Legacy community science data suggest reduced beached litter in response to a container deposit scheme at a local scale.

Marine debris is causing significant environmental harm. Legislation is being implemented to reduce litter, including schemes like container deposit legislation that incentivize the return of commonly littered items for recycling. While there is a suggestion that these schemes reduce litter, no study has examined the long-term impact on the local environment before and after implementation. This study analyses community science data from 8 years prior to the implementation of a container deposit scheme, paired with 3 years of data afterwards, to assess the scheme's effectiveness at a local scale. Although using legacy datasets limits the generalizability of the conclusions compared to dedicated studies, the findings strongly indicate that container deposit schemes effectively manage targeted containers but have little impact on overall waste abundances. Long-term datasets like these are invaluable for assessing the impact of management efforts.

Identification and characterization of the long non-coding RNA NFIA-AS2 as a novel locus for body mass index in American Indians.

BACKGROUND: Genome-wide association studies have shown that body mass index (BMI), an estimate of obesity, is highly polygenic. Individual variants typically have small effect sizes, making it challenging to identify unique loci in under-represented ethnic groups which lack statistical power due to their small sample size. Yet obesity is a major health disparity and is particularly prevalent in southwestern American Indians. Here, we identify and characterize a new locus for BMI that was detected by analyzing moderate associations with BMI obtained in a population-based sample of southwestern American Indians together with the well-powered GIANT dataset. METHODS: Genotypes for 10.5 million variants were tested for association with BMI in 5870 American Indians and 2600 variants that showed an association P < 10-3 in the American Indian sample were combined in a meta-analysis with the BMI data reported in GIANT (N = 240,608). The newly identified gene, NFIA-AS2 was functionally characterized, and the impact of its lead associated variant rs1777538 was studied both in-silico and in-vitro. RESULTS: Rs1777538 (T/C; C allele frequency = 0.16 in American Indians and 0.04 in GIANT, meta-analysis P = 5.0 × 10-7) exhibited a large effect in American Indians (1 kg/m2 decrease in BMI per copy of C allele). NFIA-AS2 was found to be a nuclear localized long non-coding RNA expressed in tissues pertinent to human obesity. Analysis of this variant in human brown preadipocytes showed that NFIA-AS2 transcripts carrying the C allele had increased RNA degradation compared to the T allele transcripts (half-lives = 9 h, 13 h respectively). During brown adipogenesis, NFIA-AS2 featured a stage-specific regulation of nearby gene expression where rs1777538 demonstrated an allelic difference in regulation in the mature adipocytes (the strongest difference was observed for L1TD1, P = 0.007). CONCLUSION: Our findings support a role for NFIA-AS2 in regulating pathways that impact BMI.

Generation of Isogenic hiPSCs with Targeted Edits at Multiple Intronic SNPs to Study the Effects of the Type 2 Diabetes Associated KCNQ1 Locus in American Indians.

The top genetic association signal for type 2 diabetes (T2D) in Southwestern American Indians maps to intron 15 of KCNQ1, an imprinted gene. We aim to understand the biology whereby variation at this locus affects T2D specifically in this genomic background. To do so, we obtained human induced pluripotent stem cells (hiPSC) derived from American Indians. Using these iPSCs, we show that imprinting of KCNQ1 and CDKN1C during pancreatic islet-like cell generation from iPSCs is consistent with known imprinting patterns in fetal pancreas and adult islets and therefore is an ideal model system to study this locus. In this report, we detail the use of allele-specific guide RNAs and CRISPR to generate isogenic hiPSCs that differ only at multiple T2D associated intronic SNPs at this locus which can be used to elucidate their functional effects. Characterization of these isogenic hiPSCs identified a few aberrant cell lines; namely cell lines with large hemizygous deletions in the putative functional region of KCNQ1 and cell lines hypomethylated at the KCNQ1OT1 promoter. Comparison of an isogenic cell line with a hemizygous deletion to the parental cell line identified CDKN1C and H19 as differentially expressed during the endocrine progenitor stage of pancreatic-islet development.

Functional variants in cytochrome b5 type A (CYB5A) are enriched in Southwest American Indian individuals and associate with obesity.

OBJECTIVE: This study aimed to identify genetic variants enriched in Southwest American Indian (SWAI) individuals that associate with BMI. METHODS: Whole genome sequencing data (n = 296) were used to identify potentially functional variants that are common in SWAI individuals (minor allele frequency ≥10%) but rare in other ethnic groups (minor allele frequency < 0.1%). Enriched variants were tested for association with BMI in 5,870 SWAI individuals. One variant was studied using a luciferase reporter, and haplotypes that included this variant were analyzed for association with various measures of obesity (n = 917-5,870), 24-hour energy expenditure (24-h EE; n = 419), and skeletal muscle biopsy expression data (n = 207). RESULTS: A 5' untranslated region variant in cytochrome b5 type A (CYB5A), rs548402150, met the enrichment criteria and associated with increased BMI (ß = 2%, p = 0.004). Functionally, rs548402150 decreased luciferase expression by 30% (p = 0.003) and correlated with decreased skeletal muscle CYB5A expression (ß = -0.5 SD, p = 0.0008). Combining rs548402150 with two splicing quantitative trait loci in CYB5A identified a haplotype carried almost exclusively in SWAI individuals that associated with increased BMI (ß = 3%, p = 0.0003) and decreased CYB5A expression, whereas the most common haplotype in all ethnic groups associated with lower BMI and percentage of body fatness, increased 24-h EE, and increased CYB5A expression. CONCLUSIONS: Further studies on the effects of CYB5A on 24-h EE and BMI may provide insights into obesity-related physiology.

Assessing established BMI variants for a role in nighttime eating behavior in robustly phenotyped Southwestern American Indians.

BACKGROUND/OBJECTIVES: Nighttime eating (NE) behavior has a genetic component and predicts weight gain. We hypothesized that some genetic variants, which affect NE would also show an effect on body mass index (BMI). We aimed to determine which known BMI variants associate with NE in Southwestern American Indians (SWAIs), who are at elevated risk for obesity. METHODS: Known BMI variants from the GIANT-UK Biobank meta-analysis (N = 700,000) were analysed in SWAIs characterized for NE during an inpatient 3-day protocol. Variants were analysed for association with NE using whole-genome sequence data from 50 SWAIs (23 cases and 27 controls) and selected variants were genotyped in an additional 32 SWAIs (13 NE cases and 19 controls). Variants associated with NE in a meta-analysis of the two SWAI samples were further analysed for association with nightly caloric intake and functionality in hypothalamus, pituitary, and adrenal tissues. RESULTS: Variants were identified where the allele that associated with increased BMI in the GIANT-UK Biobank meta-analysis (P ≤ 1 × 10-8) also had a P < 0.01 for increased NE in the SWAI meta-analysis. These variants were captured by six tagSNPs. Comparison of the nightly calorie intake by genotype and eQTL data from relevant tissues highlighted rs3753612 upstream of HCRTR1. CONCLUSIONS: Our strategy led to the HCRTR1 locus, which has previously been linked to sleep regulation and feeding. Although this is an intriguing candidate gene for NE, further studies in larger samples and different populations are required to validate the role of HCRTR1 in NE.

Identification and functional validation of genetic variants in potential miRNA target sites of established BMI genes.

OBJECTIVE: MicroRNAs (miRNAs) play an important role in posttranscriptional regulation by binding to target sites in the 3'UTR of protein-coding genes. Genetic variation within target sites may potentially disrupt the binding activity of miRNAs, thereby impacting this regulation. In the current study, we investigated whether any established BMI-associated genetic variants potentially function by altering a miRNA target site. METHODS: The genomic positions of all predicted miRNA target site seed regions were identified, and these positions were queried in the T2D Knowledge Portal for variants that associated with BMI in the GIANT UK Biobank. This in silico analysis identified ten target site variants that associated with BMI with a P value ≤ 5 × 10-8. These ten variants mapped to nine genes, FAIM2, CCDC171, ADPGK, ZNF654, MLXIP, NT5C2, SHISA4, SLC25A22, and CTNNB1. RESULTS: In vitro functional analyses showed that five of these target site variants, rs7132908 (FAIM2), rs4963153 (SLC25A22), rs9460 (ADPGK), rs11191548 (NT5C2), and rs3008747 (CCDC171), disrupted the binding activity of miRNAs to their target in an allele-specific manner. CONCLUSION: In conclusion, our study suggests that some established variants for BMI may function by altering miRNA binding to a 3'UTR target site.

Functional and association analysis of an Amerindian-derived population-specific p.(Thr280Met) variant in RBPJL, a component of the PTF1 complex.

PTF1 complex is critical for pancreatic development and maintenance of adult exocrine pancreas. As a part of our ongoing studies to identify genetic variation that contributes to type 2 diabetes (T2D) in American Indians, we analyzed variation in genes that form this complex, namely PTF1A, RBPJ, and its paralogue RBPJL. A c.839C>T (p.(Thr280Met)) variant (rs200998587:C>T, risk allele frequency = 0.03) in RBPJL, identified only in Amerindian-derived populations, associated with T2D (OR = 1.60[1.21-2.13] per Met allele, P = 0.001) and age of diabetes onset (HR = 1.40[1.14-1.72], P = 0.001). Knockdown of Rbpjl in mouse pancreatic acinar cells resulted in a significant decrease in the mRNA expression of genes encoding exocrine enzymes including Ctrb. CTRB1/2 is an established T2D locus where the protective allele associates with increased GLP-1-stimulated insulin secretion and higher expression of CTRB1/2. In vitro studies show that cells expressing the Met280 allele had lower RBPJL protein levels than cells expressing the Thr280 allele, despite having comparable levels of RNA, suggesting that the Met280 RBPJL is less stable. Additionally, luciferase assays in HEK293 cells which examined two different RBPJL responsive promoters, including the promoter for CTRB1, also identified reduced transactivation by the Met280 RBPJL. Similarly, overexpression of both Met280 and Thr280 RBPJL in mouse pancreatic acinar cells identified a significant impairment in the expression of Cel when transactivated by the Met280 RBPJL. In summary, we identified a functional, Amerindian-derived population-specific c.839C>T (p.(Thr280Met)) variant in the pancreas specific RBPJL that may modify T2D risk by regulating exocrine enzyme expression.

Differential methylation of genes in individuals exposed to maternal diabetes in utero.

AIMS/HYPOTHESIS: Individuals exposed to maternal diabetes in utero are more likely to develop metabolic and cardiovascular diseases later in life. This may be partially attributable to epigenetic regulation of gene expression. We performed an epigenome-wide association study to examine whether differential DNA methylation, a major source of epigenetic regulation, can be observed in offspring of mothers with type 2 diabetes during the pregnancy (OMD) compared with offspring of mothers with no diabetes during the pregnancy (OMND). METHODS: DNA methylation was measured in peripheral blood using the Illumina HumanMethylation450K BeadChip. A total of 423,311 CpG sites were analysed in 388 Pima Indian individuals, mean age at examination was 13.0 years, 187 of whom were OMD and 201 were OMND. Differences in methylation between OMD and OMND were assessed. RESULTS: Forty-eight differentially methylated CpG sites (with an empirical false discovery rate ≤0.05), mapping to 29 genes and ten intergenic regions, were identified. The gene with the strongest evidence was LHX3, in which six CpG sites were hypermethylated in OMD compared with OMND (p ≤ 1.1 × 10-5). Similarly, a CpG near PRDM16 was hypermethylated in OMD (1.1% higher, p = 5.6 × 10-7), where hypermethylation also predicted future diabetes risk (HR 2.12 per SD methylation increase, p = 9.7 × 10-5). Hypermethylation near AK3 and hypomethylation at PCDHGA4 and STC1 were associated with exposure to diabetes in utero (AK3: 2.5% higher, p = 7.8 × 10-6; PCDHGA4: 2.8% lower, p = 3.0 × 10-5; STC1: 2.9% lower, p = 1.6 × 10-5) and decreased insulin secretory function among offspring with normal glucose tolerance (AK3: 0.088 SD lower per SD of methylation increase, p = 0.02; PCDHGA4: 0.08 lower SD per SD of methylation decrease, p = 0.03; STC1: 0.072 SD lower per SD of methylation decrease, p = 0.05). Seventeen CpG sites were also associated with BMI (p ≤ 0.05). Pathway analysis of the genes with at least one differentially methylated CpG (p < 0.005) showed enrichment for three relevant biological pathways. CONCLUSIONS/INTERPRETATION: Intrauterine exposure to diabetes can affect methylation at multiple genomic sites. Methylation status at some of these sites can impair insulin secretion, increase body weight and increase risk of type 2 diabetes.

Analysis of SLC16A11 Variants in 12,811 American Indians: Genotype-Obesity Interaction for Type 2 Diabetes and an Association With RNASEK Expression.

Genetic variants in SLC16A11 were recently reported to be associated with type 2 diabetes in Mexican and other Latin American populations. The diabetes risk haplotype had a frequency of 50% in Native Americans from Mexico but was rare in Europeans and Africans. In the current study, we analyzed SLC16A11 in 12,811 North American Indians and found that the diabetes risk haplotype, tagged by the rs75493593 A allele, was nominally associated with type 2 diabetes (P = 0.001, odds ratio 1.11). However, there was a strong interaction with BMI (P = 5.1 × 10(-7)) such that the diabetes association was stronger in leaner individuals. rs75493593 was also strongly associated with BMI in individuals with type 2 diabetes (P = 3.4 × 10(-15)) but not in individuals without diabetes (P = 0.77). Longitudinal analyses suggest that this is due, in part, to an association of the A allele with greater weight loss following diabetes onset (P = 0.02). Analyses of global gene expression data from adipose tissue, skeletal muscle, and whole blood provide evidence that rs75493593 is associated with expression of the nearby RNASEK gene, suggesting that RNASEK expression may mediate the effect of genotype on diabetes.

ABCC8 R1420H Loss-of-Function Variant in a Southwest American Indian Community: Association With Increased Birth Weight and Doubled Risk of Type 2 Diabetes.

Missense variants in KCNJ11 and ABCC8, which encode the KIR6.2 and SUR1 subunits of the ß-cell KATP channel, have previously been implicated in type 2 diabetes, neonatal diabetes, and hyperinsulinemic hypoglycemia of infancy (HHI). To determine whether variation in these genes affects risk for type 2 diabetes or increased birth weight as a consequence of fetal hyperinsulinemia in Pima Indians, missense and common noncoding variants were analyzed in individuals living in the Gila River Indian Community. A R1420H variant in SUR1 (ABCC8) was identified in 3.3% of the population (N = 7,710). R1420H carriers had higher mean birth weights and a twofold increased risk for type 2 diabetes with a 7-year earlier onset age despite being leaner than noncarriers. One individual homozygous for R1420H was identified; retrospective review of his medical records was consistent with HHI and a diagnosis of diabetes at age 3.5 years. In vitro studies showed that the R1420H substitution decreases KATP channel activity. Identification of this loss-of-function variant in ABCC8 with a carrier frequency of 3.3% affects clinical care as homozygous inheritance and potential HHI will occur in 1/3,600 births in this American Indian population.

A genome-wide association study in American Indians implicates DNER as a susceptibility locus for type 2 diabetes.

Most genetic variants associated with type 2 diabetes mellitus (T2DM) have been identified through genome-wide association studies (GWASs) in Europeans. The current study reports a GWAS for young-onset T2DM in American Indians. Participants were selected from a longitudinal study conducted in Pima Indians and included 278 cases with diabetes with onset before 25 years of age, 295 nondiabetic controls ≥45 years of age, and 267 siblings of cases or controls. Individuals were genotyped on a ∼1M single nucleotide polymorphism (SNP) array, resulting in 453,654 SNPs with minor allele frequency >0.05. SNPs were analyzed for association in cases and controls, and a family-based association test was conducted. Tag SNPs (n = 311) were selected for 499 SNPs associated with diabetes (P < 0.0005 in case-control analyses or P < 0.0003 in family-based analyses), and these SNPs were genotyped in up to 6,834 additional Pima Indians to assess replication. Rs1861612 in DNER was associated with T2DM (odds ratio = 1.29 per copy of the T allele; P = 6.6 × 10(-8), which represents genome-wide significance accounting for the number of effectively independent SNPs analyzed). Transfection studies in murine pancreatic ß-cells suggested that DNER regulates expression of notch signaling pathway genes. These studies implicate DNER as a susceptibility gene for T2DM in American Indians.

MAP2K3 is associated with body mass index in American Indians and Caucasians and may mediate hypothalamic inflammation.

To identify genes that affect body mass index (BMI) in American Indians who are predominately of Pima Indian heritage, we previously completed a genome-wide association study in 1120 American Indians. That study also included follow-up genotyping for 9 SNPs in 2133 additional subjects. A comprehensive follow-up study has subsequently been completed where 292 SNPs were genotyped in 3562 subjects, of which 128 SNPs were assessed for replication in 3238 additional subjects. In the combined subjects (n = 6800), BMI associations for two SNPs, rs12882548 and rs11652094, approached genome-wide significance (P = 6.7 × 10(-7) and 8.1 × 10(-7), respectively). Rs12882548 is located in a gene desert on chromosome 14 and rs11652094 maps near MAP2K3. Several SNPs in the MAP2K3 region including rs11652094 were also associated with BMI in Caucasians from the GIANT consortium (P = 10(-2)-10(-5)), and the combined P-values across both American Indians and Caucasian were P = 10(-4)-10(-9). Follow-up sequencing across MAP2K3 identified several paralogous sequence variants indicating that the region may have been duplicated. MAP2K3 expression levels in adipose tissue biopsies were positively correlated with BMI, although it is unclear if this correlation is a cause or effect. In vitro studies with cloned MAP2K3 promoters suggest that MAP2K3 expression may be up-regulated during adipogenesis. Microarray analyses of mouse hypothalamus cells expressing constitutively active MAP2K3 identified several up-regulated genes involved in immune/inflammatory pathways and a gene, Hap1, thought to play a role in appetite regulation. We conclude that MAP2K3 is a reproducible obesity locus that may affect body weight via complex mechanisms involving appetite regulation and hypothalamic inflammation.

Evidence for a role of LPGAT1 in influencing BMI and percent body fat in Native Americans.

OBJECTIVE: A genome-wide association study (GWAS) was recently completed in 1120 Pima Indians to identify loci that influence BMI. Among the top 100 signals were three variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases, which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Therefore LPGAT1 was analyzed as a candidate gene for obesity in Pima Indians. DESIGN AND METHODS: Variants (n = 26) located within and adjacent to LPGAT1 including a novel 27bp deletion in the 5'-untranslated region identified by sequencing were genotyped in a population-based sample of 3,391 full-heritage Pima Indians living in the Gila River Indian Community. Replication of selected variants was assessed in a second sample of 3,327 mixed-heritage Native Americans from the same community. RESULTS: Variants with nominal associations with BMI in each of the two independent samples (tagged by rs112662024 and rs12058008) had associations of P = 1-4 × 10(-5) in the combined sample (n = 6718). A haplotype that includes the novel 27bp deletion, which does not occur in Caucasians, showed the strongest association with BMI in the full-heritage Pima Indians. In vitro functional studies provided suggestive evidence that this 27bp deletion may affect transcriptional or posttranscriptional regulation. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide. CONCLUSIONS: LPGAT1 is a novel gene that influences BMI in Native Americans.

Identification of genetic variation that determines human trehalase activity and its association with type 2 diabetes.

A prior linkage scan in Pima Indians identified a putative locus for type two diabetes (T2D) and body mass index (BMI) on chromosome 11q23-25. Association mapping across this region identified single nucleotide polymorphisms (SNPs) in the trehalase gene (TREH) that were associated with T2D. To assess the putative connection between trehalase activity and T2D, we performed a linkage study for trehalase activity in 570 Pima Indians who had measures of trehalase activity. Strong evidence of linkage of plasma trehalase activity (LOD = 7.0) was observed in the TREH locus. Four tag SNPs in TREH were genotyped in these subjects and plasma trehalase activity was highly associated with three SNPs: rs2276064, rs117619140 and rs558907 (p = 2.2 × 10(-11)-1.4 × 10(-23)), and the fourth SNP, rs10790256, was associated conditionally on these three (p = 2.9 × 10(-7)). Together, the four tag SNPs explained 51 % of the variance in plasma trehalase activity and 79 % of the variance attributed to the linked locus. These four tag SNPs were further genotyped in 828 subjects used for association mapping of T2D, and rs558907 was associated with T2D (odds ratio (OR) 1.94, p = 0.002). To assess replication of the T2D association, all four tag SNPs were additionally genotyped in two non-overlapping samples of Native Americans. Rs558907 was reproducibly associated with T2D in 2,942 full-heritage Pima Indians (OR 1.27 p = 0.03) and 3,897 "mixed" heritage Native Americans (OR 1.21, p = 0.03), and the strongest evidence for association came from combining all samples (OR 1.27 p = 1.6 × 10(-4), n = 7,667). However, among 320 longitudinally studied subjects, measures of trehalase activity from a non-diabetic exam did not predict those who would eventually develop diabetes versus those who would remain non-diabetic (hazard ratio 0.94 per SD of trehalase activity, p = 0.29). We conclude that variants in TREH control trehalase activity, and although one of these variants is also reproducibly associated with T2D, it is likely that the effect of the SNP on risk of T2D occurs by a mechanism different than affecting trehalase activity. Alternatively, TREH variants may be tagging a nearby T2D locus.

Variants in the LEPR gene are nominally associated with higher BMI and lower 24-h energy expenditure in Pima Indians.

Genome-wide association studies (GWASs) have been used to search for susceptibility genes for type 2 diabetes and obesity in the Pima Indians, a population with a high prevalence of both diseases. In these studies, a variant (rs2025804) in the LEPR gene was nominally associated with BMI in 1,082 subjects (P = 0.03 adjusted for age, sex, birth year, and family membership). Therefore the LEPR and leptin overlapping transcript (LEPROT) genes were selected for further sequencing and genotyping in larger population-based samples for association analyses with obesity-related phenotypes. Selected variants (n = 80) spanning these genes were genotyped in a sample of full-heritage Pima Indians (n = 2,842) and several common variants including rs2025804 were nominally associated with BMI (P = 0.05-0.003 adjusted for age, sex, birth year, and family membership). Four common tag variants associated with BMI in the full-heritage Pima Indian sample were genotyped in a second sample of mixed-heritage Native Americans (n = 2,969) and three of the variants showed nominal replication (P = 0.03-0.006 adjusted as above and additionally for Indian heritage). Combining both samples provided the strongest evidence for association (adjusted P = 0.0003-0.0001). A subset of these individuals (n = 403) had been metabolically characterized for predictors of obesity and the BMI risk alleles for the variants tagged by rs2025804 were also associated with lower 24-h energy expenditure (24hEE) as assessed in a human respiratory chamber (P = 0.0007 adjusted for age, sex, fat mass, fat-free mass, activity, and family membership). We conclude that common noncoding variation in the LEPR gene is associated with higher BMI and lower energy expenditure in Native Americans.

SIRT1 is associated with a decrease in acute insulin secretion and a sex specific increase in risk for type 2 diabetes in Pima Indians.

Genetic variation in SIRT1 affects obesity-related phenotypes in several populations. The purpose of this study was to determine whether variation in SIRT1 affects susceptibility to obesity or type 2 diabetes in Pima Indians, a population with very high prevalence and incidence rates of these diseases. Genotypic data from single nucleotide polymorphisms (SNPs) identified by sequencing regions of SIRT1 combined with SNPs in/near SIRT1 from a prior genome-wide association study determined that 4 tag SNPs (rs7895833, rs10509291, rs7896005, and rs4746720) could capture information across this gene and its adjacent 5' region. The tag SNPs were genotyped in a population-based sample of 3501 Pima Indians (44% had diabetes, 58% female) for association with type 2 diabetes and BMI. Metabolic trait data and adipose biopsies were available on a subset of these subjects. Two tag SNPs, rs10509291 and rs7896005, were nominally associated with type 2 diabetes (P=0.01, OR=1.25 95%CI 1.05-1.48, and P=0.02, OR=1.17 95%CI 1.02-1.34, respectively; additive P values adjusted for age, sex, birth year, and family membership), but not BMI (adjusted P values 0.52 and 0.45, respectively). Among metabolically characterized subjects with normal glucose tolerance (N=243), those carrying the diabetes risk allele (T) for rs10509291 and (G) for rs7896005 had a reduced acute insulin response (AIR) to an intravenous glucose bolus (adjusted P=0.045 and 0.035, respectively). SIRT1 expression in adipose biopsies was negatively correlated with BMI (adjusted P=0.00001). We conclude that variation in SIRT1 is nominally associated with reduced AIR and increased risk for type 2 diabetes. SIRT1 expression in adipose is correlated with BMI, but it remains unknown whether this is a cause or consequence of obesity.

Evaluation of A2BP1 as an obesity gene.

OBJECTIVE: A genome-wide association study (GWAS) in Pima Indians (n = 413) identified variation in the ataxin-2 binding protein 1 gene (A2BP1) that was associated with percent body fat. On the basis of this association and the obese phenotype of ataxin-2 knockout mice, A2BP1 was genetically and functionally analyzed to assess its potential role in human obesity. RESEARCH DESIGN AND METHODS: Variants spanning A2BP1 were genotyped in a population-based sample of 3,234 full-heritage Pima Indians, 2,843 of whom were not part of the initial GWAS study and therefore could serve as a sample to assess replication. Published GWAS data across A2BP1 were additionally analyzed in French adult (n = 1,426) and children case/control subjects (n = 1,392) (Meyre et al. Nat Genet 2009;41:157-159). Selected variants were genotyped in two additional samples of Caucasians (Amish, n = 1,149, and German children case/control subjects, n = 998) and one additional Native American (n = 2,531) sample. Small interfering RNA was used to knockdown A2bp1 message levels in mouse embryonic hypothalamus cells. RESULTS: No single variant in A2BP1 was reproducibly associated with obesity across the different populations. However, different variants within intron 1 of A2BP1 were associated with BMI in full-heritage Pima Indians (rs10500331, P = 1.9 × 10(-7)) and obesity in French Caucasian adult (rs4786847, P = 1.9 × 10(-10)) and children (rs8054147, P = 9.2 × 10(-6)) case/control subjects. Reduction of A2bp1 in mouse embryonic hypothalamus cells decreased expression of Atxn2, Insr, and Mc4r. CONCLUSIONS: Association analysis suggests that variation in A2BP1 influences obesity, and functional studies suggest that A2BP1 could potentially affect adiposity via the hypothalamic MC4R pathway.

Variants in ASK1 are associated with skeletal muscle ASK1 expression, in vivo insulin resistance, and type 2 diabetes in Pima Indians.

OBJECTIVE: Prior genome-wide association and exon array expression studies both provided suggestive evidence that apoptosis signal regulating kinase 1 (ASK1) may influence in vivo insulin action in Pima Indians. Genetic variants in or near ASK1 were analyzed to assess the role of this gene in insulin action and type 2 diabetes. RESEARCH DESIGN AND METHODS: Genotypic data from 31 variants were used to determine the linkage disequilibrium pattern across ASK1 in Pima Indians. Eight tag SNPs were initially genotyped in 3,501 full-heritage Pima Indians. Replication for association with diabetes was assessed in a second population-based sample of 3,723 Native Americans and the published DIAGRAM study. Quantitative traits were analyzed in 536 nondiabetic Native Americans, and ASK1 expression was examined in skeletal muscle of 153 nondiabetic Native Americans. RESULTS: Three tag SNPs were associated with type 2 diabetes (rs35898099, P = 0.003, odds ratio [95% CI] 1.27 [1.08-1.47]; rs1570056, P = 0.007, 1.19 [1.05-1.36]; rs7775356, P = 0.04, 1.14 [1.01-1.28]) in the full-heritage Pima Indians. The association with rs35898099 was replicated in a second sample of Native Americans (P = 0.04, 1.22 [1.01-1.47]), while that for rs1570056 was replicated in the DIAGRAM study of Caucasians (Z statistic based P = 0.026; fixed-effect model, 1.06 [1.00-1.12]). The diabetes risk allele for rs1570056 was associated with reduced insulin action as assessed by either HOMA-IR in 2,549 nondiabetic full-heritage Pima Indians (P = 0.027) or a hyperinsulinemic-euglycemic clamp among 536 nondiabetic Native Americans (P = 0.02). Real-time PCR identified a positive correlation between ASK1 expression in skeletal muscle biopsies and in vivo insulin action (P = 0.02, r = 0.23), and the risk allele for rs1570056 was associated with lower ASK1 expression (P = 0.003, r = -0.22). CONCLUSIONS: ASK1 variants may increase susceptibility to type 2 diabetes by decreasing insulin sensitivity via reduced ASK1 expression.

Analysis of the SIM1 contribution to polygenic obesity in the French population.

SIM1 (single-minded 1) haploinsufficiency is responsible for obesity in both humans and mice, but the contribution of frequent DNA variation to polygenic obesity is unknown. Sequencing of all exons, exon/intron boundaries, 870 base pairs (bp) of the putative promoter, and 1,095 bp of the 3'UTR of SIM1 gene in 143 obese children and 24 control adults identified 13 common variants. After analysis of the linkage disequilibrium (LD) structure, association study of eight variants was performed in 1,275 obese children and severely obese adults, in 1,395 control subjects, and in 578 obesity-selected pedigrees. A nominal evidence of association was found for the nonsynonymous variant P352T C/A (rs3734354) (P = 0.01, OR = 0.81 (0.70-0.95)), the +2,004 TGA -/insT SNP (rs35180395) (P = 0.02, OR = 1.21 (1.02-1.43)), the +2,215A/G TGA SNP (rs9386126) (P = 0.002, OR = 0.81 (0.71-0.93)), and pooled childhood/adult obesity. Even though transmission disequilibrium test (TDT) further supported the association of P352T and +2,004 -/inst T with obesity, none of these nominal associations remained significant after a multiple testing Bonferroni correction. Therefore, our study excludes a major contribution of SIM1 common variants in exons, 5' and 3' UTR regions in polygenic obesity susceptibility in French Europeans.

Common variation in SIM1 is reproducibly associated with BMI in Pima Indians.

OBJECTIVE: Haploinsufficiency of SIM1 is a cause of rare monogenic obesity. To assess the role of SIM1 in polygenic obesity, this gene was analyzed in the Pima Indian population, which has a high prevalence of obesity. RESEARCH DESIGN AND METHODS: SIM1 was sequenced in 96 individuals. Variants (n = 46) were genotyped in a population-based sample of 3,250 full-heritage Pima Indians and in a separate replication sample of 2,944 predominately non-full-heritage subjects from the same community. RESULTS: Variants spanning the upstream region of SIM1 through intron 8 were associated with BMI in the full-heritage Pima Indians, where the strongest associations (P approximately 10(-4) to 10(-6)) were with common variants (risk allele frequency 0.61-0.67). The difference in mean BMI between individuals homozygous for the major allele compared with homozygotes for the minor allele was approximately 2.2 kg/m(2) (P = 2 x 10(-5) for rs3213541). These associations replicated in the separate sample of subjects from the same community (P = 5 x 10(-3) for rs3213541). The strongest associations (P = 4 x 10(-7), controlled for age, sex, birth year, and heritage) were seen in the combined sample (n = 6,194). The risk allele for obesity was more common in full-heritage Pimas than in the mixed-heritage subjects. Two variants (rs3734353 and rs3213541) were also genotyped in 1,275 severely obese and 1,395 lean control subjects of French European ancestry. The Pima risk alleles were the minor alleles in the European samples, and these variants did not display any significant association (P > 0.05). CONCLUSIONS: Common variation in SIM1 is associated with BMI on a population level in Pima Indians where the risk allele is the major allele.