Mol Genet Metab. 2016 Jun;118(2):128-37 

The Arg59Trp variant in ANGPTL8 (betatrophin) is associated with total and HDL-cholesterol in American Indians and Mexican Americans and differentially affects cleavage of ANGPTL3

Johanna K. DiStefano


We previously identified a locus linked to total cholesterol (TC) concentration in Pima Indians on chromosome 19p. To characterize this locus, we genotyped >2000 SNPs in 1838 Pimas and assessed association with log(TC). We observed evidence for association with log(TC) with rs2278426 (3.5% decrease/copy of the T allele; P=5.045 x 10−6) in the ANGPTL8 (angiopoietin-like 8) gene. We replicated this association in 2413 participants of the San Antonio Mexican American Family Study (SAMAFS: 2.0% decrease per copy of the T allele; P=0.005842). In a meta-analysis of the combined data, we found the strongest estimated effect with rs2278426 (P=2.563 x 10−7). The variant T allele at rs2278426 predicts an Arg59Trp substitution and has previously been associated with LDL-C and HDL-C. In Pimas and SAMAFS participants, the T allele of rs2278426 was associated with reduced HDL-C levels (P=0.000741 and 0.00002, respectively), and the combined estimated effect for the two cohorts was −3.8% (P=8.526 x 10−8). ANGPTL8 transcript and protein levels increased in response to both glucose and insulin. The variant allele was associated with increased levels of cleaved ANGPTL3. We conclude that individuals with the variant allele may have lower TC and HDL-C levels due to increased activation of ANGPTL3 by ANGPTL8.From 1965 to 2007, the Pima Indians from the Gila River Indian Community participated in a longitudinal survey. Approximately every 2 years, each resident ≥5 years of age was invited to receive a standardized medical examination (39). In addition to a 75 g glucose tolerance test, total fasting serum cholesterol and HDL-C concentrations were measured enzymatically (40), and measurements used in the genome scan were those taken from the last available examination for each individual (21). Assay accuracy was monitored and verified by the CDC Laboratory Program Office, the College of American Pathologists Surveys Program, or the American Association of Bioanalysts. The interassay coefficient of variation was 1.97% and 4.83% for TC and HDL-C measurements, respectively.Characteristics of the Pima Indian study sample are shown in Table 1. The sample included all individuals who participated in the genome-wide linkage study (n=912) and a random sample of participants in the longitudinal Pima study, excluding first-degree relatives (n=926). Inclusion criteria for the study sample were 1) age ≥20 years, 2) total cholesterol levels available at examination, and 3) known diabetes status. Individuals taking lipid medications were excluded from the study. Approvals were obtained from the Institutional Review Boards of the National Institute of Diabetes and Digestive and Kidney Diseases, and the Translational Genomics Research Institute. NIDDK gave regular reports of study findings to the Council of the Gila River Indian Community. All subjects provided written informed consent.We validated the most strongly associated DOCK6 and ANGPTL8 markers in a Mexican American cohort using family data obtained from two studies: the San Antonio Family Heart Study (SAFHS) and the San Antonio Family Diabetes/Gallbladder Study (SAFDGS). Details of the SAFHS (41, 42) and SAFDGS (43) have been published elsewhere. Together, the SAFHS and SAFDGS comprise the San Antonio Mexican American Family Study (SAMAFS), which represents approximately 2,500 individuals from about 80 Mexican American complex pedigrees. The clinical characteristics of the individuals that are part of this replication study are reported in Table 2. Genome-wide SNP genotypic data (~1 million SNPs acquired using the Illumina platform) were available for SAMAFS individuals (44). For validation studies, we performed association analysis between selected DOCK6 and ANGPTL8 markers that were most strongly associated with fasting serum TC in the Pima population and TC concentrations in SAMAFS data. TC concentrations used in these analyses were taken from measures obtained at the most recent clinical examination (Pimas) or based on information at the last study examination (SAMAFS).To narrow the 30 cM 1-LOD support interval of linkage for fasting serum TC concentration, we first genotyped 346 SNPs in Pima Indian families who participated in the original genome scan (21). Markers common to the Caucasian (CEU), African (YRI), Chinese (CHB), and Japanese (JPT) populations available in the HapMap database ( were selected based upon physical position using a minor allele frequency ≥0.10 and r2=0.80. Mean inter-marker distance was ~132 kb. Variance components linkage analysis, as implemented in the program Merlin (46), was performed. We observed resolution of the interval, with the peak of linkage at marker rs9807915 (Supplemental Fig. 1). The region of linkage was substantially narrowed to a 1-LOD support interval (rs1054623-rs10405035) of ~15.7 cM, which corresponds to a physical distance of ~6.8 Mb (chr 19:8,492,086-15,308,898).To determine the extent to which loci previously identified in various GWAS (27, 28, 30–32) contributed to TC concentration in Pima Indians, we also genotyped 11 markers (rs6511720, rs2228671, rs688, rs2228603, rs10401969, rs16996148, rs17545624, rs4803750, rs2075650, rs4420638, and rs16979595) showing statistically significant evidence for association in previously published studies. Of the genotyped markers, only rs10401969, located in the SURP And G Patch Domain Containing 1 (SUGP1) gene, was nominally associated with fasting TC concentration (P=0.0437). None of the remaining markers showed statistically significant evidence for association with this trait in Pima Indians.We next genotyped over 1800 markers spanning the refined linkage interval and assessed the evidence for association between genetic variants and log(TC) concentration. The results from the tests of general association for TC concentration for all genotyped SNPs are shown in Fig. 1. The strongest evidence for association with TC concentration was found with marker rs4804576, located in the gene encoding dedicator of cytokinesis 6 (DOCK6), which showed an decrease of 4.0% in TC concentration per copy of the A allele (P=2.0 x 10−7). We also observed evidence for association with marker rs2278426, located in the angiopoietin-like 8 gene (ANGPTL8), which showed a 3.5% decrease in TC concentration per copy of the T allele (P=5.0 x 10−6) and rs2116876, in DOCK6, which showed a 3.8% decrease per copy of the A allele; P=1.1 x 10−6. A summary of the association findings is shown in Table 3. The rs2278426 marker was not associated with diabetes (odds ratio=0.94 per copy of the T allele, P=0.44), nor was it associated with 2-hour post-load glucose concentration in nondiabetic individuals (P=0.38).To narrow the region of linkage, we selected a panel of 346 SNPs from the HapMap database, and genotyped them in the Pima Indian families who participated in the original genome scan. Markers common to all four populations available in the HapMap database (i.e., Caucasian, African, Chinese, and Japanese) were selected based upon physical position using a minor allele frequency ≥0.10 and r2=0.80. The solid line depicts the original region of linkage and the dotted line reflects the reduced linkage interval following additional SNP genotyping.This work was supported by the National Institutes of Health HL093042 (JKD) and the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (RLH, SK). The San Antonio Family Heart Study (SAFHS) and San Antonio Family Diabetes/Gallbladder Study (SAFDGS) were supported by NIH grants P01 HL045222, R01 DK047482, and R01 DK053889. We appreciate the technical assistance of Mark Zhang, Mahdieh Khosroheidari, and Matthew Taila at TGen. We thank Dr. William C. Knowler for his support and advice, the staff of the Phoenix Epidemiology and Clinical Research Branch for their significant contribution to this study, and all Pima study volunteers and participants of the SAFHS and SAFDGS for their cooperation.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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