Ingrid B. Borecki

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease

BACKGROUND Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17‐beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10‐12) and AST (P=6.2×10‐10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk‐increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS A loss‐of‐function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.)

Heritable DNA Methylation in CD4+ Cells among Complex Families Displays Genetic and Non-Genetic Effects

DNA methylation at CpG sites is both heritable and influenced by environment, but the relative contributions of each to DNA methylation levels are unclear. We conducted a heritability analysis of CpG methylation in human CD4+ cells across 975 individuals from 163 families in the Genetics of Lipid-lowering Drugs and Diet Network (GOLDN). Based on a broad-sense heritability (H2) value threshold of 0.4, we identified 20,575 highly heritable CpGs among the 174,445 most variable autosomal CpGs (SD > 0.02). Tests for associations of heritable CpGs with genotype at 2,145,360 SNPs using 717 of 975 individuals showed that ~74% were cis-meQTLs (< 1 Mb away from the CpG), 6% of CpGs exhibited trans-meQTL associations (>1 Mb away from the CpG or located on a different chromosome), and 20% of CpGs showed no strong significant associations with genotype (based on a p-value threshold of 1e-7). Genes proximal to the genotype independent heritable CpGs were enriched for functional terms related to regulation of T cell activation. These CpGs were also among those that distinguished T cells from other blood cell lineages. Compared to genes proximal to meQTL-associated heritable CpGs, genotype independent heritable CpGs were moderately enriched in the same genomic regions that escape erasure during primordial germ cell development and could carry potential for generational transmission.

Multiethnic Exome-Wide Association Study of Subclinical Atherosclerosis

Background—The burden of subclinical atherosclerosis in asymptomatic individuals is heritable and associated with elevated risk of developing clinical coronary heart disease. We sought to identify genetic variants in protein-coding regions associated with subclinical atherosclerosis and the risk of subsequent coronary heart disease. Methods and Results—We studied a total of 25 109 European ancestry and African ancestry participants with coronary artery calcification (CAC) measured by cardiac computed tomography and 52 869 participants with common carotid intima–media thickness measured by ultrasonography within the CHARGE Consortium (Cohorts for Heart and Aging Research in Genomic Epidemiology). Participants were genotyped for 247 870 DNA sequence variants (231 539 in exons) across the genome. A meta-analysis of exome-wide association studies was performed across cohorts for CAC and carotid intima–media thickness. APOB p.Arg3527Gln was associated with 4-fold excess CAC (P=3×10−10). The APOE &egr;2 allele (p.Arg176Cys) was associated with both 22.3% reduced CAC (P=1×10−12) and 1.4% reduced carotid intima–media thickness (P=4×10−14) in carriers compared with noncarriers. In secondary analyses conditioning on low-density lipoprotein cholesterol concentration, the &egr;2 protective association with CAC, although attenuated, remained strongly significant. Additionally, the presence of &egr;2 was associated with reduced risk for coronary heart disease (odds ratio 0.77; P=1×10−11). Conclusions—Exome-wide association meta-analysis demonstrates that protein-coding variants in APOB and APOE associate with subclinical atherosclerosis. APOE &egr;2 represents the first significant association for multiple subclinical atherosclerosis traits across multiple ethnicities, as well as clinical coronary heart disease.

Rare Variation in TET2 Is Associated with Clinically Relevant Prostate Carcinoma in African-Americans

Background: Common variants have been associated with prostate cancer risk. Unfortunately, few are reproducibly linked to aggressive disease, the phenotype of greatest clinical relevance. One possible explanation is that rare genetic variants underlie a significant proportion of the risk for aggressive disease. Method: To identify such variants, we performed a two-stage approach using whole-exome sequencing followed by targeted sequencing of 800 genes in 652 aggressive prostate cancer patients and 752 disease-free controls in both African and European Americans. In each population, we tested rare variants for association using two gene-based aggregation tests. We established a study-wide significance threshold of 3.125 × 10−5 to correct for multiple testing. Results: TET2 in African Americans was associated with aggressive disease, with 24.4% of cases harboring a rare deleterious variant compared with 9.6% of controls (FET P = 1.84 × 10−5, OR = 3.0; SKAT-O P = 2.74 × 10−5). We report 8 additional genes with suggestive evidence of association, including the DNA repair genes PARP2 and MSH6. Finally, we observed an excess of rare truncation variants in 5 genes, including the DNA repair genes MSH6, BRCA1, and BRCA2. This adds to the growing body of evidence that DNA repair pathway defects may influence susceptibility to aggressive prostate cancer. Conclusions: Our findings suggest that rare variants influence risk of clinically relevant prostate cancer and, if validated, could serve to identify men for screening, prophylaxis, and treatment. Impact: This study provides evidence that rare variants in TET2 may help identify African American men at increased risk for clinically relevant prostate cancer. Cancer Epidemiol Biomarkers Prev; 25(11); 1456–63. ©2016 AACR.

A family-specific linkage analysis of blood lipid response to fenofibrate in the Genetics of Lipid Lowering Drug and Diet Network.

Cost-effective identification of novel pharmacogenetic variants remains a pressing need in the field. Using data from the Genetics of Lipid Lowering Drugs and Diet Network, we identified genomic regions of relevance to fenofibrate response in a sample of 173 families. Our approach included a multipoint linkage scan, followed by selection of the families showing evidence of linkage. We identified a strong signal for changes in LDL-cholesterol (LDL-C) on chromosome 7 (peak logarithm of odds score=4.76) in the full sample (n=821). The signal for LDL-C response remained even after adjusting for baseline LDL-C. Restricting analyses only to the families contributing to the linkage signal for LDL-C (N=19), we observed a peak logarithm of odds score of 5.17 for chromosome 7. Two genes under this peak (ABCB4 and CD36) were of biological interest. These results suggest that linked family analyses might be a useful approach to gene discovery in the presence of a complex (e.g. multigenic) phenotype.

A high throughput, functional screen of human Body Mass Index GWAS loci using tissue-specific RNAi Drosophila melanogaster crosses

Human GWAS of obesity have been successful in identifying loci associated with adiposity, but for the most part, these are non-coding SNPs whose function, or even whose gene of action, is unknown. To help identify the genes on which these human BMI loci may be operating, we conducted a high throughput screen in Drosophila melanogaster. Starting with 78 BMI loci from two recently published GWAS meta-analyses, we identified fly orthologs of all nearby genes (± 250KB). We crossed RNAi knockdown lines of each gene with flies containing tissue-specific drivers to knock down (KD) the expression of the genes only in the brain and the fat body. We then raised the flies on a control diet and compared the amount of fat/triglyceride in the tissue-specific KD group compared to the driver-only control flies. 16 of the 78 BMI GWAS loci could not be screened with this approach, as no gene in the 500-kb region had a fly ortholog. Of the remaining 62 GWAS loci testable in the fly, we found a significant fat phenotype in the KD flies for at least one gene for 26 loci (42%) even after correcting for multiple comparisons. By contrast, the rate of significant fat phenotypes in RNAi KD found in a recent genome-wide Drosophila screen (Pospisilik et al. (2010) is ~5%. More interestingly, for 10 of the 26 positive regions, we found that the nearest gene was not the one that showed a significant phenotype in the fly. Specifically, our screen suggests that for the 10 human BMI SNPs rs11057405, rs205262, rs9925964, rs9914578, rs2287019, rs11688816, rs13107325, rs7164727, rs17724992, and rs299412, the functional genes may NOT be the nearest ones (CLIP1, C6orf106, KAT8, SMG6, QPCTL, EHBP1, SLC39A8, ADPGK /ADPGK-AS1, PGPEP1, KCTD15, respectively), but instead, the specific nearby cis genes are the functional target (namely: ZCCHC8, VPS33A, RSRC2; SPDEF, NUDT3; PAGR1; SETD1, VKORC1; SGSM2, SRR; VASP, SIX5; OTX1; BANK1; ARIH1; ELL; CHST8, respectively). The study also suggests further functional experiments to elucidate mechanism of action for genes evolutionarily conserved for fat storage.

An exome-wide sequencing study of lipid response to high-fat meal and fenofibrate in Caucasians from the GOLDN cohort

Our understanding of genetic influences on the response of lipids to specific interventions is limited. In this study, we sought to elucidate effects of rare genetic variants on lipid response to a high-fat meal challenge and fenofibrate (FFB) therapy in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) cohort using an exome-wide sequencing-based association study. Our results showed that the rare coding variants in ITGA7, SIPA1L2, and CEP72 are significantly associated with fasting LDL cholesterol response to FFB (P = 1.24E-07), triglyceride postprandial area under the increase (AUI) (P = 2.31E-06), and triglyceride postprandial AUI response to FFB (P = 1.88E-06), respectively. We sought to replicate the association for SIPA1L2 in the Heredity and Phenotype Intervention (HAPI) Heart Study, which included a high-fat meal challenge but not FFB treatment. The associated rare variants in GOLDN were not observed in the HAPI Heart study, and thus the gene-based result was not replicated. For functional validation, we found that gene transcript level of SIPA1L2 is associated with triglyceride postprandial AUI (P < 0.05) in GOLDN. Our study suggests unique genetic mechanisms contributing to the lipid response to the high-fat meal challenge and FFB therapy.

A common haplotype lowers SPI1 (PU.1) expression in myeloid cells and delays age at onset for Alzheimer's disease

In this study we used age at onset of Alzheimer’s disease (AD), cerebrospinal fluid (CSF) biomarkers, and eQTL datasets to fine map AD-associated GWAS loci and investigate the underlying mechanisms. In a genome-wide survival analysis of 40,255 samples, eight of the previously reported AD risk loci are significantly (p < 5×10−8) or suggestively (p < 1×10−5) associated with age at onset-defined survival and a further fourteen novel loci reached suggestive significance. One third (8/22) of these SNPs are cis-eQTLs in monocytes and/or macrophages, including rs7930318 associated with expression of MS4A4A and MS4A6A. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, shows association with higher age at onset of AD (p=8.40×10−6), higher CSF levels of Aβ42 (p=1.2×10−4), and lower expression of SPI1 in monocytes (p = 1.50×10−105) and macrophages (p = 6.41×10−87). SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability is enriched within the SPI1 cistromes of monocytes and macrophages, implicating a myeloid PU.1 target gene network in the etiology of AD. Finally, experimentally altered PU.1 levels are correlated with phagocytic activity of BV2 mouse microglial cells and specific changes in the expression of multiple myeloid-expressed genes, including the mouse orthologs of AD risk genes, MS4A4A and MS4A6A. Our results collectively suggest that lower SPI1 expression reduces AD risk by modulating myeloid cell gene expression and function.