Comprehensive Gene Panel Testing for Hearing Loss in Children: Understanding Factors Influencing Diagnostic Yield.

OBJECTIVE: To evaluate factors influencing the diagnostic yield of comprehensive gene panel testing (CGPT) for hearing loss (HL) in children and to understand the characteristics of undiagnosed probands. STUDY DESIGN: This was a retrospective cohort study of 474 probands with childhood-onset HL who underwent CGPT between 2016 and 2020 at a single center. Main outcomes and measures included the association between clinical variables and diagnostic yield and the genetic and clinical characteristics of undiagnosed probands. RESULTS: The overall diagnostic yield was 44% (209/474) with causative variants involving 41 genes. While the diagnostic yield was high in the probands with congenital, bilateral, and severe HL, it was low in those with unilateral, noncongenital, or mild HL; cochlear nerve deficiency; preterm birth; neonatal intensive care unit admittance; certain ancestry; and developmental delay. Follow-up studies on 49 probands with initially inconclusive CGPT results changed the diagnostic status to likely positive or negative outcomes in 39 of them (80%). Reflex to exome sequencing on 128 undiagnosed probands by CGPT revealed diagnostic findings in 8 individuals, 5 of whom had developmental delays. The remaining 255 probands were undiagnosed, with 173 (173/255) having only a single variant in the gene(s) associated with autosomal recessive HL and 28% (48/173) having a matched phenotype. CONCLUSION: CGPT efficiently identifies the genetic etiologies of HL in children. CGPT-undiagnosed probands may benefit from follow-up studies or expanded testing.

Long-read sequencing for molecular diagnostics in constitutional genetic disorders.

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings.

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

Genome sequencing increases diagnostic yield in clinically diagnosed Alagille syndrome patients with previously negative test results.

PURPOSE: Detection of all major classes of genomic variants in a single test would decrease cost and increase the efficiency of genomic diagnostics. Genome sequencing (GS) has the potential to provide this level of comprehensive detection. We sought to demonstrate the utility of GS in the molecular diagnosis of 18 patients with clinically defined Alagille syndrome (ALGS), who had a negative or inconclusive result by standard-of-care testing. METHODS: We performed GS on 16 pathogenic variant-negative probands and two probands with inconclusive results (of 406 ALGS probands) and analyzed the data for sequence, copy-number, and structural variants in JAG1 and NOTCH2. RESULTS: GS identified four novel pathogenic alterations including a copy-neutral inversion, a partial deletion, and a promoter variant in JAG1, and a partial NOTCH2 deletion, for an additional diagnostic yield of 0.9%. Furthermore, GS resolved two complex rearrangements, resulting in identification of a pathogenic variant in 97.5% (n = 396/406) of patients after GS. CONCLUSION: GS provided an increased diagnostic yield for individuals with clinically defined ALGS who had prior negative or incomplete genetic testing by other methods. Our results show that GS can detect all major classes of variants and has potential to become a single first-tier diagnostic test for Mendelian disorders.

Protein-elongating mutations in MYH11 are implicated in a dominantly inherited smooth muscle dysmotility syndrome with severe esophageal, gastric, and intestinal disease.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

Identification of Polycystic Kidney Disease 1 Like 1 Gene Variants in Children With Biliary Atresia Splenic Malformation Syndrome.

Biliary atresia (BA) is the most common cause of end-stage liver disease in children and the primary indication for pediatric liver transplantation, yet underlying etiologies remain unknown. Approximately 10% of infants affected by BA exhibit various laterality defects (heterotaxy) including splenic abnormalities and complex cardiac malformations-a distinctive subgroup commonly referred to as the biliary atresia splenic malformation (BASM) syndrome. We hypothesized that genetic factors linking laterality features with the etiopathogenesis of BA in BASM patients could be identified through whole-exome sequencing (WES) of an affected cohort. DNA specimens from 67 BASM subjects, including 58 patient-parent trios, from the National Institute of Diabetes and Digestive and Kidney Diseases-supported Childhood Liver Disease Research Network (ChiLDReN) underwent WES. Candidate gene variants derived from a prespecified set of 2,016 genes associated with ciliary dysgenesis and/or dysfunction or cholestasis were prioritized according to pathogenicity, population frequency, and mode of inheritance. Five BASM subjects harbored rare and potentially deleterious biallelic variants in polycystic kidney disease 1 like 1 (PKD1L1), a gene associated with ciliary calcium signaling and embryonic laterality determination in fish, mice, and humans. Heterozygous PKD1L1 variants were found in 3 additional subjects. Immunohistochemical analysis of liver from the one BASM subject available revealed decreased PKD1L1 expression in bile duct epithelium when compared to normal livers and livers affected by other noncholestatic diseases. Conclusion: WES identified biallelic and heterozygous PKD1L1 variants of interest in 8 BASM subjects from the ChiLDReN data set; the dual roles for PKD1L1 in laterality determination and ciliary function suggest that PKD1L1 is a biologically plausible, cholangiocyte-expressed candidate gene for the BASM syndrome.

Alagille syndrome mutation update: Comprehensive overview of JAG1 and NOTCH2 mutation frequencies and insight into missense variant classification.

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single-center study, which includes 401 probands and 111 affected family members amassed over a 27-year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.

Association of Enterovirus D68 with Acute Flaccid Myelitis, Philadelphia, Pennsylvania, USA, 2009-2018.

Acute flaccid myelitis (AFM) is a polio-like disease that results in paralysis in previously healthy persons. Although the definitive cause of AFM remains unconfirmed, enterovirus D68 (EV-D68) is suspected based on 2014 data demonstrating an increase in AFM cases concomitant with an EV-D68 outbreak. We examined the prevalence in children and the molecular evolution of EV-D68 for 2009-2018 in Philadelphia, Pennsylvania, USA. We detected widespread EV-D68 circulation in 2009, rare detections in 2010 and 2011, and then biennial circulation, only in even years, during 2012-2018. Prevalence of EV-D68 significantly correlated with AFM cases during this period. Finally, whole-genome sequencing revealed early detection of the B1 clade in 2009 and continued evolution of the B3 clade from 2016 to 2018. These data reinforce the need to improve surveillance programs for nonpolio enterovirus to identify possible AFM triggers and predict disease prevalence to better prepare for future outbreaks.

High-throughput single-molecule mapping links subtelomeric variants and long-range haplotypes with specific telomeres.

Accurate maps and DNA sequences for human subtelomere regions, along with detailed knowledge of subtelomere variation and long-range telomere-terminal haplotypes in individuals, are critical for understanding telomere function and its roles in human biology. Here, we use a highly automated whole genome mapping technology in nano-channel arrays to analyze large terminal human chromosome segments extending from chromosome-specific subtelomere sequences through subtelomeric repeat regions to terminal (TTAGGG)n repeat tracts. We establish detailed maps for subtelomere gap regions in the human reference sequence, detect many new large subtelomeric variants and demonstrate the feasibility of long-range haplotyping through segmentally duplicated subtelomere regions. These features make the method a uniquely valuable new tool for improving the quality of genome assemblies in complex DNA regions. Based on single molecule mapping of telomere-terminal DNA fragments, we provide proof of principle for a novel method to estimate telomere lengths linked to distinguishable telomeric haplotypes; this single-telomere genotyping method may ultimately enable delineation of human cis elements involved in telomere length regulation.

Utility and limitations of exome sequencing as a genetic diagnostic tool for children with hearing loss.

PURPOSE: Hearing loss (HL) is the most common sensory disorder in children. Prompt molecular diagnosis may guide screening and management, especially in syndromic cases when HL is the single presenting feature. Exome sequencing (ES) is an appealing diagnostic tool for HL as the genetic causes are highly heterogeneous. METHODS: ES was performed on a prospective cohort of 43 probands with HL. Sequence data were analyzed for primary and secondary findings. Capture and coverage analysis was performed for genes and variants associated with HL. RESULTS: The diagnostic rate using ES was 37.2%, compared with 15.8% for the clinical HL panel. Secondary findings were discovered in three patients. For 247 genes associated with HL, 94.7% of the exons were targeted for capture and 81.7% of these exons were covered at 20× or greater. Further analysis of 454 randomly selected HL-associated variants showed that 89% were targeted for capture and 75% were covered at a read depth of at least 20×. CONCLUSION: ES has an improved yield compared with clinical testing and may capture diagnoses not initially considered due to subtle clinical phenotypes. Technical challenges were identified, including inadequate capture and coverage of HL genes. Additional considerations of ES include secondary findings, cost, and turnaround time.

Utility and limitations of exome sequencing in the molecular diagnosis of pediatric inherited platelet disorders.

Inherited platelet disorders (IPD) are a heterogeneous group of rare disorders that affect platelet number and function and often predispose to other significant medical complications. In spite of the identification of over 50 IPD disease-associated genes, a molecular diagnosis is only identified in a minority (10%) of affected patients without a clinically suspected etiology. We studied a cohort of 21 pediatric patients with suspected IPDs by exome sequencing (ES) to: (1) examine the performance of the exome test for IPD genes, (2) determine if this exome-wide diagnostic test provided a higher diagnostic yield than has been previously reported, (3) to evaluate the frequency of variants of uncertain significance identified, and (4) to identify candidate variants for functional evaluation in patients with an uncertain or negative diagnosis. We established a high priority gene list of 53 genes, evaluated exome capture kit performance, and determined the coverage for these genes and disease-related variants. We identified likely disease causing variants in 5 of the 21 probands (23.8%) and variants of uncertain significance in 52% of patients studied. In conclusion, ES has the potential to molecularly diagnose causes of IPD, and to identify candidate genes for functional evaluation. Robust exome sequencing also requires that coverage of genes known to be associated with clinical findings of interest need to be carefully examined and supplemented if necessary. Clinicians who undertake ES should understand the limitations of the test and the full significance of results that may be returned.

Flexible robust binder-free carbon nanotube membranes for solid state and microcapacitor application.

We present a liquid phase post synthesis self-assemble protocol that transforms trillions of carbon nanotubes (CNTs) in powder form into densely packed flexible, robust and binder-free macroscopic membranes with a hierarchical pore structure. We employ charge transfer engineering to spontaneously disperse the CNTs in a liquid medium. The processing protocol has limited or no impact on the intrinsic properties of the CNTs. As the thickness of the CNT membrane is increased, we observed a gradual transition from high flexibility to buckling and brittleness in the flexural properties of the membranes. The binder-free CNT membranes have bulk mass density greater than that of water (1.0 g cm-3). We correlate the mass of the CNTs in the membrane to the thickness of the membrane and obtained a bulk mass density of ∼1.11 g cm-3 ± 0.03 g cm-3. We demonstrate the use of the CNT membranes as electrode in a pristine and oxidized single/stacked solid-state capacitor as well as pristine interdigitated microcapacitor that show time constant of ∼32 ms with no degradation in performance even after 10 000 cycles. The capacitors show very good temperature dependence over a wide range of temperatures with good cycling performance up to 90 °C. The specific capacitance of the pseudocapacitive CNT electrode at room temperature was 72 F g-1 and increased to 100 F g-1 at 70 °C. The leakage current of bipolar stacked solid state capacitor was ∼100 nA cm-2 at 2.5 V when held for 72 h.

Utility and limitations of exome sequencing as a genetic diagnostic tool for conditions associated with pediatric sudden cardiac arrest/sudden cardiac death.

BACKGROUND: Conditions associated with sudden cardiac arrest/death (SCA/D) in youth often have a genetic etiology. While SCA/D is uncommon, a pro-active family screening approach may identify these inherited structural and electrical abnormalities prior to symptomatic events and allow appropriate surveillance and treatment. This study investigated the diagnostic utility of exome sequencing (ES) by evaluating the capture and coverage of genes related to SCA/D. METHODS: Samples from 102 individuals (13 with known molecular etiologies for SCA/D, 30 individuals without known molecular etiologies for SCA/D and 59 with other conditions) were analyzed following exome capture and sequencing at an average read depth of 100X. Reads were mapped to human genome GRCh37 using Novoalign, and post-processing and analysis was done using Picard and GATK. A total of 103 genes (2,190 exons) related to SCA/D were used as a primary filter. An additional 100 random variants within the targeted genes associated with SCA/D were also selected and evaluated for depth of sequencing and coverage. Although the primary objective was to evaluate the adequacy of depth of sequencing and coverage of targeted SCA/D genes and not for primary diagnosis, all patients who had SCA/D (known or unknown molecular etiologies) were evaluated with the project's variant analysis pipeline to determine if the molecular etiologies could be successfully identified. RESULTS: The majority of exons (97.6 %) were captured and fully covered on average at minimum of 20x sequencing depth. The proportion of unique genomic positions reported within poorly covered exons remained small (4 %). Exonic regions with less coverage reflect the need to enrich these areas to improve coverage. Despite limitations in coverage, we identified 100 % of cases with a prior known molecular etiology for SCA/D, and analysis of an additional 30 individuals with SCA/D but no known molecular etiology revealed a diagnostic answer in 5/30 (17 %). We also demonstrated 95 % of 100 randomly selected reported variants within our targeted genes would have been picked up on ES based on our coverage analysis. CONCLUSIONS: ES is a helpful clinical diagnostic tool for SCA/D given its potential to successfully identify a molecular diagnosis, but clinicians should be aware of limitations of available platforms from technical and diagnostic perspectives.

Compound heterozygous mutations in NEK8 in siblings with end-stage renal disease with hepatic and cardiac anomalies.

We studied two brothers who presented in the newborn period with cardiac, renal, and hepatic anomalies that were initially suggestive of ALGS, although no mutations in JAG1 or NOTCH2 were identified. Exome sequencing demonstrated compound heterozygous mutations in the NEK8 gene (Never in mitosis A-related Kinase 8), a ciliary kinase indispensable for cardiac and renal development based on murine studies. The mutations included a c.2069_2070insC variant (p.Ter693LeufsTer86), and a c.1043C>T variant (p.Thr348Met) in the highly conserved RCC1 (Regulation of Chromosome Condensation 1) domain. The RCC1 domain is crucial for localization of the NEK8 protein to the centrosomes and cilia. Mutations in NEK8 have been previously reported in three fetuses (from a single family) with renal-hepatic-pancreatic dysplasia 2 (RHPD2), similar to Ivemark syndrome, and in three individuals with nephronophthisis (NPHP9). This is the third report of disease-causing mutations in the NEK8 gene in humans and only the second describing multi-organ involvement. The clinical features we describe differ from those in the previously published report in that (1) a pancreatic phenotype was not observed in the individuals reported here, (2) there were more prominent cardiac findings, (consistent with observations in murine models), and (3) we observed bile duct hypoplasia rather than ductal plate malformation. The patients reported here expand our understanding of the NEK8-associated phenotype. Our findings highlight the variable phenotypic expressivity and the spectrum of clinical manifestations due to mutations in the NEK8 gene.

Heterozygous deletion of FOXA2 segregates with disease in a family with heterotaxy, panhypopituitarism, and biliary atresia.

Biliary atresia (BA) is a pediatric cholangiopathy with unknown etiology occurring in isolated and syndromic forms. Laterality defects affecting the cardiovascular and gastrointestinal systems are the most common features present in syndromic BA. Most cases are sporadic, although reports of familial cases have led to the hypothesis of genetic susceptibility in some patients. We identified a child with BA, malrotation, and interrupted inferior vena cava whose father presented with situs inversus, polysplenia, panhypopituitarism, and mildly dysmorphic facial features. Chromosomal microarray analysis demonstrated a 277 kb heterozygous deletion on chromosome 20, which included a single gene, FOXA2, in the proband and her father. This deletion was confirmed to be de novo in the father. The proband and her father share a common diagnosis of heterotaxy, but they also each presented with a variety of other issues. Further genetic screening revealed that the proband carried an additional protein-altering polymorphism (rs1904589; p.His165Arg) in the NODAL gene that is not present in the father, and this variant has been shown to decrease expression of the gene. As FOXA2 can be a regulator of NODAL expression, we propose that haploinsufficiency for FOXA2 combined with a decreased expression of NODAL is the likely cause for syndromic BA in this proband.

Loci influencing blood pressure identified using a cardiovascular gene-centric array.

Blood pressure (BP) is a heritable determinant of risk for cardiovascular disease (CVD). To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP) and pulse pressure (PP), we genotyped ∼50 000 single-nucleotide polymorphisms (SNPs) that capture variation in ∼2100 candidate genes for cardiovascular phenotypes in 61 619 individuals of European ancestry from cohort studies in the USA and Europe. We identified novel associations between rs347591 and SBP (chromosome 3p25.3, in an intron of HRH1) and between rs2169137 and DBP (chromosome1q32.1 in an intron of MDM4) and between rs2014408 and SBP (chromosome 11p15 in an intron of SOX6), previously reported to be associated with MAP. We also confirmed 10 previously known loci associated with SBP, DBP, MAP or PP (ADRB1, ATP2B1, SH2B3/ATXN2, CSK, CYP17A1, FURIN, HFE, LSP1, MTHFR, SOX6) at array-wide significance (P < 2.4 × 10(-6)). We then replicated these associations in an independent set of 65 886 individuals of European ancestry. The findings from expression QTL (eQTL) analysis showed associations of SNPs in the MDM4 region with MDM4 expression. We did not find any evidence of association of the two novel SNPs in MDM4 and HRH1 with sequelae of high BP including coronary artery disease (CAD), left ventricular hypertrophy (LVH) or stroke. In summary, we identified two novel loci associated with BP and confirmed multiple previously reported associations. Our findings extend our understanding of genes involved in BP regulation, some of which may eventually provide new targets for therapeutic intervention.

Large-scale gene-centric meta-analysis across 32 studies identifies multiple lipid loci.

Genome-wide association studies (GWASs) have identified many SNPs underlying variations in plasma-lipid levels. We explore whether additional loci associated with plasma-lipid phenotypes, such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TGs), can be identified by a dense gene-centric approach. Our meta-analysis of 32 studies in 66,240 individuals of European ancestry was based on the custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) covering ∼2,000 candidate genes. SNP-lipid associations were replicated either in a cohort comprising an additional 24,736 samples or within the Global Lipid Genetic Consortium. We identified four, six, ten, and four unreported SNPs in established lipid genes for HDL-C, LDL-C, TC, and TGs, respectively. We also identified several lipid-related SNPs in previously unreported genes: DGAT2, HCAR2, GPIHBP1, PPARG, and FTO for HDL-C; SOCS3, APOH, SPTY2D1, BRCA2, and VLDLR for LDL-C; SOCS3, UGT1A1, BRCA2, UBE3B, FCGR2A, CHUK, and INSIG2 for TC; and SERPINF2, C4B, GCK, GATA4, INSR, and LPAL2 for TGs. The proportion of explained phenotypic variance in the subset of studies providing individual-level data was 9.9% for HDL-C, 9.5% for LDL-C, 10.3% for TC, and 8.0% for TGs. This large meta-analysis of lipid phenotypes with the use of a dense gene-centric approach identified multiple SNPs not previously described in established lipid genes and several previously unknown loci. The explained phenotypic variance from this approach was comparable to that from a meta-analysis of GWAS data, suggesting that a focused genotyping approach can further increase the understanding of heritability of plasma lipids.

Large-scale gene-centric meta-analysis across 39 studies identifies type 2 diabetes loci.

To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.

Meta-analysis of Dense Genecentric Association Studies Reveals Common and Uncommon Variants Associated with Height.

Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10(-6)), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10(-8)). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10(-11)). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait.