | Mentch, Frank D | Müller, Martina | O’Reilly, Paul F | Prokopenko, Inga | Rivadeneira, Fernando | Steegers, Eric A P | Sunyer, Jordi | Tiesler, Carla | Yaghootkar, Hanieh | Breteler, Monique M B | Debette, Stephanie | Fornage, Myriam | Gudnason, Vilmundur | Launer, Lenore J | van der Lugt, Aad | Mosley, Thomas H | Seshadri, Sudha | Smith, Albert V | Vernooij, Meike W | Blakemore, Alexandra IF | Chiavacci, Rosetta M | Feenstra, Bjarke | Fernandez-Benet, Julio | Grant, Struan F A | Hartikainen, Anna-Liisa | van der Heijden, Albert J | Iñiguez, Carmen | Lathrop, Mark | McArdle, Wendy L | Mølgaard, Anne | Newnham, John P | Palmer, Lyle J | Palotie, Aarno | Pouta, Annneli | Ring, Susan M | Sovio, Ulla | Standl, Marie | Uitterlinden, Andre G | Wichmann, H-Erich | Vissing, Nadja Hawwa | DeCarli, Charles | van Duijn, Cornelia M | McCarthy, Mark I | Koppelman, Gerard H. | Estivill, Xavier | Hattersley, Andrew T | Melbye, Mads | Bisgaard, Hans | Pennell, Craig E | Widen, Elisabeth | Hakonarson, Hakon | Smith, George Davey | Heinrich, Joachim | Jarvelin, Marjo-Riitta | Jaddoe, Vincent W V
Nature genetics  2012;44(5):532-538.
To identify genetic variants associated with head circumference in infancy, we performed a meta-analysis of seven genome-wide association (GWA) studies (N=10,768 from European ancestry enrolled in pregnancy/birth cohorts) and followed up three lead signals in six replication studies (combined N=19,089). Rs7980687 on chromosome 12q24 (P=8.1×10−9), and rs1042725 on chromosome 12q15 (P=2.8×10−10) were robustly associated with head circumference in infancy. Although these loci have previously been associated with adult height1, their effects on infant head circumference were largely independent of height (P=3.8×10−7 for rs7980687, P=1.3×10−7 for rs1042725 after adjustment for infant height). A third signal, rs11655470 on chromosome 17q21, showed suggestive evidence of association with head circumference (P=3.9×10−6). SNPs correlated to the 17q21 signal show genome-wide association with adult intra cranial volume2, Parkinson’s disease and other neurodegenerative diseases3-5, indicating that a common genetic variant in this region might link early brain growth with neurological disease in later life.
doi:10.1038/ng.2238
PMCID: PMC3773913  PMID: 22504419
16.  Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis 
Human Molecular Genetics  2012;21(13):2899-2911.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation–prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation–prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation–prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.
doi:10.1093/hmg/dds116
PMCID: PMC3373238  PMID: 22454397
17.  The missense variation landscape of FTO, MC4R and TMEM18 in obese children of African ancestry 
Obesity (Silver Spring, Md.)  2013;21(1):159-163.
Common variation at the loci harboring FTO, MC4R and TMEM18 is consistently reported as being statistically the most strongly associated with obesity. We investigated if these loci also harbor rarer missense variants that confer substantially higher risk of common childhood obesity in African American (AA) children. We sequenced the exons of FTO, MC4R and TMEM18 in an initial subset of our cohort i.e. 200 obese (BMI≥95th percentile) and 200 lean AA children (BMI≤5th percentile). Any missense exonic variants that were uncovered went on to be further genotyped in a further 768 obese and 768 lean (BMI≤50th percentile) children of the same ethnicity. A number of exonic variants were observed from our sequencing effort: seven in FTO, of which four were non-synonymous (A163T, G182A, M400V and A405V), thirteen in MC4R, of which six were non-synonymous (V103I, N123S, S136A, F202L, N240S and I251L) and four in TMEM18, of which two were non-synonymous (P2S and V113L). Follow-up genotyping of these missense variants revealed only one significant difference in allele frequency between cases and controls, namely with N240S in MC4R(Fisher's Exact P = 0.0001). In summary, moderately rare missense variants within the FTO, MC4R and TMEM18 genes observed in our study did not confer risk of common childhood obesity in African Americans except for a degree of evidence for one known loss-of-function variant in MC4R.
doi:10.1002/oby.20147
PMCID: PMC3605748  PMID: 23505181
Obesity; Pediatrics; Genomics
18.  Copy Number Variations in Alternative Splicing Gene Networks Impact Lifespan 
PLoS ONE  2013;8(1):e53846.
Longevity has a strong genetic component evidenced by family-based studies. Lipoprotein metabolism, FOXO proteins, and insulin/IGF-1 signaling pathways in model systems have shown polygenic variations predisposing to shorter lifespan. To test the hypothesis that rare variants could influence lifespan, we compared the rates of CNVs in healthy children (0–18 years of age) with individuals 67 years or older. CNVs at a significantly higher frequency in the pediatric cohort were considered risk variants impacting lifespan, while those enriched in the geriatric cohort were considered longevity protective variants. We performed a whole-genome CNV analysis on 7,313 children and 2,701 adults of European ancestry genotyped with 302,108 SNP probes. Positive findings were evaluated in an independent cohort of 2,079 pediatric and 4,692 geriatric subjects. We detected 8 deletions and 10 duplications that were enriched in the pediatric group (P = 3.33×10−8–1.6×10−2 unadjusted), while only one duplication was enriched in the geriatric cohort (P = 6.3×10−4). Population stratification correction resulted in 5 deletions and 3 duplications remaining significant (P = 5.16×10−5–4.26×10−2) in the replication cohort. Three deletions and four duplications were significant combined (combined P = 3.7×10−4−3.9×10−2). All associated loci were experimentally validated using qPCR. Evaluation of these genes for pathway enrichment demonstrated ∼50% are involved in alternative splicing (P = 0.0077 Benjamini and Hochberg corrected). We conclude that genetic variations disrupting RNA splicing could have long-term biological effects impacting lifespan.
doi:10.1371/journal.pone.0053846
PMCID: PMC3559729  PMID: 23382853
19.  Common variants at five new loci associated with early-onset inflammatory bowel disease 
Nature Genetics  2009;41(12):1335-1340.
The inflammatory bowel diseases (IBD) Crohn’s disease and ulcerative colitis are common causes of morbidity in children and young adults in the western world. Here we report the results of a genome-wide association study in early-onset IBD involving 3,426 affected individuals and 11,963 genetically matched controls recruited through international collaborations in Europe and North America, thereby extending the results from a previous study of 1,011 individuals with early-onset IBD1. We have identified five new regions associated with early-onset IBD susceptibility, including 16p11 near the cytokine gene IL27 (rs8049439, P = 2.41 × 10−9), 22q12 (rs2412973, P = 1.55 × 10−9), 10q22 (rs1250550, P = 5.63 × 10−9), 2q37 (rs4676410, P = 3.64 × 10−8) and 19q13.11 (rs10500264, P = 4.26 × 10−10). Our scan also detected associations at 23 of 32 loci previously implicated in adult-onset Crohn’s disease and at 8 of 17 loci implicated in adult-onset ulcerative colitis, highlighting the close pathogenetic relationship between early- and adult-onset IBD.
doi:10.1038/ng.489
PMCID: PMC3267927  PMID: 19915574
20.  A Genome-Wide Meta-Analysis of Six Type 1 Diabetes Cohorts Identifies Multiple Associated Loci 
PLoS Genetics  2011;7(9):e1002293.
Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5.66×10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3.50×10−9) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8.06×10−9) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.
Author Summary
Despite the fact that there is clearly a large genetic component to type 1 diabetes (T1D), uncovering the genes contributing to this disease has proven challenging. However, in the past three years there has been relatively major progress in this regard, with advances in genetic screening technologies allowing investigators to scan the genome for variants conferring risk for disease without prior hypotheses. Such genome-wide association studies have revealed multiple regions of the genome to be robustly and consistently associated with T1D. More recent findings have been a consequence of combining of multiple datasets from independent investigators in meta-analyses, which have more power to pick up additional variants contributing to the trait. In the current study, we describe the largest meta-analysis of T1D genome-wide genotyped datasets to date, which combines six large studies. As a consequence, we have uncovered three new signals residing at the chromosomal locations 13q22, 2p23, and 6q27, which went on to be replicated in independent sample sets. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.
doi:10.1371/journal.pgen.1002293
PMCID: PMC3183083  PMID: 21980299
21.  Examination of All Type 2 Diabetes GWAS Loci Reveals HHEX-IDE as a Locus Influencing Pediatric BMI 
Diabetes  2009;59(3):751-755.
OBJECTIVE
A number of studies have found that BMI in early life influences the risk of developing type 2 diabetes later in life. Our goal was to investigate if any type 2 diabetes variants uncovered through genome-wide association studies (GWAS) impact BMI in childhood.
RESEARCH DESIGN AND METHODS
Using data from an ongoing GWAS of pediatric BMI in our cohort, we investigated the association of pediatric BMI with 20 single nucleotide polymorphisms at 18 type 2 diabetes loci uncovered through GWAS, consisting of ADAMTS9, CDC123-CAMK1D, CDKAL1, CDKN2A/B, EXT2, FTO, HHEX-IDE, IGF2BP2, the intragenic region on 11p12, JAZF1, KCNQ1, LOC387761, MTNR1B, NOTCH2, SLC30A8, TCF7L2, THADA, and TSPAN8-LGR5. We randomly partitioned our cohort exactly in half in order to have a discovery cohort (n = 3,592) and a replication cohort (n = 3,592).
RESULTS
Our data show that the major type 2 diabetes risk–conferring G allele of rs7923837 at the HHEX-IDE locus was associated with higher pediatric BMI in both the discovery (P = 0.0013 and survived correction for 20 tests) and replication (P = 0.023) sets (combined P = 1.01 × 10−4). Association was not detected with any other known type 2 diabetes loci uncovered to date through GWAS except for the well-established FTO.
CONCLUSIONS
Our data show that the same genetic HHEX-IDE variant, which is associated with type 2 diabetes from previous studies, also influences pediatric BMI.
doi:10.2337/db09-0972
PMCID: PMC2828649  PMID: 19933996
22.  Duplication of the SLIT3 Locus on 5q35.1 Predisposes to Major Depressive Disorder 
PLoS ONE  2010;5(12):e15463.
Major depressive disorder (MDD) is a common psychiatric and behavioral disorder. To discover novel variants conferring risk to MDD, we conducted a whole-genome scan of copy number variation (CNV), including 1,693 MDD cases and 4,506 controls genotyped on the Perlegen 600K platform. The most significant locus was observed on 5q35.1, harboring the SLIT3 gene (P = 2×10−3). Extending the controls with 30,000 subjects typed on the Illumina 550 k array, we found the CNV to remain exclusive to MDD cases (P = 3.2×10−9). Duplication was observed in 5 unrelated MDD cases encompassing 646 kb with highly similar breakpoints. SLIT3 is integral to repulsive axon guidance based on binding to Roundabout receptors. Duplication of 5q35.1 is a highly penetrant variation accounting for 0.7% of the subset of 647 cases harboring large CNVs, using a threshold of a minimum of 10 SNPs and 100 kb. This study leverages a large dataset of MDD cases and controls for the analysis of CNVs with matched platform and ethnicity. SLIT3 duplication is a novel association which explains a definitive proportion of the largely unknown etiology of MDD.
doi:10.1371/journal.pone.0015463
PMCID: PMC2995745  PMID: 21152026
23.  Common variants in HSPB7 and FRMD4B associated with advanced heart failure 
Background
Heart failure results from abnormalities in multiple biological processes that contribute to cardiac dysfunction. We tested the hypothesis that inherited variation in genes of known importance to cardiovascular biology would thus contribute to heart failure risk.
Methods and Results
We utilized the ITMAT/Broad/CARe (IBC) cardiovascular SNP-array to screen referral populations of advanced heart failure patients for variants in ~2,000 genes of predicted importance to cardiovascular biology. Our design was a two-stage case-control study. In Stage 1, genotypes in Caucasian heart failure patients (n=1,590; ejection fraction 32±16%) were compared to those in unaffected controls (n=577; ejection fraction 67±8%) recruited from the same referral centers. Associations were tested for independent replication in Stage 2 (n=308 cases, 2,314 controls). Two intronic SNPs showed replicated associations with all-cause heart failure: rs1739843 in HSPB7 (combined P=3.09×10−6) and rs6787362 in FRMD4B (P=6.09×10−6). For both SNPs the minor allele was protective. In subgroup analyses, rs1739843 associated with both ischemic and nonischemic heart failure, whereas rs6787362 associated principally with ischemic heart failure. Linkage disequilibrium surrounding rs1739843 suggested that the causal variant resides in a region containing HSPB7 and a neighboring gene, CLCNKA, whereas the causal variant near rs6787362 is probably within FRMD4B. Allele frequencies for these SNPs were substantially different in African Americans (n=635 cases, 714 controls) and showed no association with heart failure in this population.
Conclusions
Our findings identify regions containing HSPB7 and FRMD4B as novel susceptibility loci for advanced heart failure. More broadly, in an era of genome-wide association studies, we demonstrate how knowledge of candidate genes can be leveraged as a complementary strategy to discern the genetics of complex disorders.
doi:10.1161/CIRCGENETICS.109.898395
PMCID: PMC2957840  PMID: 20124441
cardiomyopathy; genetics; heart failure
24.  Examination of Type 2 Diabetes Loci Implicates CDKAL1 as a Birth Weight Gene 
Diabetes  2009;58(10):2414-2418.
OBJECTIVE
A number of studies have found that reduced birth weight is associated with type 2 diabetes later in life; however, the underlying mechanism for this correlation remains unresolved. Recently, association has been demonstrated between low birth weight and single nucleotide polymorphisms (SNPs) at the CDKAL1 and HHEX-IDE loci, regions that were previously implicated in the pathogenesis of type 2 diabetes. In order to investigate whether type 2 diabetes risk–conferring alleles associate with low birth weight in our Caucasian childhood cohort, we examined the effects of 20 such loci on this trait.
RESEARCH DESIGN AND METHODS
Using data from an ongoing genome-wide association study in our cohort of 5,465 Caucasian children with recorded birth weights, we investigated the association of the previously reported type 2 diabetes–associated variation at 20 loci including TCF7L2, HHEX-IDE, PPARG, KCNJ11, SLC30A8, IGF2BP2, CDKAL1, CDKN2A/2B, and JAZF1 with birth weight.
RESULTS
Our data show that the minor allele of rs7756992 (P = 8 × 10−5) at the CDKAL1 locus is strongly associated with lower birth weight, whereas a perfect surrogate for variation previously implicated for the trait at the same locus only yielded nominally significant association (P = 0.01; r2 rs7756992 = 0.677). However, association was not detected with any of the other type 2 diabetes loci studied.
CONCLUSIONS
We observe association between lower birth weight and type 2 diabetes risk–conferring alleles at the CDKAL1 locus. Our data show that the same genetic locus that has been identified as a marker for type 2 diabetes in previous studies also influences birth weight.
doi:10.2337/db09-0506
PMCID: PMC2750235  PMID: 19592620
25.  Common genetic variants on 5p14.1 associate with autism spectrum disorders 
Nature  2009;459(7246):528-533.
Autism spectrum disorders (ASDs) represent a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal communication, impairment of social interaction, and restricted and repetitive patterns of interests and behaviour. To identify common genetic risk factors underlying ASDs, here we present the results of genome-wide association studies on a cohort of 780 families (3,101 subjects) with affected children, and a second cohort of 1,204 affected subjects and 6,491 control subjects, all of whom were of European ancestry. Six single nucleotide polymorphisms between cadherin 10 (CDH10) and cadherin 9 (CDH9)—two genes encoding neuronal cell-adhesion molecules—revealed strong association signals, with the most significant SNP being rs4307059 (P = 3.4 × 10−8, odds ratio = 1.19). These signals were replicated in two independent cohorts, with combined P values ranging from 7.4 × 10−8 to 2.1 × 10−10. Our results implicate neuronal cell-adhesion molecules in the pathogenesis of ASDs, and represent, to our knowledge, the first demonstration of genome-wide significant association of common variants with susceptibility to ASDs.
doi:10.1038/nature07999
PMCID: PMC2943511  PMID: 19404256

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