Multiple Hepatic Regulatory Variants at the GALNT2 GWAS Locus Associated with High-Density Lipoprotein Cholesterol.

Genome-wide association studies (GWASs) have identified more than 150 loci associated with blood lipid and cholesterol levels; however, the functional and molecular mechanisms for many associations are unknown. We examined the functional regulatory effects of candidate variants at the GALNT2 locus associated with high-density lipoprotein cholesterol (HDL-C). Fine-mapping and conditional analyses in the METSIM study identified a single locus harboring 25 noncoding variants (r(2) > 0.7 with the lead GWAS variants) strongly associated with total cholesterol in medium-sized HDL (e.g., rs17315646, p = 3.5 × 10(-12)). We used luciferase reporter assays in HepG2 cells to test all 25 variants for allelic differences in regulatory enhancer activity. rs2281721 showed allelic differences in transcriptional activity (75-fold [T] versus 27-fold [C] more than the empty-vector control), as did a separate 780-bp segment containing rs4846913, rs2144300, and rs6143660 (49-fold [AT(-) haplotype] versus 16-fold [CC(+) haplotype] more). Using electrophoretic mobility shift assays, we observed differential CEBPB binding to rs4846913, and we confirmed this binding in a native chromatin context by performing chromatin-immunoprecipitation (ChIP) assays in HepG2 and Huh-7 cell lines of differing genotypes. Additionally, sequence reads in HepG2 DNase-I-hypersensitivity and CEBPB ChIP-seq signals spanning rs4846913 showed significant allelic imbalance. Allelic-expression-imbalance assays performed with RNA from primary human hepatocyte samples and expression-quantitative-trait-locus (eQTL) data in human subcutaneous adipose tissue samples confirmed that alleles associated with increased HDL-C are associated with a modest increase in GALNT2 expression. Together, these data suggest that at least rs4846913 and rs2281721 play key roles in influencing GALNT2 expression at this HDL-C locus.

Genetic association with lipids in Filipinos: waist circumference modifies an APOA5 effect on triglyceride levels.

Blood levels of lipoprotein cholesterol and triglycerides (TGs) are highly heritable and are major risk factors for cardiovascular disease (CVD). Approximately 100 lipid-associated loci have been identified in populations of European ancestry. We performed a genome-wide association study of lipid traits in 1,782 Filipino women from the Cebu Longitudinal Health and Nutrition Survey, and tested for evidence of interactions with waist circumference. We conducted additional association and interaction analyses in 1,719 of their young adult offspring. Genome-wide significant associations (P < 5 × 10⁻8) were detected at APOE for low density lipoprotein cholesterol and total cholesterol, and at APOA5 for TGs. Suggestive associations (P < 10⁻6) were detected at GCKR for TGs, and at CETP and TOM1 for high density lipoprotein cholesterol. Our data also supported the existence of allelic heterogeneity at APOA5, CETP, LIPC, and APOE. The secondary signal (Gly185Cys) at APOA5 exhibited a single nucleotide polymorphism (SNP)-by-waist circumference interaction affecting TGs (Pinteraction = 1.6 × 10⁻4), manifested by stronger SNP effects as waist circumference increased. These findings provide the first evidence that central obesity may accentuate the effect of the TG-increasing allele of the APOA5 signal, emphasizing that CVD risk could be reduced by central obesity control.

Increased uptake of [¹²³I]meta-iodobenzylguanidine, [¹8F]fluorodopamine, and [³H]norepinephrine in mouse pheochromocytoma cells and tumors after treatment with the histone deacetylase inhibitors.

[¹³¹I]meta-iodobenzylguanidine ([¹³¹I]MIBG) is the most commonly used treatment for metastatic pheochromocytoma and paraganglioma. It enters the chromaffin cells via the membrane norepinephrine transporter; however, its success has been modest. We studied the ability of histone deacetylase (HDAC) inhibitors to enhance [¹²³I]MIBG uptake by tumors in a mouse metastatic pheochromocytoma model. HDAC inhibitors are known to arrest growth, induce differentiation and apoptosis in various cancer cells, and further inhibit tumor growth. We report the in vitro and in vivo effects of two HDAC inhibitors, romidepsin and trichostatin A, on the uptake of [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine in a mouse model of metastatic pheochromocytoma. The effects of both inhibitors on norepinephrine transporter activity were assessed in mouse pheochromocytoma (MPC) cells by using the transporter-blocking agent desipramine and the vesicular-blocking agent reserpine. HDAC inhibitors increased [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine uptake through the norepinephrine transporter in MPC cells. In vivo, inhibitor treatment resulted in significantly increased uptake of [(18)F]fluorodopamine positron emission tomography (PET) in pheochromocytoma liver metastases (19.1 ± 3.2% injected dose per gram of tumor (%ID/g) compared to liver metastases in pretreatment scans 5.9 ± 0.6%; P<0.001). Biodistribution analysis after inhibitors treatment confirmed the PET results. The uptake of [(123)I]MIBG was significantly increased in liver metastases 9.5 ± 1.1% compared to 3.19 ± 0.4% in untreated control liver metastases (P<0.05). We found that HDAC inhibitors caused an increase in the amount of norepinephrine transporter expressed in tumors. HDAC inhibitors may enhance the therapeutic efficacy of [(131)I]MIBG treatment in patients with advanced malignant pheochromocytoma and paraganglioma.

Genome-wide association study of anthropometric traits and evidence of interactions with age and study year in Filipino women.

Increased values of multiple adiposity-related anthropometric traits are important risk factors for many common complex diseases. We performed a genome-wide association (GWA) study for four quantitative traits related to body size and adiposity (BMI, weight, waist circumference, and height) in a cohort of 1,792 adult Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). This is the first GWA study of anthropometric traits in Filipinos, a population experiencing a rapid transition into a more obesogenic environment. In addition to identifying suggestive evidence of additional single-nucleotide polymorphism (SNP) association signals (P < 10(-5)), we replicated (P < 0.05, same direction of additive effect) associations previously reported in European populations of both BMI and weight with MC4R and FTO, of BMI with BDNF, and of height with EFEMP1, ZBTB38, and NPPC, but none with waist circumference. We also replicated loci reported in Japanese or Korean populations as associated with BMI (OTOL1) and height (HIST1H1PS2, C14orf145, GPC5). A difference in local linkage disequilibrium (LD) between European and Asian populations suggests a narrowed association region for BDNF, while still including a proposed functional nonsynonymous amino acid substitution variant (rs6265, Val66Met). Finally, we observed significant evidence (P < 0.0042) for age-by-genotype interactions influencing BMI for rs17782313 (MC4R) and rs9939609 (FTO), and for a study year-by-genotype interaction for rs4923461 (BDNF). Our results show that several genetic risk factors are associated with anthropometric traits in Filipinos and provide further insight into the effects of BDNF, FTO, and MC4R on BMI.

Genome-wide association study of homocysteine levels in Filipinos provides evidence for CPS1 in women and a stronger MTHFR effect in young adults.

Plasma homocysteine (Hcy) level is associated with cardiovascular disease and may play an etiologic role in vascular damage, a precursor for atherosclerosis. We performed a genome-wide association study for Hcy in 1786 unrelated Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). The most strongly associated single-nucleotide polymorphism (SNP) (rs7422339, P = 4.7 x 10(-13)) encodes Thr1405Asn in the gene CPS1 and explained 3.0% of variation in the Hcy level. The widely studied MTHFR C677T SNP (rs1801133) was also highly significant (P = 8.7 x 10(-10)) and explained 1.6% of the trait variation. We also genotyped these two SNPs in 1679 CLHNS young adult offspring. The MTHFR C677T SNP was strongly associated with Hcy (P = 1.9 x 10(-26)) and explained approximately 5.1% of the variation in the offspring. In contrast, the CPS1 variant was significant only in females (P = 0.11 in all; P = 0.0087 in females). Combined analysis of all samples confirmed that the MTHFR variant was more strongly associated with Hcy in the offspring (interaction P = 1.2 x 10(-5)). Furthermore, although there was evidence for a positive synergistic effect between the CPS1 and MTHFR SNPs in the offspring (interaction P = 0.0046), there was no significant evidence for an interaction in the mothers (P = 0.55). These data confirm a recent finding that CPS1 is a locus influencing Hcy levels in women and suggest that genetic effects on Hcy may differ across developmental stages.

Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes.

Genome-wide association (GWA) studies have identified multiple loci at which common variants modestly but reproducibly influence risk of type 2 diabetes (T2D). Established associations to common and rare variants explain only a small proportion of the heritability of T2D. As previously published analyses had limited power to identify variants with modest effects, we carried out meta-analysis of three T2D GWA scans comprising 10,128 individuals of European descent and approximately 2.2 million SNPs (directly genotyped and imputed), followed by replication testing in an independent sample with an effective sample size of up to 53,975. We detected at least six previously unknown loci with robust evidence for association, including the JAZF1 (P = 5.0 x 10(-14)), CDC123-CAMK1D (P = 1.2 x 10(-10)), TSPAN8-LGR5 (P = 1.1 x 10(-9)), THADA (P = 1.1 x 10(-9)), ADAMTS9 (P = 1.2 x 10(-8)) and NOTCH2 (P = 4.1 x 10(-8)) gene regions. Our results illustrate the value of large discovery and follow-up samples for gaining further insights into the inherited basis of T2D.

Association of FTO with obesity-related traits in the Cebu Longitudinal Health and Nutrition Survey (CLHNS) Cohort.

OBJECTIVE: The underlying genetic component of obesity-related traits is not well understood, and there is limited evidence to support genetic association shared across multiple studies, populations, and environmental contexts. The present study investigated the association between candidate variants and obesity-related traits in a sample of 1,886 adult Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS) cohort. RESEARCH DESIGN AND METHODS: We selected and genotyped 19 single nucleotide polymorphisms in 10 genes (ADRB2, ADRB3, FTO, GNB3, INSIG2, LEPR, PPARG, TNF, UCP2, and UCP3) that had been previously reported to be associated with an obesity-related quantitative trait. RESULTS: We observed evidence for association of the A allele of rs9939609 (FTO intron 1) with increased BMI (P = 0.0072 before multiple test correction), baseline BMI (P = 0.0015), longitudinal BMI based on eight surveys from 1983 to 2005 (P = 0.000029), waist circumference (P = 0.0094), and weight (P = 0.021). The increase in average BMI was approximately 0.4 for each additional A allele. We also observed association of the ADRB3 Trp64Arg variant with BMI, waist circumference, percent body fat, weight, fat mass, arm fat area, and arm muscle area (P < 0.05), although the direction of effect is inconsistent with the majority of previous reports. CONCLUSIONS: Our study confirms that FTO is a common obesity susceptibility gene in Filipinos, with an effect size similar to that seen in samples of European origin.

Comparison of ENCODE region SNPs between Cebu Filipino and Asian HapMap samples.

Patterns of linkage disequilibrium (LD) act as the framework for designing efficient association studies; these patterns are being studied and catalogued by The International HapMap Project. The current study assessed the transferability of tag SNPs chosen from HapMap panels to a cohort of 80 individuals from metro Cebu, Philippines, who participated in the Cebu Longitudinal Health and Nutrition Survey (CLHNS). The analyses focused on 627 single nucleotide polymorphisms (SNPs) in the central 40 kb within each of the 10 HapMap ENCODE regions. The similarity between the genetic variants in Cebu Filipino samples and HapMap panels was examined using allele frequency estimates, measures of pairwise linkage disequilibrium (LD), and haplotype frequency estimates. For these measures, strong correlations were observed between the Cebu Filipino samples and the Asian panels from HapMap, with the strongest correlations observed with the Han Chinese from Beijing (CHB) panel. Tag SNPs selected using the HapMap CHB panel were particularly effective at representing the genetic variation in Cebu Filipino samples. These results suggest that the HapMap data will be an effective resource for future studies in Cebu Filipino samples.