Cardiovascular disease (CVD) is the leading cause of death worldwide. Elevated low density lipoprotein cholesterol (LDL) and fibrinogen are major independent risk factors for CVD. Understanding their genetic basis may identify novel therapeutic targets to lower their levels and treat or prevent CVD.
Isolated founder populations can enable discovery of novel disease-associated variants enriched in these populations through genetic drift. Although very rare in the cosmopolitan populations, these novel variants can inform biology relevant to all humans.
To identify novel rare drifted alleles associated with CVD risk factors, we performed whole exome (n= 4,725) and genome sequencing (n=1113) on well phenotyped Amish subjects. We identified a strong novel association (p = 3.3E-18) between a missense SNP (N352S) in B4GALT1 and LDL. Each 352S allele is associated with a 14.7 mg/dl lower LDL, and has a frequency of 6% in the Amish while extremely rare in the general population. In addition, this SNP was associated with a 20% lower fibrinogen level (p= 5.0E-4), two-fold lower aspartate transaminase (AST) level (p= 3.0E-8), and 50% higher creatine kinase level (CPK) (p=0.02). These observations are consistent with coagulopathy and myopathy phenotypes reported in patients homozygous for rare protein-truncating mutations in B4GALT1. Notably, the 13 Amish 352S homozygotes appear to be healthy adults without any obvious phenotypic sequelae.
The enzyme encoded by B4GALT1 plays a critical role in processing of N-linked oligosaccharide moieties in glycoproteins. The N352S substitution resides very close to the catalytic site of the enzyme. Alterations in glycosylation have been reported to impair intracellular trafficking of various glycoproteins including the LDL receptor, and may also affect various circulating glycoproteins such as apolipoprotein B and fibrinogen.
As expected for a missense SNP we found no difference in the protein expression between the wild type and the mutant forms expressed in cultured Cos-7 cells. We are currently testing the hypothesis that the 352S allele will cause lower enzymatic activity. Morpholino knocked down b4galt1 zebrafish embryos had lower LDL levels, and that was reversed by injecting wild type b4galt1 mRNA. More functional assays are underway to better understand the biological impact of the protein glycosylation pathway on CVD risk factors and its therapeutic opportunities.

Lipoprotein(a), Lp(a), is a heritable, independent risk factor for coronary heart disease. Deep whole-genome sequencing permits an assessment of the full spectrum of genomic variation contributing to Lp(a) variation. Here, we associate variation in 30x whole-genome sequences from 2,255 Estonians from the Estonian Biobank and 2,937 African Americans from the Jackson Heart Study (JHS), with Lp(a) cholesterol (Lp(a)-Ch).
We replicate prior SNV associations at the LPA locus (min(p) = 1.2e-74) and observe two novel loci: SORT1 (min(p)=4.7e-23) and CETP (min(p)=3.0e-9) . Direct analysis of structural polymorphisms provides a new estimate of KIV2-CN (Mean:40,SD:7), higher than reported estimates from qPCR. We observe a strong negative association between Lp(a)-Ch and KIV2-CN in JHS (B=-0.05 SD per CN, p=9.3e-84) and Estonia (B=-0.05 SD per CN, p=1.5e-70). Step-wise conditional analyses at the LPA locus, after conditioning on KIV2-CN, identifies four independent SNVs in Estonia and three independent SNVs in JHS. Interaction analysis between variants at the LPA locus and KIV2-CN identifies 11 independent variants with significance in both studies. Mixed-model SKAT burden analysis using rare (MAF < 1%) variants identifies significance at LPA in aggregated nonsynonymous variants (p= 0.0095), and in aggregated HepG2 non-coding enhancer variants linked to LPA via gene expression (p= 0.0022).