Publications

Cigarette smoke is a strong risk factor for obesity and cardiovascular disease. The effect of genetic variants involved in nicotine metabolism on obesity or body composition has not been well studied. Though many genetic variants have previously been associated with adiposity or body fat distribution, a single variant usually confers a minimal individual risk. The goal of this study is to evaluate the joint association of multiple variants involved in cigarette smoke or nicotine dependence with obesity-related phenotypes in American Indians. To achieve this goal, we genotyped 61 tagSNPs in seven genes encoding nicotine acetylcholine receptors (nAChRs) in 3,665 American Indians participating in the Strong Heart Family Study. Single SNP association with obesity-related traits was tested using family-based association, adjusting for traditional risk factors including smoking. Joint association of all SNPs in the seven nAChRs genes were examined by gene-family analysis based on weighted truncated product method (TPM). Multiple testing was controlled by false discovery rate (FDR). Results demonstrate that multiple SNPs showed weak individual association with one or more measures of obesity, but none survived correction for multiple testing. However, gene-family analysis revealed significant associations with waist circumference (p = 0.0001) and waist-to-hip ratio (p = 0.0001), but not body mass index (p = 0.20) and percent body fat (p = 0.29), indicating that genetic variants are jointly associated with abdominal, but not general, obesity among American Indians. The observed combined genetic effect is independent of cigarette smoking per se. In conclusion, multiple variants in the nAChR gene family are jointly associated with abdominal obesity in American Indians, independent of general obesity and cigarette smoking per se.

BACKGROUND: In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown.

OBJECTIVE: The objective of this study is to compare BDNF concentrations of subjects with loss-of-function (LOF) and gain-of-function (GOF) MC4R variants with those of controls with common sequence MC4R.

METHODS: Circulating BDNF was measured in two cohorts with known MC4R sequence: 148 subjects of Pima Indian heritage ((mean±s.d.): age, 15.7±6.5 years; body mass index z-scores (BMI-Z), 1.63±1.03) and 69 subjects of Hispanic heritage (10.8±3.6 years; BMI-Z, 1.57±1.07). MC4R variants were characterized in vitro by cell surface expression, receptor binding and cyclic AMP response after agonist administration. BDNF single-nucleotide polymorphisms (SNPs) rs12291186, rs6265 and rs7124442 were also genotyped.

RESULTS: In the Pima cohort, no significant differences in serum BDNF was observed for 43 LOF subjects versus 65 LOF-matched controls (age, sex and BMI matched; P=0.29) or 20 GOF subjects versus 20 GOF-matched controls (P=0.40). Serum BDNF was significantly associated with genotype for BDNF rs12291186 (P=0.006) and rs6265 (P=0.009), but not rs7124442 (P=0.99); BDNF SNPs did not interact with MC4R status to predict serum BDNF. In the Hispanic cohort, plasma BDNF was not significantly different among 21 LOF subjects, 20 GOF subjects and 28 controls (P=0.79); plasma BDNF was not predicted by BDNF genotype or BDNF-x-MC4R genotype interaction.

CONCLUSIONS: Circulating BDNF concentrations were not significantly associated with MC4R functional status, suggesting that peripheral BDNF does not directly reflect hypothalamic BDNF secretion and/or that MC4R signaling is not a significant regulator of the bulk of BDNF expression in humans.

BACKGROUND: The association between human blood DNA global methylation and global hydroxymethylation has not been evaluated in population-based studies. No studies have evaluated environmental determinants of global DNA hydroxymethylation, including exposure to metals.

OBJECTIVE: We evaluated the association between global DNA methylation and global DNA hydroxymethylation in 48 Strong Heart Study participants for which selected metals had been measured in urine at baseline and DNA was available from 1989-1991 (visit 1) and 1998-1999 (visit 3).

METHODS: We measured the percentage of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in samples using capture and detection antibodies followed by colorimetric quantification. We explored the association of participant characteristics (i.e., age, adiposity, smoking, and metal exposure) with both global DNA methylation and global DNA hydroxymethylation.

RESULTS: The Spearman's correlation coefficient for 5-mC and 5-hmC levels was 0.32 (p = 0.03) at visit 1 and 0.54 (p < 0.001) at visit 3. Trends for both epigenetic modifications were consistent across potential determinants. In cross-sectional analyses, the odds ratios of methylated and hydroxymethylated DNA were 1.56 (95% CI: 0.95, 2.57) and 1.76 (95% CI: 1.07, 2.88), respectively, for the comparison of participants above and below the median percentage of dimethylarsinate. The corresponding odds ratios were 1.64 (95% CI: 1.02, 2.65) and 1.16 (95% CI: 0.70, 1.94), respectively, for the comparison of participants above and below the median cadmium level. Arsenic exposure and metabolism were consistently associated with both epigenetic markers in cross-sectional and prospective analyses. The positive correlation of 5-mC and 5-hmC levels was confirmed in an independent study population.

CONCLUSIONS: Our findings support that both epigenetic measures are related at the population level. The consistent trends in the associations between these two epigenetic modifications and the characteristics evaluated, especially arsenic exposure and metabolism, suggest the need for understanding which of the two measures is a better biomarker for environmental epigenetic effects in future large-scale epidemiologic studies.

BACKGROUND: Blood pressure (BP) is a complex trait, with a heritability of 30 to 40%. Several genome wide associated BP loci explain only a small fraction of the phenotypic variation. Family studies can provide an important tool for gene discovery by utilizing trait and genetic transmission information among relative-pairs. We have previously described a quantitative trait locus at chromosome 17q25.3 influencing systolic BP in American Indians of the Strong Heart Family Study (SHFS). This locus has been reported to associate with variation in BP traits in family studies of Europeans, African Americans and Hispanics.

METHODS: To follow-up persuasive linkage findings at this locus, we performed comprehensive genotyping in the 1-LOD unit support interval region surrounding this QTL using a multi-step strategy. We first genotyped 1,334 single nucleotide polymorphisms (SNPs) in 928 individuals from families that showed evidence of linkage for BP. We then genotyped a second panel of 306 SNPs in all SHFS participants (N = 3,807) for genes that displayed the strongest evidence of association in the region, and, in a third step, included additional genotyping to better cover the genes of interest and to interrogate plausible candidate genes in the region.

RESULTS: Three genes had multiple SNPs marginally associated with systolic BP (TBC1D16, HRNBP3 and AZI1). In BQTN analysis, used to estimate the posterior probability that any variant in each gene had an effect on the phenotype, AZI1 showed the most prominent findings (posterior probability of 0.66). Importantly, upon correction for multiple testing, none of our study findings could be distinguished from chance.

CONCLUSION: Our findings demonstrate the difficulty of follow-up studies of linkage studies for complex traits, particularly in the context of low powered studies and rare variants underlying linkage peaks.

The prevalence of type 2 diabetes (T2D) is rising rapidly and in Mexicans is  19%. T2D is affected by both environmental and genetic factors. Although specific genes have been implicated in T2D risk few of these findings are confirmed in studies of Mexican subjects. Our aim was to replicate associations of 39 single nucleotide polymorphisms (SNPs) from 10 genes with T2D-related phenotypes in a community-based Mexican cohort. Unrelated individuals (n = 259) living in southeastern Mexico were enrolled in the study based at the University of Yucatan School of Medicine in Merida. Phenotypes measured included anthropometric measurements, circulating levels of adipose tissue endocrine factors (leptin, adiponectin, pro-inflammatory cytokines), and insulin, glucose, and blood pressure. Association analyses were conducted by measured genotype analysis implemented in SOLAR, adapted for unrelated individuals. SNP Minor allele frequencies ranged from 2.2 to 48.6%. Nominal associations were found for CNR1, SLC30A8, GCK, and PCSK1 SNPs with systolic blood pressure, insulin and glucose, and for CNR1, SLC30A8, KCNJ11, and PCSK1 SNPs with adiponectin and leptin (p < 0.05). P-values greater than 0.0014 were considered significant. Association of SNPs rs10485170 of CNR1 and rs5215 of KCNJ11 with adiponectin and leptin, respectively, reached near significance (p = 0.002). Significant association (p = 0.001) was observed between plasma leptin and rs5219 of KCNJ11.

BACKGROUND: Coronary heart disease (CHD) is a leading cause of morbidity and mortality in African Americans. However, there is a paucity of studies assessing genetic determinants of CHD in African Americans. We examined the association of published variants in CHD loci with incident CHD, attempted to fine map these loci, and characterize novel variants influencing CHD risk in African Americans.

METHODS AND RESULTS: Up to 8,201 African Americans (including 546 first CHD events) were genotyped using the MetaboChip array in the Atherosclerosis Risk in Communities (ARIC) study and Women's Health Initiative (WHI). We tested associations using Cox proportional hazard models in sex- and study-stratified analyses and combined results using meta-analysis. Among 44 validated CHD loci available in the array, we replicated and fine-mapped the SORT1 locus, and showed same direction of effects as reported in studies of individuals of European ancestry for SNPs in 22 additional published loci. We also identified a SNP achieving array wide significance (MYC: rs2070583, allele frequency 0.02, P = 8.1 × 10(-8)), but the association did not replicate in an additional 8,059 African Americans (577 events) from the WHI, HealthABC and GeneSTAR studies, and in a meta-analysis of 5 cohort studies of European ancestry (24,024 individuals including 1,570 cases of MI and 2,406 cases of CHD) from the CHARGE Consortium.

CONCLUSIONS: Our findings suggest that some CHD loci previously identified in individuals of European ancestry may be relevant to incident CHD in African Americans.

Domestic cats have a unique breeding history and can be used as models for human hereditary and infectious diseases. In the current era of genome-wide association studies, insights regarding linkage disequilibrium (LD) are essential for efficient association studies. The objective of this study is to investigate the extent of LD in the domestic cat, Felis silvestris catus, particularly within its breeds. A custom illumina GoldenGate Assay consisting of 1536 single nucleotide polymorphisms (SNPs) equally divided over ten 1 Mb chromosomal regions was developed, and genotyped across 18 globally recognized cat breeds and two distinct random bred populations. The pair-wise LD descriptive measure (r(2)) was calculated between the SNPs in each region and within each population independently. LD decay was estimated by determining the non-linear least-squares of all pair-wise estimates as a function of distance using established models. The point of 50% decay of r(2) was used to compare the extent of LD between breeds. The longest extent of LD was observed in the Burmese breed, where the distance at which r(2) ≈ 0.25 was ∼380 kb, comparable to several horse and dog breeds. The shortest extent of LD was found in the Siberian breed, with an r(2) ≈ 0.25 at approximately 17 kb, comparable to random bred cats and human populations. A comprehensive haplotype analysis was also conducted. The haplotype structure of each region within each breed mirrored the LD estimates. The LD of cat breeds largely reflects the breeds' population history and breeding strategies. Understanding LD in diverse populations will contribute to an efficient use of the newly developed SNP array for the cat in the design of genome-wide association studies, as well as to the interpretation of results for the fine mapping of disease and phenotypic traits.

Infection with Epstein-Barr virus (EBV) is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV) nuclear antigen-1 (EBNA-1) IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA) region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10(-15) for SNPs rs477515 and rs2516049). Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis) also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen.

BACKGROUND: Atherosclerosis is the underlying cause of cardiovascular disease, the leading cause of morbidity and mortality in all American populations, including American Indians. Genetic factors play an important role in the pathogenesis of atherosclerosis. Although a single-nucleotide polymorphism (SNP) may explain only a small portion of variability in disease, the joint effect of multiple variants in a pathway on disease susceptibility could be large.

METHODS AND RESULTS: Using a gene-family analysis, we investigated the joint associations of 61 tag SNPs in 7 nicotinic acetylcholine receptor genes with subclinical atherosclerosis, as measured by carotid intima-media thickness and plaque score, in 3665 American Indians from 94 families recruited by the Strong Heart Family Study (SHFS). Although multiple SNPs showed marginal association with intima-media thickness and plaque score individually, only a few survived adjustments for multiple testing. However, simultaneously modeling of the joint effect of all 61 SNPs in 7 nicotinic acetylcholine receptor genes revealed significant association of the nicotinic acetylcholine receptor gene family with both intima-media thickness and plaque score independent of known coronary risk factors.

CONCLUSIONS: Genetic variants in the nicotinic acetylcholine receptor gene family jointly contribute to subclinical atherosclerosis in American Indians who participated in the SHFS. These variants may influence the susceptibility of atherosclerosis through pathways other than cigarette smoking per se.

BACKGROUND: Arsenic (III) methyltransferase (AS3MT) has been related to urine arsenic metabolites in association studies. Other genes might also play roles in arsenic metabolism and excretion.

OBJECTIVE: We evaluated genetic determinants of urine arsenic metabolites in American Indian adults from the Strong Heart Study (SHS).

METHODS: We evaluated heritability of urine arsenic metabolites [percent inorganic arsenic (%iAs), percent monomethylarsonate (%MMA), and percent dimethylarsinate (%DMA)] in 2,907 SHS participants with urine arsenic measurements and at least one relative within the cohort. We conducted a preliminary linkage analysis in a subset of 487 participants with available genotypes on approximately 400 short tandem repeat markers using a general pedigree variance component approach for localizing quantitative trait loci (QTL).

RESULTS: The medians (interquartile ranges) for %iAs, %MMA, and %DMA were 7.7% (5.4-10.7%), 13.6% (10.5-17.1%), and 78.4% (72.5-83.1%), respectively. The estimated heritability was 53% for %iAs, 50% for %MMA, and 59% for %DMA. After adjustment for sex, age, smoking, body mass index, alcohol consumption, region, and total urine arsenic concentrations, LOD [logarithm (to the base of 10) of the odds] scores indicated suggestive evidence for genetic linkage with QTLs influencing urine arsenic metabolites on chromosomes 5 (LOD = 2.03 for %iAs), 9 (LOD = 2.05 for %iAs and 2.10 for %MMA), and 11 (LOD = 1.94 for %iAs). A peak for %DMA on chromosome 10 within 2 Mb of AS3MT had an LOD of 1.80.

CONCLUSIONS: This population-based family study in American Indian communities supports a genetic contribution to variation in the distribution of arsenic metabolites in urine and, potentially, the involvement of genes other than AS3MT.