Genome-wide meta-analysis of variant-by-diuretic interactions as modulators of lipid traits in persons of European and African ancestry.

Hypertension (HTN) is a significant risk factor for cardiovascular morbidity and mortality. Metabolic abnormalities, including adverse cholesterol and triglycerides (TG) profiles, are frequent comorbid findings with HTN and contribute to cardiovascular disease. Diuretics, which are used to treat HTN and heart failure, have been associated with worsening of fasting lipid concentrations. Genome-wide meta-analyses with 39,710 European-ancestry (EA) individuals and 9925 African-ancestry (AA) individuals were performed to identify genetic variants that modify the effect of loop or thiazide diuretic use on blood lipid concentrations. Both longitudinal and cross sectional data were used to compute cohort-specific interaction results, which were then combined through meta-analysis in each ancestry. These ancestry-specific results were further combined through trans-ancestry meta-analysis. Analysis of EA data identified two genome-wide significant (p < 5 × 10-8) loci with single nucleotide variant (SNV)-loop diuretic interaction on TG concentrations (including COL11A1). Analysis of AA data identified one genome-wide significant locus adjacent to BMP2 with SNV-loop diuretic interaction on TG concentrations. Trans-ancestry analysis strengthened evidence of association for SNV-loop diuretic interaction at two loci (KIAA1217 and BAALC). There were few significant SNV-thiazide diuretic interaction associations on TG concentrations and for either diuretic on cholesterol concentrations. Several promising loci were identified that may implicate biologic pathways that contribute to adverse metabolic side effects from diuretic therapy.

Whole-genome copy number variation analysis in anophthalmia and microphthalmia.

Anophthalmia/microphthalmia (A/M) represent severe developmental ocular malformations. Currently, mutations in known genes explain less than 40% of A/M cases. We performed whole-genome copy number variation analysis in 60 patients affected with isolated or syndromic A/M. Pathogenic deletions of 3q26 (SOX2) were identified in four independent patients with syndromic microphthalmia. Other variants of interest included regions with a known role in human disease (likely pathogenic) as well as novel rearrangements (uncertain significance). A 2.2-Mb duplication of 3q29 in a patient with non-syndromic anophthalmia and an 877-kb duplication of 11p13 (PAX6) and a 1.4-Mb deletion of 17q11.2 (NF1) in two independent probands with syndromic microphthalmia and other ocular defects were identified; while ocular anomalies have been previously associated with 3q29 duplications, PAX6 duplications, and NF1 mutations in some cases, the ocular phenotypes observed here are more severe than previously reported. Three novel regions of possible interest included a 2q14.2 duplication which cosegregated with microphthalmia/microcornea and congenital cataracts in one family, and 2q21 and 15q26 duplications in two additional cases; each of these regions contains genes that are active during vertebrate ocular development. Overall, this study identified causative copy number mutations and regions with a possible role in ocular disease in 17% of A/M cases.

Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency.

Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.

Comparison of genome sequencing and clinical genotyping for pharmacogenes.

We compared whole exome sequencing (WES, n = 176 patients) and whole genome sequencing (WGS, n = 68) and clinical genotyping (DMET array-based approach) for interrogating 13 genes with Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. We focused on 127 CPIC important variants: 103 single nucleotide variations (SNV), 21 insertion/deletions (Indel), HLA-B alleles, and two CYP2D6 structural variations. WES and WGS provided interrogation of nonoverlapping sets of 115 SNV/Indels with call rate >98%. Among 68 loci interrogated by both WES and DMET, 64 loci (94.1%, confidence interval [CI]: 85.6-98.4%) showed no discrepant genotyping calls. Among 66 loci interrogated by both WGS and DMET, 63 loci (95.5%, CI: 87.2-99.0%) showed no discrepant genotyping calls. In conclusion, even without optimization to interrogate pharmacogenetic variants, WES and WGS displayed potential to provide reliable interrogation of most pharmacogenes and further validation of genome sequencing in a clinical lab setting is warranted.

Lack of association between a polymorphism of the aldosterone synthase gene and left ventricular structure.

BACKGROUND: Cardiac growth and function may be modulated in part by trophic effects of neurohormones. Specifically, aldosterone has been shown to stimulate the growth of cardiac myocytes and the accumulation of cardiac extracellular matrix proteins. Moreover, a variant of the aldosterone synthase gene (a cytosine/thymidine exchange at position -344 in the transcriptional regulatory region) has been associated with enlargement and disturbed filling of the left ventricle (LV) in a small sample of young white adults. The aim of the present study was to reinvestigate the implications of aldosterone synthase -344C/T allele status for serum aldosterone levels, blood pressure, and LV structure and function in large population-based samples. METHODS AND RESULTS: Individuals who participated in the echocardiographic substudy of the third MONICA (MONitoring trends and determinants in CArdiovascular disease) survey (n=1445) or in the second follow-up of the first MONICA survey (n=562) were studied by standardized anthropometric, echocardiographic, and biochemical measurements as well as genotyping for aldosterone synthase -344C/T allele status. In both surveys, the distribution of sex, age, arterial blood pressure, and body mass index was homogeneous in the aldosterone synthase genotype groups. Echocardiographic LV wall thicknesses, dimensions, and mass indexes were not significantly associated with a specific aldosterone synthase genotype. Likewise, no association was detectable with echocardiographic measures of LV systolic or diastolic function. Data were consistent in both samples and not materially different in subgroups defined by age, sex, or intake of antihypertensive medication. Finally, no significant association was observed for aldosterone synthase allele status and serum aldosterone levels in the group of 562 individuals. CONCLUSIONS: The data are not in favor of a significant contribution of the C/T exchange at position -344 in the aldosterone synthase transcriptional regulatory region to the variability of serum aldosterone levels, blood pressure, or cardiac size or function as found in 2 white population-based samples.

The associations of body size and body composition with left ventricular mass: impacts for indexation in adults.

OBJECTIVES: We investigated the relationship between body size, body composition and left ventricular mass (LVM) in adults, and assessed the impact of different indexations of LVM on its associations with gender, adiposity and blood pressure. BACKGROUND: The best way to normalize LVM for body size to appropriately distinguish physiologic adaptation from morbid heart morphology was discussed. METHODS: We undertook a community survey of 653 men and 718 women, aged 25 to 74 years. Lean body mass (LBM) was determined by bioelectric impedance analyses and LVM was assessed by two-dimensional guided M-mode echocardiography. RESULTS: After traditional indexations to body height, body height2.7, or body surface area, men had higher LVM than women (p < 0.001). These gender differences disappeared (p > 0.05) when LVM was indexed to LBM. The type of indexation also modified the strength of the association between adiposity and LVM. The estimated impact of body fat on LVM indexed to LBM was less than half that obtained with traditional indexations. In contrast, the magnitude of the associations of blood pressure with LVM was entirely independent of the type of indexation. CONCLUSIONS: This study showed the prominent influence of body composition on adult heart size. Indexation for LBM removed gender differences for LVM and reduced the impact of adiposity, but left the effects of blood pressure unchanged. We suggest that this approach be used for clinical and research applications.

Clinical, genetic and environmental factors associated with congenital vertebral malformations.

Congenital vertebral malformations (CVM) pose a significant health problem because they can be associated with spinal deformities, such as congenital scoliosis and kyphosis, in addition to various syndromes and other congenital malformations. Additional information remains to be learned regarding the natural history of congenital scoliosis and related health problems. Although significant progress has been made in understanding the process of somite formation, which gives rise to vertebral bodies, there is a wide gap in our understanding of how genetic factors contribute to CVM development. Maternal diabetes during pregnancy most commonly contributes to the occurrence of CVM, followed by other factors such as hypoxia and anticonvulsant medications. This review highlights several emerging clinical issues related to CVM, including pulmonary and orthopedic outcome in congenital scoliosis. Recent breakthroughs in genetics related to gene and environment interactions associated with CVM development are discussed. The Klippel-Feil syndrome which is associated with cervical segmentation abnormalities is illustrated as an example in which animal models, such as the zebrafish, can be utilized to provide functional evidence of pathogenicity of identified mutations.

QTL-based association analyses reveal novel genes influencing pleiotropy of metabolic syndrome (MetS).

OBJECTIVE: Metabolic Syndrome (MetS) is a phenotype cluster predisposing to type 2 diabetes and cardiovascular disease. We conducted a study to elucidate the genetic basis underlying linkage signals for multiple representative traits of MetS that we had previously identified at two significant QTLs on chromosomes 3q27 and 17p12. DESIGN AND METHODS: We performed QTL-specific genomic and transcriptomic analyses in 1,137 individuals from 85 extended families that contributed to the original linkage. We tested in SOLAR association of MetS phenotypes with QTL-specific haplotype-tagging SNPs as well as transcriptional profiles of peripheral blood mononuclear cells (PBMCs). RESULTS: SNPs significantly associated with MetS phenotypes under the prior hypothesis of linkage mapped to seven genes at 3q27 and seven at 17p12. Prioritization based on biologic relevance, SNP association, and expression analyses identified two genes: insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) at 3q27 and tumor necrosis factor receptor 13B (TNFRSF13B) at 17p12. Prioritized genes could influence cell-cell adhesion and adipocyte differentiation, insulin/glucose responsiveness, cytokine effectiveness, plasma lipid levels, and lipoprotein densities. CONCLUSIONS: Using an approach combining genomic, transcriptomic, and bioinformatic data we identified novel candidate genes for MetS.

Whole-exome sequencing and an iPSC-derived cardiomyocyte model provides a powerful platform for gene discovery in left ventricular hypertrophy.

RATIONALE: Left ventricular hypertrophy (LVH) is a heritable predictor of cardiovascular disease, particularly in blacks. OBJECTIVE: Determine the feasibility of combining evidence from two distinct but complementary experimental approaches to identify novel genetic predictors of increased LV mass. METHODS: Whole-exome sequencing (WES) was conducted in seven African-American sibling trios ascertained on high average familial LV mass indexed to height (LVMHT) using Illumina HiSeq technology. Identified missense or nonsense (MS/NS) mutations were examined for association with LVMHT using linear mixed models adjusted for age, sex, body weight, and familial relationship. To functionally assess WES findings, human induced pluripotent stem cell-derived cardiomyocytes (induced pluripotent stem cell-CM) were stimulated to induce hypertrophy; mRNA sequencing (RNA-seq) was used to determine gene expression differences associated with hypertrophy onset. Statistically significant findings under both experimental approaches identified LVH candidate genes. Candidate genes were further prioritized by seven supportive criteria that included additional association tests (two criteria), regional linkage evidence in the larger HyperGEN cohort (one criterion), and publically available gene and variant based annotations (four criteria). RESULTS: WES reads covered 91% of the target capture region (of size 37.2 MB) with an average coverage of 65×. WES identified 31,426 MS/NS mutations among the 21 individuals. A total of 295 MS/NS variants in 265 genes were associated with LVMHT with q-value <0.25. Of the 265 WES genes, 44 were differentially expressed (P < 0.05) in hypertrophied cells. Among the 44 candidate genes identified, 5, including HLA-B, HTT, MTSS1, SLC5A12, and THBS1, met 3 of 7 supporting criteria. THBS1 encodes an adhesive glycoprotein that promotes matrix preservation in pressure-overload LVH. THBS1 gene expression was 34% higher in hypertrophied cells (P = 0.0003) and a predicted conserved and damaging NS variant in exon 13 (A2099G) was significantly associated with LVHMT (P = 4 × 10(-6)). CONCLUSION: Combining evidence from cutting-edge genetic and cellular experiments can enable identification of novel LVH risk loci.

Concordance of DMET plus genotyping results with those of orthogonal genotyping methods.

There are several hurdles to the clinical implementation of pharmacogenetics. One approach is to employ pre-prescription genotyping, involving interrogation of multiple pharmacogenetic variants using a high-throughput platform. We compared the performance of the Drug Metabolizing Enzymes and Transporters (DMET) Plus array (1,931 variants in 225 genes) with that of orthogonal genotyping methods in 220 pediatric patients. A total of 1,692 variants had call rates >98% and were in Hardy-Weinberg equilibrium. Of these, 259 were genotyped by at least one independent method, and a total of 19,942 single-nucleotide polymorphism (SNP)-patient sample pairs were evaluated. The concordance rate was 99.9%, with only 28 genotype discordances observed. For the genes deemed most likely to be clinically relevant (TPMT, CYP2D6, CYP2C19, CYP2C9, VKORC1, DPYD, UGT1A1, and SLCO1B1), a total of 3,799 SNP-patient sample pairs were evaluable and had a concordance rate of 99.96%. We conclude that the DMET Plus array performs well with primary patient samples, with the results in good concordance with those of several lower-throughput genotyping methods.

Genome-wide meta-analyses of smoking behaviors in African Americans.

The identification and exploration of genetic loci that influence smoking behaviors have been conducted primarily in populations of the European ancestry. Here we report results of the first genome-wide association study meta-analysis of smoking behavior in African Americans in the Study of Tobacco in Minority Populations Genetics Consortium (n = 32,389). We identified one non-coding single-nucleotide polymorphism (SNP; rs2036527[A]) on chromosome 15q25.1 associated with smoking quantity (cigarettes per day), which exceeded genome-wide significance (ß = 0.040, s.e. = 0.007, P = 1.84 × 10(-8)). This variant is present in the 5'-distal enhancer region of the CHRNA5 gene and defines the primary index signal reported in studies of the European ancestry. No other SNP reached genome-wide significance for smoking initiation (SI, ever vs never smoking), age of SI, or smoking cessation (SC, former vs current smoking). Informative associations that approached genome-wide significance included three modestly correlated variants, at 15q25.1 within PSMA4, CHRNA5 and CHRNA3 for smoking quantity, which are associated with a second signal previously reported in studies in European ancestry populations, and a signal represented by three SNPs in the SPOCK2 gene on chr10q22.1. The association at 15q25.1 confirms this region as an important susceptibility locus for smoking quantity in men and women of African ancestry. Larger studies will be needed to validate the suggestive loci that did not reach genome-wide significance and further elucidate the contribution of genetic variation to disparities in cigarette consumption, SC and smoking-attributable disease between African Americans and European Americans.

Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

The future of genetic case-control studies.

The case-control study design has been a veritable workhorse in epidemiological research since its inception and acceptance as a valid and valued field of inquiry. The reasons for this owe to the simplicity of the required sampling and the (potential) ease of analysis and interpretation of results. Unfortunately, there are a number of problems that plague the use of the case-control design in assessing relationships between genetic variation and disease susceptibility in the population at large. Many of these problems are entirely analogous to problems that inhere in applications of the case-control design in nongenetic settings. These problems include stratification, the assessment of statistical significance, heterogeneity, and the interpretation of multiple outcomes or phenotypic information. In this chapter we describe 10 problems thought to plague genetic case-control studies and offer potential solutions to each. Many of our proposed solutions require the use of multiple DNA markers to accommodate the genetic background of the individuals sampled as cases and controls. It is hoped that our discussions and proposals will spark further debate about the analysis and ultimate utility of the case-control study in genetic epidemiology research.

Prevalence of left ventricular diastolic dysfunction in the community. Results from a Doppler echocardiographic-based survey of a population sample.

AIMS: The prevalence of left ventricular diastolic abnormalities in the general population is largely unclear. Thus, the aim of this study was, firstly, to identify abnormal diastolic function by echocardiography in an age-stratified population-based European sample (MONICA Augsburg, n=1274, 25 to 75 years, mean 51+/-14) and, secondly, to analyse clinical and anthropometric parameters associated with diastolic abnormalities. METHODS AND RESULTS: The overall prevalence of diastolic abnormalities, as defined by the European Study Group on Diastolic Heart Failure (i.e. age dependent isovolumic relaxation time (92-105 ms) and early (E-wave) and late (A-wave) left ventricular filling (E/A-ratio, 1-0.5)) was 11.1%. When only subjects treated with diuretics or with left atrial enlargement were considered (suggesting diastolic dysfunction) the prevalence was 3.1%. The prevalence of diastolic abnormalities varied according to age: from 2.8% in individuals aged 25-35 years to 15.8% among those older than 65 years (P<0.01). Significantly higher rates of diastolic abnormalities were observed in men as compared to women (13.8% vs 8.6%, P<0.01). Independent predictors of diastolic abnormalities were arterial hypertension, evidence of left ventricular (LV) hypertrophy, and coronary artery disease. Interestingly, in the absence of these predisposing conditions, diastolic abnormalities (4.3%) or diastolic dysfunction (1.1%) were rare, even in subjects older than 50 years of age (4.6%) and (1.2%), respectively. In addition to these factors, diastolic dysfunction was related to high body mass index, high body fat mass, and diabetes mellitus. CONCLUSION: The prevalences of diastolic abnormalities and diastolic dysfunction are higher than that of systolic dysfunction and are increased (despite age-dependent diagnostic criteria) in the elderly. However, in the absence of risk factors for diastolic abnormalities or diastolic dysfunction, namely LV hypertrophy, arterial hypertension, coronary artery disease, obesity and diabetes the condition is rare even in elderly subjects. These data allow speculation on whether diastolic heart failure may be prevented by improved implementation of measures directed against predisposing conditions.

Localization of a small genomic region associated with elevated ACE.

Defining the relationship between multiple polymorphisms in a small genomic region and an underlying quantitative trait locus (QTL) represents a major challenge in human genetics. Pedigree analyses have shown that angiotensin I-converting enzyme (ACE) levels are influenced by a QTL located within or close to the ACE gene and most likely resides in the 3' region of this locus. We genotyped seven polymorphisms spanning 13 kb in the 3' end of ACE in 159 Afro-Caribbean subjects to evaluate the linkage disequilibrium between these sites and to narrow the genomic region associated with an elevated ACE level using a cladistic analysis. The linkage disequilibrium measurement D' and a haplotype tree revealed three distinct haplotype segments, presumably because of recombination. The value of the linkage disequilibrium parameter p(excess) was highest for site 22982, which is located in the middle segment. A series of nested, cladistic analyses confirmed that the other two regions are unlikely to be the ACE-linked QTL and that the variant resides in the middle region. Analyses of the same polymorphisms in 98 unrelated Europeans in the Monitoring Trends and Determinants in Cardiovascular Diseases (MONICA) study resulted in fewer haplotypes than were observed among the Afro-Caribbean subjects, suggesting that populations with greater genetic diversity may be especially informative for fine-scale mapping.