Enhancer deletion and allelic effects define a regulatory molecular mechanism at the VLDLR cholesterol GWAS locus.

Total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are heritable risk factors for cardiovascular disease, yet the molecular mechanisms underlying the majority of blood lipid-associated genome-wide association studies signals remain elusive. One association signal is located in intron 3 of VLDLR; rs3780181-A is a risk allele associated (P ≤ 2 × 10-9) with increased TC and LDL-C. We investigated variants, genes and mechanisms underlying this association signal. We used a functional genetic approach to show that the intronic region spanning rs3780181 exhibited 1.6-7.6-fold enhancer activity in human HepG2 hepatocyte, THP-1 monocyte and Simpson-Golabi-Behmel Syndrome (SGBS) preadipocyte cells and that the rs3780181-A risk allele showed significantly less enhancer activity compared with the G allele, consistent with the direction of an expression quantitative trait locus in liver. In addition, rs3780181 alleles showed differential binding to multiple nuclear proteins, including stronger IRF2 binding to the rs3780181 G allele. We used a CRISPR-cas9 approach to delete 475 and 663 bp of the putative enhancer element in HEK293T kidney cells; compared to expression of mock-edited cell lines, the homozygous enhancer deletion cell lines showed 1.2-fold significantly (P < 0.04) decreased expression of VLDLR, as well as 1.5-fold decreased expression of SMARCA2, located 388 kb away. Together, these results identify an enhancer of VLDLR expression and suggest that altered binding of one or more factors bound to rs3780181 alleles decreases enhancer activity and reduces at least VLDLR expression, leading to increased TC and LDL-C.

Identification and functional analysis of glycemic trait loci in the China Health and Nutrition Survey.

To identify genetic contributions to type 2 diabetes (T2D) and related glycemic traits (fasting glucose, fasting insulin, and HbA1c), we conducted genome-wide association analyses (GWAS) in up to 7,178 Chinese subjects from nine provinces in the China Health and Nutrition Survey (CHNS). We examined patterns of population structure within CHNS and found that allele frequencies differed across provinces, consistent with genetic drift and population substructure. We further validated 32 previously described T2D- and glycemic trait-loci, including G6PC2 and SIX3-SIX2 associated with fasting glucose. At G6PC2, we replicated a known fasting glucose-associated variant (rs34177044) and identified a second signal (rs2232326), a low-frequency (4%), probably damaging missense variant (S324P). A variant within the lead fasting glucose-associated signal at SIX3-SIX2 co-localized with pancreatic islet expression quantitative trait loci (eQTL) for SIX3, SIX2, and three noncoding transcripts. To identify variants functionally responsible for the fasting glucose association at SIX3-SIX2, we tested five candidate variants for allelic differences in regulatory function. The rs12712928-C allele, associated with higher fasting glucose and lower transcript expression level, showed lower transcriptional activity in reporter assays and increased binding to GABP compared to the rs12712928-G, suggesting that rs12712928-C contributes to elevated fasting glucose levels by disrupting an islet enhancer, resulting in reduced gene expression. Taken together, these analyses identified multiple loci associated with glycemic traits across China, and suggest a regulatory mechanism at the SIX3-SIX2 fasting glucose GWAS locus.

Four novel mutations of the BCKDHA, BCKDHB and DBT genes in Iranian patients with maple syrup urine disease.

BACKGROUND: Maple syrup urine disease (MSUD) is a rare metabolic autosomal recessive disorder caused by dysfunction of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex. Mutations in the BCKDHA, BCKDHB and DBT genes are responsible for MSUD. The current study analyzed seven Iranian MSUD patients genetically and explored probable correlations between their genotype and phenotype. METHODS: The panel of genes, including BCKDHA, BCKDHB and DBT, was evaluated, using routine the polymerase chain reaction (PCR)-sequencing method. In addition, protein modeling (homology and threading modeling) of the deduced novel mutations was performed. The resulting structures were then analyzed, using state-of-the-art bioinformatics tools to better understand the structural and functional effects caused by mutations. RESULTS: Seven mutations were detected in seven patients, including four novel pathogenic mutations in BCKDHA (c.1198delA, c.629C>T), BCKDHB (c.652C>T) and DBT (c.1150A>G) genes. Molecular modeling of the novel mutations revealed clear changes in the molecular energy levels and stereochemical traits of the modeled proteins, which may be indicative of strong correlations with the functional modifications of the genes. Structural deficiencies were compatible with the observed phenotypes. CONCLUSIONS: Any type of MSUD can show heterogeneous clinical manifestations in different ethnic groups. Comprehensive molecular investigations would be necessary for differential diagnosis.

Trans-ancestry Fine Mapping and Molecular Assays Identify Regulatory Variants at the ANGPTL8 HDL-C GWAS Locus.

Recent genome-wide association studies (GWAS) have identified variants associated with high-density lipoprotein cholesterol (HDL-C) located in or near the ANGPTL8 gene. Given the extensive sharing of GWAS loci across populations, we hypothesized that at least one shared variant at this locus affects HDL-C. The HDL-C-associated variants are coincident with expression quantitative trait loci for ANGPTL8 and DOCK6 in subcutaneous adipose tissue; however, only ANGPTL8 expression levels are associated with HDL-C levels. We identified a 400-bp promoter region of ANGPTL8 and enhancer regions within 5 kb that contribute to regulating expression in liver and adipose. To identify variants functionally responsible for the HDL-C association, we performed fine-mapping analyses and selected 13 candidate variants that overlap putative regulatory regions to test for allelic differences in regulatory function. Of these variants, rs12463177-G increased transcriptional activity (1.5-fold, P = 0.004) and showed differential protein binding. Six additional variants (rs17699089, rs200788077, rs56322906, rs3760782, rs737337, and rs3745683) showed evidence of allelic differences in transcriptional activity and/or protein binding. Taken together, these data suggest a regulatory mechanism at the ANGPTL8 HDL-C GWAS locus involving tissue-selective expression and at least one functional variant.