Genome-wide association studies have identified numerous variants associated with lipid levels; yet, the majority are located in non-coding regions with unclear mechanisms. In the Insulin Resistance Atherosclerosis Family Study (IRASFS), heritability estimates suggest a strong genetic basis: low-density lipoprotein (LDL, h2 = 0.50), high-density lipoprotein (HDL, h2 = 0.57), total cholesterol (TC, h2 = 0.53), and triglyceride (TG, h2 = 0.42) levels. Exome sequencing of 1,205 Mexican Americans (90 pedigrees) from the IRASFS identified 548,889 variants and association and linkage analyses with lipid levels were performed. One genome-wide significant signal was detected in APOA5 with TG (rs651821, PTG = 3.67 × 10−10, LODTG = 2.36, MAF = 14.2%). In addition, two correlated SNPs (r2 = 1.0) rs189547099 (PTG = 6.31 × 10−08, LODTG = 3.13, MAF = 0.50%) and chr4:157997598 (PTG = 6.31 × 10−08, LODTG = 3.13, MAF = 0.50%) reached exome-wide significance (P < 9.11 × 10−08). rs189547099 is an intronic SNP in FNIP2 and SNP chr4:157997598 is intronic in GLRB. Linkage analysis revealed 46 SNPs with a LOD > 3 with the strongest signal at rs1141070 (LODLDL = 4.30, PLDL = 0.33, MAF = 21.6%) in DFFB. A total of 53 nominally associated variants (P < 5.00 × 10−05, MAF ≥ 1.0%) were selected for replication in six Mexican-American cohorts (N = 3,280). The strongest signal observed was a synonymous variant (rs1160983, PLDL = 4.44 × 10−17, MAF = 2.7%) in TOMM40. Beyond primary findings, previously reported lipid loci were fine-mapped using exome sequencing in IRASFS. These results support that exome sequencing complements and extends insights into the genetics of lipid levels.

Linkage studies of complex genetic diseases have been largely replaced by Genome-Wide Association studies (GWAS), due in part to limited success in complex trait discovery. However, recent interest in rare and low-frequency variants motivates reexamination of family-based methods. In this study we investigated the performance of two-point linkage analysis for over 1.6 million SNPs combined with single variant association analysis to identify high impact variants which are both strongly linked and associated with cardiometabolic traits in up to 1 414 Hispanics from the Insulin Resistance Atherosclerosis Family Study (IRASFS). Evaluation of all 50 phenotypes yielded 83 557 000 LOD scores with 9 214 LOD scores ≥ 3.0, 845 ≥ 4.0, and 89 ≥ 5.0, with a maximal LOD score of 6.49 (rs12956744 in the LAMA1 gene for TNFα receptor 2). Twenty-seven variants were associated with p < 0.005 as well as having a LOD score > 4, including variants in the NFIB gene under a linkage peak with TNFα receptor 2 levels on chromosome 9. Linkage regions of interest included a broad peak (31Mb) on chromosome 1q with acute insulin response (max LOD = 5.37). This region was previously documented with type 2 diabetes in family-based studies, providing support for the validity of these results. Overall, we have demonstrated the utility of two-point linkage and association in comprehensive genome-wide array-based SNP genotypes.

Summary Family-based methods are a potentially powerful tool to identify trait-defining genetic variants in extended families, particularly when used to complement conventional association analysis. We utilized two-point linkage analysis and single variant association analysis to evaluate whole exome sequencing (WES) data from 1,205 Hispanic Americans (78 families) from the Insulin Resistance Atherosclerosis Family Study. WES identified 211,612 variants above the minor allele frequency threshold of ≥0.005. These variants were tested for linkage and/or association with 50 cardiometabolic traits after quality control checks. Two-point linkage analysis yielded 10,580,600 LOD scores with 1,148 LOD scores ≥3, 183 LOD scores ≥4, and 29 LOD scores ≥5. The maximal novel LOD score was 5.50 for rs2289043:T>C, in UNC5C with subcutaneous adipose tissue volume. Association analysis identified 13 variants attaining genome-wide significance (p<5×10-08), with the strongest association between rs651821:C>T in APOA5, and triglyceride levels (p=3.67×10-10). Overall, there was a 5.2-fold increase in the number of informative variants detected by WES compared to exome chip analysis in this population, nearly 30% of which were novel variants relative to dbSNP build 138. Thus, integration of results from two-point linkage and single-variant association analysis from WES data enabled identification of novel signals potentially contributing to cardiometabolic traits.

Objective Adiponectin is found in human serum in three groups of multimers (high, medium, and low molecular weight). Previously, we reported two ethnic-specific variants in ADIPOQ, G45R (Hispanic Americans) and R55C (African Americans). Although carriers of both variants had mean adiponectin levels ≤20% of those of non-carriers, they were not clinically different from non-carriers. To compare carriers of both variants and non-carriers, relative quantification of adiponectin isoforms to total adiponectin was performed on serum samples. Methods The multimeric patterns of serum adiponectin in G45R carriers (n=23), R55C carriers (n=3), and Hispanic and African American non-carriers (n=84 and 44, respectively) from the IRAS Family Study were explored using native western blotting and densitometry. Results Serum samples from carriers showed an absence of the high molecular weight (HMW) isoform and a marked reduction in the medium molecular weight isoform but an approximate two-fold increase in the amount of the low molecular weight isoform (LMW). Thus, individuals making only LMW adiponectin are metabolically normal. Conclusions The results contrast with the proposed biological importance of the HMW multimer. This suggests that the LMW isoform may functionally compensate for some of the loss/reduction of the higher-order multimers in carriers of the G45R and R55C mutations.