Bayesian Quantitative Trait Nucleotide Analysis Research Articles

Variants in CPT1A, FADS1, and FADS2 are Associated with Higher Levels of Estimated Plasma and Erythrocyte Delta-5 Desaturases in Alaskan Eskimos

The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10−28 and 10−5). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10−75 and 10−7) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.

Resequencing ofIRS2reveals rare variants for obesity but not fasting glucose homeostasis in Hispanic children

Our objective was to resequence insulin receptor substrate 2 (IRS2) to identify variants associated with obesity- and diabetes-related traits in Hispanic children. Exonic and intronic segments, 5' and 3' flanking regions of IRS2 (∼14.5 kb), were bidirectionally sequenced for single nucleotide polymorphism (SNP) discovery in 934 Hispanic children using 3730XL DNA Sequencers. Additionally, 15 SNPs derived from Illumina HumanOmni1-Quad BeadChips were analyzed. Measured genotype analysis tested associations between SNPs and obesity and diabetes-related traits. Bayesian quantitative trait nucleotide analysis was used to statistically infer the most likely functional polymorphisms. A total of 140 SNPs were identified with minor allele frequencies (MAF) ranging from 0.001 to 0.47. Forty-two of the 70 coding SNPs result in nonsynonymous amino acid substitutions relative to the consensus sequence; 28 SNPs were detected in the promoter, 12 in introns, 28 in the 3'-UTR, and 2 in the 5'-UTR. Two insertion/deletions (indels) were detected. Ten independent rare SNPs (MAF = 0.001-0.009) were associated with obesity-related traits (P = 0.01-0.00002). SNP 10510452_139 in the promoter region was shown to have a high posterior probability (P = 0.77-0.86) of influencing BMI, fat mass, and waist circumference in Hispanic children. SNP 10510452_139 contributed between 2 and 4% of the population variance in body weight and composition. None of the SNPs or indels were associated with diabetes-related traits or accounted for a previously identified quantitative trait locus on chromosome 13 for fasting serum glucose. Rare but not common IRS2 variants may play a role in the regulation of body weight but not an essential role in fasting glucose homeostasis in Hispanic children.

Genetic variation in APOJ, LPL, and TNFRSF10B affects plasma fatty acid distribution in Alaskan Eskimos

Alterations in plasma fatty acid distribution are linked to metabolic abnormalities related to type 2 diabetes and cardiovascular disease. The aim of this study was to investigate genetic factors influencing plasma fatty acid distribution in Alaskan Eskimos from the Genetics of Coronary Artery Disease in Alaska Natives (GOCADAN) study. Fatty acids in plasma were measured by gas chromatography in 761 related individuals (>35 y of age). Quantitative genetic analyses showed that fatty acid distribution is significantly heritable (P < 0.001), with heritabilities ranging from 0.33 to 0.55. A genome-wide scan for plasma fatty acids identified a 20-cM region on chromosome 8 (p12-p21) with a quantitative trait locus for monounsaturated fatty acids (logarithm of odds score = 3.8). The same region had a quantitative trait locus for polyunsaturated fatty acids (logarithm of odds score = 2.6). We genotyped single nucleotide polymorphisms (SNPs) in candidate genes in 8p12-p21 and found a significant association between fatty acids and SNPs in apolipoprotein J (APOJ), lipoprotein lipase (LPL), macrophage scavenger receptor 1 (MSR1), and tumor necrosis factor receptor superfamily member 10b (TNFRSF10B). A Bayesian quantitative trait nucleotide analysis based on a measured genotype model showed that SNPs in LPL, TNFRSF10B, and APOJ had strong statistical evidence of a functional effect (posterior probability > or =75%) on plasma fatty acid distribution. The results indicate that there is strong genetic influence on plasma fatty acid distribution and that genetic variation in APOJ, LPL, and TNFRSF10B may play a role. The GOCADAN study was registered at www.clinicaltrials.gov as NCT00006192.

Evidence that multiple genetic variants of MC4R play a functional role in the regulation of energy expenditure and appetite in Hispanic children

Melanocortin-4-receptor (MC4R) haploinsufficiency is the most common form of monogenic obesity; however, the frequency of MC4R variants and their functional effects in general populations remain uncertain. The aim was to identify and characterize the effects of MC4R variants in Hispanic children. MC4R was resequenced in 376 parents, and the identified single nucleotide polymorphisms (SNPs) were genotyped in 613 parents and 1016 children from the Viva la Familia cohort. Measured genotype analysis (MGA) tested associations between SNPs and phenotypes. Bayesian quantitative trait nucleotide (BQTN) analysis was used to infer the most likely functional polymorphisms influencing obesity-related traits. Seven rare SNPs in coding and 18 SNPs in flanking regions of MC4R were identified. MGA showed suggestive associations between MC4R variants and body size, adiposity, glucose, insulin, leptin, ghrelin, energy expenditure, physical activity, and food intake. BQTN analysis identified SNP 1704 in a predicted micro-RNA target sequence in the downstream flanking region of MC4R as a strong, probable functional variant influencing total, sedentary, and moderate activities with posterior probabilities of 1.0. SNP 2132 was identified as a variant with a high probability (1.0) of exerting a functional effect on total energy expenditure and sleeping metabolic rate. SNP rs34114122 was selected as having likely functional effects on the appetite hormone ghrelin, with a posterior probability of 0.81. This comprehensive investigation provides strong evidence that MC4R genetic variants are likely to play a functional role in the regulation of weight, not only through energy intake but through energy expenditure.

Quantitative Trait Nucleotide Analysis Using Bayesian Model Selection

Although much attention has been given to statistical genetic methods for the initial localization and fine mapping of quantitative trait loci (QTLs), little methodological work has been done to date on the problem of statistically identifying the most likely functional polymorphisms using sequence data. In this paper we provide a general statistical genetic framework, called Bayesian quantitative trait nucleotide (BQTN) analysis, for assessing the likely functional status of genetic variants. The approach requires the initial enumeration of all genetic variants in a set of resequenced individuals. These polymorphisms are then typed in a large number of individuals (potentially in families), and marker variation is related to quantitative phenotypic variation using Bayesian model selection and averaging. For each sequence variant a posterior probability of effect is obtained and can be used to prioritize additional molecular functional experiments. An example of this quantitative nucleotide analysis is provided using the GAW12 simulated data. The results show that the BQTN method may be useful for choosing the most likely functional variants within a gene (or set of genes). We also include instructions on how to use our computer program, SOLAR, for association analysis and BQTN analysis.

Quantitative Trait Nucleotide Analysis Using Bayesian Model Selection

Although much attention has been given to statistical genetic methods for the initial localization and fine mapping of quantitative trait loci (QTLs), little methodological work has been done to date on the problem of statistically identifying the most likely functional polymorphisms using sequence data. In this paper we provide a general statistical genetic framework, called Bayesian quantitative trait nucleotide (BQTN) analysis, for assessing the likely functional status of genetic variants. The approach requires the initial enumeration of all genetic variants in a set of resequenced individuals. These polymorphisms are then typed in a large number of individuals (potentially in families), and marker variation is related to quantitative phenotypic variation using Bayesian model selection and averaging. For each sequence variant a posterior probability of effect is obtained and can be used to prioritize additional molecular functional experiments. An example of this quantitative nucleotide analysis is provided using the GAW12 simulated data. The results show that the BQTN method may be useful for choosing the most likely functional variants within a gene (or set of genes). We also include instructions on how to use our computer program, SOLAR, for association analysis and BQTN analysis.

Association between variants in the genes for adiponectin and its receptors with insulin resistance syndrome (IRS)-related phenotypes in Mexican Americans

The aim of this study was to examine whether genetic variation in ADIPOQ, ADIPOR1 and ADIPOR2 may contribute to increased susceptibility to components of the insulin resistance syndrome (IRS). We genotyped single-nucleotide polymorphisms (SNPs) in ADIPOQ, ADIPOR1 and ADIPOR2 in Mexican American subjects (N=439) and performed an association analysis of IRS-related traits. Of the eight SNPs examined in the ADIPOQ gene, rs4632532 and rs182052 exhibited significant associations with BMI (p=0.029 and p=0.032), fasting specific insulin (p=0.023 and p=0.026), sum of skin folds (SS) (p=0.0089 and p=0.0084) and homeostasis model assessment of insulin sensitivity (HOMA-%S) (p=0.015 and p=0.016). Two other SNPs, rs266729 and rs2241767, were significantly associated with SS (p=0.036 and p=0.013). SNP rs7539542 of ADIPOR1 was significantly associated with BMI, SS and waist circumference (p=0.025, p=0.047 and p=0.0062). Fourteen of the ADIPOR2 SNPs were found to be significantly (p<0.05) associated with fasting plasma triglyceride concentrations. Four of these SNPs (rs10848569, rs929434, rs3809266 and rs12342) were in high pairwise linkage disequilibrium (r (2)=0.99) and were strongly associated with fasting triglyceride levels (p=0.00029, p=0.00016, p=0.00027 and p=0.00021). Adjusting for the effects of BMI and HOMA-%S on triglyceride concentrations increased significance to p=0.000060 for SNP rs929434. Bayesian quantitative trait nucleotide analysis was used to examine all possible models of gene action. Again, SNP rs929434 provided the strongest statistical evidence of an effect on triglyceride concentrations. These results provide evidence for association of SNPs in ADIPOQ and its receptors with multiple IRS-related phenotypes. Specifically, several genetic variants in ADIPOR2 were strongly associated with decreased triglyceride levels.

Genetic variation in selenoprotein S influences inflammatory response

Chronic inflammation has a pathological role in many common diseases and is influenced by both genetic and environmental factors. Here we assess the role of genetic variation in selenoprotein S (SEPS1, also called SELS or SELENOS), a gene involved in stress response in the endoplasmic reticulum and inflammation control. After resequencing SEPS1, we genotyped 13 SNPs in 522 individuals from 92 families. As inflammation biomarkers, we measured plasma levels of IL-6, IL-1beta and TNF-alpha. Bayesian quantitative trait nucleotide analysis identified associations between SEPS1 polymorphisms and all three proinflammatory cytokines. One promoter variant, -105G --> A, showed strong evidence for an association with each cytokine (multivariate P = 0.0000002). Functional analysis of this polymorphism showed that the A variant significantly impaired SEPS1 expression after exposure to endoplasmic reticulum stress agents (P = 0.00006). Furthermore, suppression of SEPS1 by short interfering RNA in macrophage cells increased the release of IL-6 and TNF-alpha. To investigate further the significance of the observed associations, we genotyped -105G --> A in 419 Mexican American individuals from 23 families for replication. This analysis confirmed a significant association with both TNF-alpha (P = 0.0049) and IL-1beta (P = 0.0101). These results provide a direct mechanistic link between SEPS1 and the production of inflammatory cytokines and suggest that SEPS1 has a role in mediating inflammation.

Effect of F7 Gene Promoter Polymorphisms on Factor VII Antigenic Levels.

Genetic variation in the F7 gene has been shown in several studies to influence quantitative variation in Factor VII antigenic levels. Previously in the GAIT Study, we comprehensively resequenced the F7 gene and obtained strong evidence of multiple functional variants. Two independent sets of highly correlated promoter polymorphisms appeared to contain at least two of these functional variants. However, the high degree of linkage disequilibrium within these two clusters precluded statistical identification of the most likely functional polymorphisms. In an attempt to further disentangle these highly correlated single nucleotide polymorphisms (SNPs), we performed an independent association study on 706 unrelated Spanish subjects enriched for arterial disease. Inclusion criteria included subjects with ischemic stroke or acute coronary artery disease and subjects without a personal history of arterial disease and from the same geographical area. Of these, 179 had acute coronary artery disease, 231 had ischemic stroke and 296 were subjects without personal history of arterial thromboembolic disease. For each individual, we measured Factor VII antigenic levels (FVIIag) using a standard ELISA method. Using an ABI 3100 automated sequencing platform, we typed each individuals for six promoter polymorphisms. These polymorphisms included SNPs observed in the 5′ proximal promoter region at positions −670, −630, −402, −401, and −323. As in our original observations, we observed two tight clusters of SNPs, {−670, −630, −402} and {−401, −323}, that exhibited strong intraset linkage disequilibrium.Robust measured genotype analysis, as implemented in the computer program, SOLAR, was used to assess the association between the F7 SNPs and FVIIag levels. Bayesian quantitative trait nucleotide analysis was used to calculate the Bayes Factors (which represent the relative evidence for one SNP over another)for each possible SNP within a linkage disequilibrium cluster. All analyses allowed for covariate effects such as sex and age. Each of the six promoter polymorphisms showed strong statistical association with levels of FVIIag. The observed associations were stable when analysis was limited to only normal subjects or to only subjects showing prior arterial disease. In Table 1, the variants and local sequence of each polymorphism is detailed as is the p-value showing the statistical evidence of association for each polymorphism with FVIIAg levels. Bayesian analysis suggested that the −402 and −323 polymorphisms best predicted the FVIIag phenotype (p value:3.2×10−18). Our results confirm in an Spanish population-based association study that these F7 promoter polymorphisms are in substantial linkage disequilibrium and show a strong association with levels of FVIIag, While the linkage disequilibrium within each of these SNP sets is very high and precludes unequivocal statistical identification of the true functional variants, our method has provided additional support for the −402 and −323 polymorphisms as being the most likely functional sites. This additional evidence now allows us to prioritize these polymorphisms for more expensive gold-standard molecular functional analyses.Table 1Nucleotide siteVariantSequenceP-value−670A/CccA/Cgcc4.8×10–8−630A/GaaA/Gca4.4× 10–7−401G/AcgG/Agt2.8×10–8−402G/TcgG/Tct7.7×10–14−323Ins 0/10agCCTATATCCTg4.6×10–14

Confirmation of a New Statistical Method for Comprehensively Dissecting QTLs Using In Vitro Transcription, the Model Factor VII Gene and a Novel Modification of the G-Free Cassette.

Thrombosis is a genetically complex disorder that plays a prominent pathogenic role in myocardial infarction, stroke and pulmonary embolism. Because these distinct endpoints of cardiovascular disease are the most common cause of death worldwide, the goal of investigators in several countries is to comprehensively catalogue human DNA polymorphisms influencing thrombosis susceptibility. It is now generally accepted that accomplishing this goal will require the use of linkage analysis and individuals from extended-pedigrees, even though previous investigations -- which predominantly were based on association analyses and case/control candidate gene studies -- resulted in several notable discoveries including the factor V Leiden mutation. In this regard, we recently completed the first genome linkage screen for DNA regions affecting normal variance of factor VII activity (fVII:C) levels in the 21 families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Only one quantitative trait locus (QTL) (LOD score=3.2; p-value=0.022) was identified and localized to the region of chr13q containing the fVII gene, F7. We performed the first comprehensive variation scan of this locus by resequencing the F7 genes in 40 unrelated GAIT subjects. The 49 polymorphisms identified were then genotyped in the full GAIT Project. Using a newly developed BQTN (Bayesian Quantitative Trait Nucleotide) method to estimate the posterior probability of a given variant being functional, we identified strong evidence for the functionality of 2 rare variants, including a novel SNP in intron 7 (C9830T) and a previously known SNP in the coding region of exon 8 (A294V). We also found 2 clusters of promoter (P) and intron 1 (I1) polymorphisms, designated C1: {A-630C, G-402A, T700C, G707A} and C2 {C-2989A, G-401T, -323ins0/10, T-122C, G73A, T835C}, within which all variants exhibit strong linkage disequilibrium (LD) and posterior probabilities of >90% for affecting fVII:C levels. In this study, we will use in vitro transcription to test the statistical findings of the BQTN analysis and, if confirmed, to specifically delineate the functional variant(s) in each cluster. We are creating G-free cassette (GfC) reporter constructs representing the natural P/I1 haplotypes statistically predicted as functional and 10 pairs of constructs where each pair represents the 2 alleles of each of the 10 variants in C1 and C2. All constructs have the same size P (0.9kb) and I1 (1kb) fragments ligated 5′ and 3′ of the GfC, respectively. Because C1 and C2 contain P and I1 variants, a new 120bp GfC was made to match the size of exon 1a and to approximate the native spacing of these polymorphisms in vivo. Once these constructs are sequenced, we will measure and compare their rate of transcriptional initiation using mouse liver nuclear extracts. Because of the strong LD among these P and I1 polymorphisms, population studies alone essentially only analyze the natural haplotypes that are present. The in vitro assays developed in this study will allow the specific influence of each polymorphism to be investigated in the context of a near native configuration of these known regulatory regions.