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Statins are widely prescribed to lower plasma low-density lipoprotein (LDL) cholesterol levels. They also modestly reduce plasma triglyceride (TG), an independent cardiovascular disease risk factor, in most people. The mechanism and inter-individual variability of TG statin response is poorly understood. We measured statin-induced gene expression changes in lymphoblastoid cell lines derived from 150 participants of a simvastatin clinical trial and identified 23 genes (false discovery rate, FDR=15%) with expression changes correlated with plasma TG response. The correlation of insulin-induced gene 1 (INSIG1) expression changes with TG response (rho=0.32, q=0.11) was driven by men (interaction P=0.0055). rs73161338 was associated with INSIG1 expression changes (P=5.4 × 10-5) and TG response in two statin clinical trials (P=0.0048), predominantly in men. A combined model including INSIG1 expression level and splicing changes accounted for 29.5% of plasma TG statin response variance in men (P=5.6 × 10-6). Our results suggest that INSIG1 variation may contribute to statin-induced changes in plasma TG in a sex-specific manner.

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Variation in the expression level and activity of genes involved in drug disposition and action ('pharmacogenes') can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-sequencing (RNA-seq) to determine the expression levels and alternative splicing of 389 Pharmacogenomics Research Network pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines, which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20-45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with GTEx data yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use.

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Moyamoya disease (MMD) is a progressive vasculopathy characterized by occlusion of the terminal portion of the internal carotid arteries and its branches, and the formation of compensatory moyamoya collateral vessels. Homozygous mutations in GUCY1A3 have been reported as a cause of MMD and achalasia. Probands (n = 96) from unrelated families underwent sequencing of GUCY1A3. Functional studies were performed to confirm the pathogenicity of identified GUCY1A3 variants. Two affected individuals from the unrelated families were found to have compound heterozygous mutations in GUCY1A3. MM041 was diagnosed with achalasia at 4 years of age, hypertension and MMD at 18 years of age. MM149 was diagnosed with MMD and hypertension at the age of 20 months. Both individuals carry one allele that is predicted to lead to haploinsufficiency and a second allele that is predicted to produce a mutated protein. Biochemical studies of one of these alleles, GUCY1A3 Cys517Tyr, showed that the mutant protein (a subunit of soluble guanylate cyclase) has a significantly blunted signaling response with exposure to nitric oxide (NO). GUCY1A3 missense and haploinsufficiency mutations disrupt NO signaling leading to MMD and hypertension, with or without achalasia.

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Marfan syndrome (MFS) due to mutations in FBN1 is a known cause of thoracic aortic aneurysms and acute aortic dissections (TAAD) associated with pleiotropic manifestations. Genetic predisposition to TAAD can also be inherited in families in the absence of syndromic features, termed familial TAAD (FTAAD), and several causative genes have been identified to date. FBN1 mutations can also be identified in FTAAD families, but the frequency of these mutations has not been established. We performed exome sequencing of 183 FTAAD families and identified pathogenic FBN1 variants in five (2.7%) of these families. We also identified eight additional FBN1 rare variants that could not be unequivocally classified as disease-causing in six families. FBN1 sequencing should be considered in individuals with FTAAD even without significant systemic features of MFS.

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This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

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Autism spectrum disorder (ASD) has a strong but complex genetic component. Here we report on the resequencing of 64 candidate neurodevelopmental disorder risk genes in 5,979 individuals: 3,486 probands and 2,493 unaffected siblings. We find a strong burden of de novo point mutations for these genes and specifically implicate nine genes. These include CHD2 and SYNGAP1, genes previously reported in related disorders, and novel genes TRIP12 and PAX5. We also show that mutation carriers generally have lower IQs and enrichment for seizures. These data begin to distinguish genetically distinct subtypes of autism important for aetiological classification and future therapeutics.

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We describe here the design and initial implementation of the eMERGE-PGx project. eMERGE-PGx, a partnership of the Electronic Medical Records and Genomics Network and the Pharmacogenomics Research Network, has three objectives: (i) to deploy PGRNseq, a next-generation sequencing platform assessing sequence variation in 84 proposed pharmacogenes, in nearly 9,000 patients likely to be prescribed drugs of interest in a 1- to 3-year time frame across several clinical sites; (ii) to integrate well-established clinically validated pharmacogenetic genotypes into the electronic health record with associated clinical decision support and to assess process and clinical outcomes of implementation; and (iii) to develop a repository of pharmacogenetic variants of unknown significance linked to a repository of electronic health record-based clinical phenotype data for ongoing pharmacogenomics discovery. We describe site-specific project implementation and anticipated products, including genetic variant and phenotype data repositories, novel variant association studies, clinical decision support modules, clinical and process outcomes, approaches to managing incidental findings, and patient and clinician education methods.

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Efforts to define the genetic architecture underlying variable statin response have met with limited success, possibly because previous studies were limited to effect based on a single dose. We leveraged electronic medical records (EMRs) to extract potency (ED50) and efficacy (Emax) of statin dose-response curves and tested them for association with 144 preselected variants. Two large biobanks were used to construct dose-response curves for 2,026 and 2,252 subjects on simvastatin and atorvastatin, respectively. Atorvastatin was more efficacious, was more potent, and demonstrated less interindividual variability than simvastatin. A pharmacodynamic variant emerging from randomized trials (PRDM16) was associated with Emax for both. For atorvastatin, Emax was 51.7 mg/dl in subjects homozygous for the minor allele vs. 75.0 mg/dl for those homozygous for the major allele. We also identified several loci associated with ED50. The extraction of rigorously defined traits from EMRs for pharmacogenetic studies represents a promising approach to further understand the genetic factors contributing to drug response.

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Marked prolongation of the QT interval and polymorphic ventricular tachycardia following medication (drug-induced long QT syndrome, diLQTS) is a severe adverse drug reaction (ADR) that phenocopies congenital long QT syndrome (cLQTS) and is one of the leading causes for drug withdrawal and relabeling. We evaluated the frequency of rare non-synonymous variants in genes contributing to the maintenance of heart rhythm in cases of diLQTS using targeted capture coupled to next-generation sequencing. Eleven of 31 diLQTS subjects (36%) carried a novel missense mutation in genes with known congenital arrhythmia associations or with a known cLQTS mutation. In the 26 Caucasian subjects, 23% carried a highly conserved rare variant predicted to be deleterious to protein function in these genes compared with only 2-4% in public databases (P<0.003). We conclude that the rare variation in genes responsible for congenital arrhythmia syndromes is frequent in diLQTS. Our findings demonstrate that diLQTS is a pharmacogenomic syndrome predisposed by rare genetic variants.

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Toll-like receptor (TLR)-mediated innate immune responses are important in early host defense. Using a candidate gene approach, we previously identified genetic variation within TLR1 that is associated with hyper-responsiveness to a TLR1/2 agonist in vitro and with death and organ dysfunction in patients with sepsis. Here we report a genome-wide association study (GWAS) designed to identify genetic loci controlling whole blood cytokine responses to the TLR1/2 lipopeptide agonist, Pam(3)CSK(4) (N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)(4)) ex vivo. We identified a very strong association (P<1 × 10(-27)) between genetic variation within the TLR10/1/6 locus on chromosome 4, and Pam(3)CSK(4)-induced cytokine responses. This was the predominant association explaining over 35% of the population variance for this phenotype. Notably, strong associations were observed within TLR10, suggesting that genetic variation in TLR10 may influence bacterial lipoprotein-induced responses. These findings establish the TLR10/1/6 locus as the dominant common genetic factor controlling interindividual variability in Pam(3)CSK(4)-induced whole blood responses in the healthy population.

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Signatures of natural selection occur throughout the human genome and can be detected at the sequence level. We have re-sequenced ABCE1, a host candidate gene essential for HIV-1 capsid assembly, in European- (n=23) and African-descent (Yoruban; n=24) reference populations for genetic variation discovery. We identified an excess of rare genetic variation in Yoruban samples, and the resulting Tajima's D was low (-2.27). The trend of excess rare variation persisted in flanking candidate genes ANAPC10 and OTUD4, suggesting that this pattern of positive selection can be detected across the 184.5 kb examined on chromosome 4. Owing to ABCE1's role in HIV-1 replication, we re-sequenced the candidate gene in three small cohorts of HIV-1-infected or resistant individuals. We were able to confirm the excess of rare genetic variation among HIV-1-positive African-American individuals (n=53; Tajima's D=-2.34). These results highlight the potential importance of ABCE1's role in infectious diseases such as HIV-1.

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BACKGROUND: Associations between common F7 haplotypes, plasma factor VII (FVII) levels, and cardiovascular risk have recently been reported in population studies involving predominantly European men. METHODS: We assessed associations between F7 haplotypes and cardiovascular risk in two US population-based studies: a case-control study of these alleles related to a decreased risk of arterial thrombotic outcomes such as myocardial infarction (MI) in young-to-middle-aged women (n = 671), and a cohort study of cardiovascular disease risk factors in young women (n = 1040). RESULTS: The high-expression F7 haplotype B (containing the promoter variant allele -402A) was associated with an increased FVII level among controls, but not with MI risk. Women carrying a> or =1 copy of the low FVII expression level haplotype C (containing the -401T/-323del/-122C and Gln353 alleles) had decreased FVII levels and decreased risk of MI (odds ratio 0.54, 95% CI 0.31-0.93) compared with women homozygous for the most common haplotype A. Haplotype C was also associated with a decreased body mass index (BMI) and an increased high-density lipoprotein (HDL) cholesterol level, but not with MI risk after adjustment for these metabolic risk factors. In a cohort study composed of young US women, individuals homozygous for haplotype C had a lower BMI and lower systolic blood pressure, but the association between the F7 haplotype and HDL cholesterol was not confirmed. CONCLUSION: Common FVII haplotypes may contribute to the risk of MI in women, but the mechanisms appear complex. The association between F7 haplotypes and MI susceptibility may be mediated in part through an influence on atherogenic risk factors such as BMI.

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BACKGROUND: Previous genotype-phenotype association studies of fibrinogen have been limited by incomplete knowledge of genomic sequence variation within and between major ethnic groups in FGB, FGA, and FGG. METHODS: We characterized the linkage disequilibrium patterns and haplotype structure across the human fibrinogen gene locus in European- and African-American populations. We analyzed the association between common polymorphisms in the fibrinogen genes and circulating levels of both 'functional' fibrinogen (measured by the Clauss clotting rate method) and total fibrinogen (measured by immunonephelometry) in a large, multi-center, bi-racial cohort of young US adults. RESULTS: A common haplotype tagged by the A minor allele of the well-studied FGB-455 G/A promoter polymorphism (FGB 1437) was confirmed to be strongly associated with increased plasma fibrinogen levels. Two non-coding variants specific to African-American chromosomes, FGA 3845 A and FGG 5729 G, were each associated with lower plasma fibrinogen levels. In European-Americans, a common haplotype tagged by FGA Thr312Ala and several other variant alleles across the fibrinogen gene locus was strongly associated with decreased fibrinogen levels as measured by functional assay, but not by immunoassay. Overall, common polymorphisms within the three fibrinogen genes explain < 2% of the variability in plasma fibrinogen concentration. CONCLUSIONS: In young adults, fibrinogen multi-locus genotypes are associated with plasma fibrinogen levels. The specific single nucleotide polymorphism and haplotype patterns for these associations differ according to population and also according to phenotypic assay. It is likely that a substantial proportion of the heritable component of plasma fibrinogen concentration is due to genetic variation outside the three fibrinogen genes.

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The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association.

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More than a million genetic markers in the form of single nucleotide polymorphisms are now available for use in genotype-phenotype studies in humans. The application of new strategies for representational cloning and sequencing from genomes combined with the mining of high-quality sequence variations in clone overlaps of genomic and/or cDNA sequences has played an important role in generating this new resource. The focus of variation analysis is now shifting from the identification of new markers to their typing in populations, and novel typing strategies are rapidly emerging. Assay readouts on oligonucleotide arrays, in microtiter plates, gels, flow cytometers and mass spectrometers have all been developed, but decreasing cost and increasing throughput of DNA typing remain key if high-density genetic maps are to be applied on a large scale.

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This unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A Support Protocol describes the preparation of modified biotin- and digoxigenin-labeled oligonucleotide primers.

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