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Diabetic nephropathy accounts for most of the excess mortality in individuals with diabetes, but the molecular mechanisms by which nephropathy develops are largely unknown. Here we tested cytosine methylation levels at 397,063 genomic CpG sites for association with decline in the estimated glomerular filtration rate (eGFR) over a six year period in 181 diabetic Pima Indians. Methylation levels at 77 sites showed significant association with eGFR decline after correction for multiple comparisons. A model including methylation level at two probes (cg25799291 and cg22253401) improved prediction of eGFR decline in addition to baseline eGFR and the albumin to creatinine ratio with the percent of variance explained significantly improving from 23.1% to 42.2%. Cg22253401 was also significantly associated with eGFR decline in a case-control study derived from the Chronic Renal Insufficiency Cohort. Probes at which methylation significantly associated with eGFR decline were localized to gene regulatory regions and enriched for genes with metabolic functions and apoptosis. Three of the 77 probes that were associated with eGFR decline in blood samples showed directionally consistent and significant association with fibrosis in microdissected human kidney tissue, after correction for multiple comparisons. Thus, cytosine methylation levels may provide biomarkers of disease progression in diabetic nephropathy and epigenetic variations contribute to the development of diabetic kidney disease.

Prevalence of diabetes and obesity in Mexican Pima Indians is low, while prevalence is high in Pima Indians in the United States (US); although lifestyle likely accounts for much of the difference, the role of genetic factors is not well-explored. To examine this, we genotyped 359 single nucleotide polymorphisms, including established type 2 diabetes and obesity variants from genome-wide association studies (GWAS) and 96 random markers, in 342 Mexican Pimas. A multimarker risk score of obesity variants was associated with body mass index (BMI; β = 0.81 kg/m(2) per SD, P = 0.0066). The mean value of the score was lower in Mexican Pimas than in US Pimas (P = 4.3×10(−11)), and differences in allele frequencies at established loci could account for ~7% of the population difference in BMI; however, the difference in risk scores was consistent with evolutionary neutrality given genetic distance. To identify loci potentially under recent natural selection, allele frequencies at 283 variants were compared between US and Mexican Pimas, accounting for genetic distance. The largest differences were seen at HLA markers (e.g., rs9271720, difference = 0.75, P = 8.7×10(−9)); genetic distances at HLA were greater than at random markers (P = 1.6×10(−46)). Analyses of GWAS data in 937 US Pimas also showed sharing of alleles identical by descent at HLA that exceeds its genomic expectation (P = 7.0×10(−10)). These results suggest that, in addition to the widely-recognized balancing selection at HLA, recent directional selection may also occur, resulting in marked allelic differentiation between closely related populations.

OBJECTIVES The risk of early-onset type 2 diabetes associated with the severity of obesity in youth is not well understood. This study aims to determine metabolic alterations and type 2 diabetes risk among American Indian children who are obese or severely obese. METHODS Incidence rates of diabetes before 20 years (youth-onset) and 45 years were computed in 2728 children who were from 5 to

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

AIMS/HYPOTHESIS: Elevated 2-h plasma glucose concentration (2 h-PG) during a 75 g OGTT predict the development of type 2 diabetes mellitus. However, 1-h plasma glucose concentra-tion (1 h-PG) is associated with insulin secretion and may be a better predictor of type 2 diabetes. We aimed to investigate the association between 1 h-PG and 2 h-PG using gold standard methods for measuring insulin secretion and action. We also compared 1 h-PG and 2 h-PG as predictors of type 2 diabetes mellitus. METHODS: This analysis included adult volunteers without diabetes, predominantly Native Americans of Southwestern her-itage, who were involved in a longitudinal epidemiological study from 1965 to 2007, with a baseline OGTT that included measurement of 1 h-PG. Group 1 (n = 716) underwent an IVGTT and hyperinsulinaemic–euglycaemic clamp for mea-surement of acute insulin response (AIR) and insulin-stimulated glucose disposal (M), respectively. Some members of Group 1 (n = 490 of 716) and members of a second, larger, group (Group 2; n = 1946) were followed-up to assess the development of type 2 diabetes (median 9.0 and 12.8 years follow-up, respectively). RESULTS: Compared with 2 h-PG (r = −0.281), 1 h-PG (r = −0.384) was more closely associated with AIR, whereas, compared with 1 h-PG (r = −0.340), 2 h-PG (r = −0.408) was more closely associated with M. Measures of 1 h-PG and 2 h-PG had similar abilities to predict type 2 diabetes, which did not change when both were included in the model. A 1 h-PG cut-off of 9.3 mmol/l provided similar levels of sensi-tivity and specificity as a 2 h-PG cut-off of 7.8 mmol/l; the latter is used to define impaired glucose tolerance, a recognised predictor of type 2 diabetes mellitus. CONCLUSIONS/INTERPRETATION: The 1 h-PG was associated with important physiological predictors of type 2 diabetes and was as effective as 2 h-PG for predicting type 2 diabetes mellitus. The 1 h-PG is, therefore, an alternative method of identifying individuals with an elevated risk of type 2 diabetes mellitus.

Background Underlying blood pressure is that observed in the absence of antihypertensive treatment or, among those treated, the estimate of that which would be observed without treatment. This study aims to examine the relationships between diabetes or obesity and underlying systolic blood pressure adjusted for antihypertensive treatment by several methods. Methods Data from two population studies were analyzed—an American Indian community in Arizona and the National Health and Nutrition Examination Surveys. Antihypertensive treatment was accounted for using: no adjustment; antihypertensive use as a covariate; blood pressure dichotomized into normotension and hypertension; addition of a fixed treatment effect; non-parametric algorithm; and censored normal regression. Results The magnitude of association at each time point differed by adjustment method particularly where there was a difference in prevalence of antihypertensive use between people with and without diabetes or obesity. The common methods of ignoring antihypertensive treatment or including it as a covariate in a regression model underestimated the effects of diabetes and obesity on underlying blood pressure, compared to the recommended method of the censored normal regression. Conclusion Proper accounting for antihypertensive treatment is needed in interpreting variables that affect blood pressure.

Increased urine albumin excretion is highly prevalent in Hispanics/Latinos. Previous studies have found an association between urine albumin excretion and Amerindian ancestry in Hispanic/Latino populations. Admixture between racial/ethnic groups creates long-range linkage disequilibrium between variants with different allelic frequencies in the founding populations and it can be used to localize genes. Hispanic/Latino genomes are an admixture of European, African, and Amerindian ancestries. We leveraged this admixture to identify associations between urine albumin excretion (urine albumin-to-creatinine ratio [UACR]) and genomic regions harboring variants with highly differentiated allele frequencies among the ancestral populations. Admixture mapping analysis of 12,212 Hispanic Community Health Study/Study of Latinos participants, using a linear mixed model, identified three novel genome-wide significant signals on chromosomes 2, 11, and 16. The admixture mapping signal identified on chromosome 2, spanning q11.2–14.1 and not previously reported for UACR, is driven by a difference between Amerindian ancestry and the other two ancestries (P

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.

SummaryBackground and Objectives Most studies describing childhood obesity in the United States are based on cross-sectional surveys and do not include substantial numbers of American Indians (AI). Secular trends in height and weight reflect general health status. This study describes weight trends and transitions among AI children over a 43-year period. Methods Anthropometric data were obtained from a prospective study conducted in a southwestern US AI population (1965 through 2007). For cross-sectional analysis, 12 377 observations were available from 6529 children across four birth cohorts (1955–1964, 1965–1974, 1975–1984, 1985–1994). Participants were stratified into three age groups: pre- (5–9 years), early (10–13) and late (14–17) adolescence. Longitudinal analyses included 1737 children with one exam in each age group. Results In early and late adolescence, weight increased across birth cohorts. Prevalence of obesity among pre-adolescents was 17.5% (95% CI, 15.1%–19.9%) in the 1955–1964 cohort and 33.7% (95% CI, 30.1%–36.4%) in the 1985–1994 cohort. 74% of children overweight in pre-adolescence in the 1985–1994 cohort became obese by late adolescence; in the 1955–1964 cohort, only 43% made this transition. Conclusions This study describes the rising prevalence of childhood obesity. Children obese in pre-adolescence remained obese in late adolescence, stressing the need for early intervention.

A single multimode interference waveguide is used to create distinct spectral spot patterns on two liquid-core waveguides on an optofluidic chip. This device is used for multiplexed detection of antibiotic-resistant plasmids with single nucleic acid sensitivity.

Aims Type 2 diabetes is a heterogeneous disease and may manifest from multiple disease pathways. We examined insulin secretion and insulin resistance across two ethnicities with particularly high risk for diabetes yet with widely different distributions of weight class. Materials and methods In this population-based, cross-sectional study, Pima Indians from Southwestern United States (n = 865) and Asian Indians from Chennai, India (n = 2374) had a 75-g oral glucose tolerance test. We analysed differences in plasma glucose, plasma insulin, insulin resistance (HOMA-IR), and insulin secretion (ΔI0-30 /ΔG0-30 ) across categories of body mass index (BMI) and glycemic status per American Diabetes Association criteria. Results Pima Indians were younger (mean 27.4 ± SD 6.6, Asian: 33.9 ± 6.7 years) and had higher BMI (33.6 ± 8.1, Asian: 25.7 ± 4.9 kg/m2 ). Among normal weight participants (mean BMI: Pima 22.4 SE 0.2; Asian 22.2 SE 0.06 kg/m2 ), fasting glucose was higher in Asian Indians (5.2 vs Pima: 4.8 mmol/L, P = .003), adjusted for age and sex. Pima Indians were three times as insulin resistant as Asian Indians (HOMA-IR: 7.7 SE 0.1, Asian: 2.5 SE 0.07), while Asian Indians had three times less insulin secretion (Pima: 2.8 SE 1.0 vs Asian: 0.9 SE 1.0 pmol/mmol), a pattern evident across age, BMI, and glycemic strata. Conclusions Metabolic differences between Pima and Asian Indians suggest heterogeneous pathways of type 2 diabetes in the early natural history of disease, with emphasis of insulin resistance in Pima Indians and emphasis of poor insulin secretion in Asian Indians.

A prior genome-wide association study in 7701 Pima Indians utilized a custom Axiom array (Affymetrix) which contained 474,196 SNPs. These SNPs tagged 92% of common variants (R2≥0.85, minor allele frequency ≥0.05) identified in the Pima Indian genome. SNP rs79788804, which tags 38 additional SNPs in intron 15 of KCNQ1, achieved genome-wide significance in its association with type 2 diabetes (T2D; OR=1.31, p=2 x10-8). A 3 SNP haplotype (rs79788804, rs2237892 and rs2299620) with >1 maternally-derived alleles strongly associated with T2D (ORM=2.05, p=2x10-12). ENCODE and Islet regulome databases mapped putative strong enhancers to 4 regions in intron 15 with multiple epigenetic marks and transcription factor binding sites. To validate these regulatory regions, we tested gene activation from the putative enhancers using CRISPR-CAS9-based acetyltransferase (dCas9p300) in human pancreatic beta cells (EndoC-βH3). Guide RNA specific acetylation by dCas9p300 at a 341 bp region spanning rs2299620, rs2237896, rs2237897 and rs74046911 resulted in a 1.5 fold gene activation for KCNQ1 and nearby gene TRPM5 (both p=0.001). A chromatin immunoprecipitation-qPCR assay confirmed that dCas9P300 acetylated chromatin at this targeted region. We further assessed the effects of 5 transcription factors (NKX2.2, MAFB, NKX6.1, FOXA2, PDX1) on transcriptional activity under control of these 4 enhancer regions. Mouse pancreatic β-cells (NIT1) were co-transfected with a vector expressing each transcription factor and a luciferase reporter construct containing the KCNQ1 SNPs. NKX2.2 had a 5.5 fold activation for the construct containing the same 341 bp KCNQ1 region, as compared to a 4.1 fold activation for the construct containing KCNQ1 promoter alone (p=0.02). Our data suggested that a 341 bp region containing rs2299620, rs2237896, rs2237897 and rs74046911 may harbor functional variant(s) which affect expression of KCNQ1 and TRPM5. Disclosure Y.L. Muller: None. S.E. Day: None. S. Kobes: None. W.C. Knowler: None. R.L. Hanson: None. C. Bogardus: None. L.J. Baier: None. Funding National Institutes of Health

Epidemiologic studies in many populations have shown that birth weight is associated with risk of type 2 diabetes (T2D) in adulthood. In a Southwestern American Indian population, lower and higher birth weight groups are at higher risk for T2D than those with normal birth weight. We performed a genome-wide association study (GWAS) of birth weight in 3700 Southwestern American Indians (2037 female; 1663 male). 496,190 SNPs (allele frequency>1%) were directly genotyped using an Axiom array designed to capture common variation in this community (Affymetrix; Santa Clara, CA). Birth weight was ascertained from Arizona state and medical records. T2D was determined according to American Diabetes Association criteria at a research exam or during clinical care. Birth weight data were normalized separately by sex and analyzed for genetic associations using a mixed model (SOLAR-Eclipse; Catonsville, MD) accounting for genetic relationships (based on genetic markers among all pairs of individuals) and adjusted for birth year and the 1st 5 genetic principal components. We identified 41 variants suggestively associated (p 5 of the 41 variants are also nominally associated with T2D (p Disclosure L.E. Wedekind: None. W. Hsueh: None. S. Kobes: None. M.T. Olaiya: None. W.C. Knowler: None. L.J. Baier: None. R.L. Hanson: None.

Background: Knowing vulnerable periods of growth for predicting type 2 diabetes (T2D) incidence may help focus interventions for prevention. Using life course data, we identified critical growth periods in childhood that predict T2D risk. Methods: This analysis of a longitudinal study of American Indians, aged ≥ 5 years, comprised subjects representing three growth periods (pre-, early, and late adolescence). Age-sex-height-adjusted mean attained weight and weight velocity (change/year) were computed in each growth period. Subjects were followed from the end of each growth period until T2D diagnosis or their last research examination. Poisson regression was used to model the effects of the mean attained weight or weight velocity in a growth period on T2D incidence. Results: There were 290 incident T2D cases among 2100 subjects representing the pre-adolescence growth period, 315 cases among 1558 in the early adolescence period, and 380 cases among 1418 in the late adolescence period. In each growth period, the mean attained weight significantly predicted T2D incidence, especially in the first 5 years of follow-up (Figure, A-C). There were significant effects of weight velocity in pre-adolescence and modest effects in late adolescence on T2D incidence (Figure, D-F). Attained weight pre-adolescence had the strongest effect on T2D incidence. Conclusions: Pre-adolescence is an especially vulnerable period for predicting later T2D. Disclosure M.T. Olaiya: None. W.C. Knowler: None. R.L. Hanson: None. S. Kobes: None. R. Nelson: None. M. Sinha: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Although hundreds of genome-wide association studies-implicated loci have been reported for adult obesity-related traits, less is known about the genetics specific for early-onset obesity and with only a few studies conducted in non-European populations to date. Searching for additional genetic variants associated with childhood obesity, we performed a trans-ancestral meta-analysis of 30 studies consisting of up to 13 005 cases (≥95th percentile of body mass index (BMI) achieved 2–18 years old) and 15 599 controls (consistently

Identification of high-impact loss-of-function variants for common disease is challenging because these variants are typically rare and thus statistical power is low when sample sizes are limited. In this study, we utilized whole-exome variant detection, variant prediction programs, tissue expression data, and case-control analysis to identify potentially highly penetrant obesity-susceptibility variants for prioritization for functional testing. Whole-exome sequencing was performed on DNA samples collected from a longitudinal, population-based study of American Indians (N=5432; maximum BMI recorded at ≥18 years). Among the 1 million biallelic SNVs detected in this cohort, those that were exonic or splicing, have minor allele frequency (MAF) ≤0.05, predicted to be damaging (CADD score ≥15), and located in genes expressed in hypothalamus were selected for analysis. These selection criteria resulted in 94,444 SNVs, and odds ratios were calculated for them by comparing the group of individuals with BMI in the top 5th percentile for this population (N=271, BMI ≥53.13 kg/m2) vs. those below the median (N=2,717, BMI Disclosure C. Koroglu Altok: None. P. Piaggi: None. M. Traurig: None. R.L. Hanson: None. W.C. Knowler: None. H. Kim: Employee; Self; Regeneron Pharmaceuticals. N. Gosalia: Employee; Self; Regeneron Pharmaceuticals. C.V. Van Hout: Employee; Self; Regeneron Pharmaceuticals. B. Ye: Employee; Self; Regeneron Pharmaceuticals. A.R. Shuldiner: Employee; Self; Regeneron Pharmaceuticals. Stock/Shareholder; Self; Rhythm Pharmaceuticals, Inc. C. Bogardus: None. L.J. Baier: None. Funding National Institutes of Health

South Asians have a high burden of diabetes at younger ages. We compared diabetes risk in South Asians aged 20-44 years living in India (Delhi and Chennai) and Pakistan (Karachi) with Pima Indians, a population with the world’s highest incidence of diabetes. We analyzed prospective cohort data from the Pima Indian Study (Pima, n=1852), and the Center for Cardiometabolic Risk Reduction in South Asia Study (CARRS, South Asians, n=6676). We defined diabetes as fasting glucose ≥126 mg/dl, HbA1c ≥ 6.5%, or medication use. At baseline, on average, the South Asians were less obese than Pima (BMI, kg/m2: 24.4 vs. 33.8; Waist, cms 82.5 vs.107). Age-standardized diabetes incidence (cases/1000 person-years, 95% CI) was lower in South Asians than in Pima (Men: 14.2, 12.2-16.2 vs. 37.3, 31.8-42.8; Women: 14.8, 13.0-16.5 vs. 46.1, 41.2-51.1). Incidence among 20-24 year old were 80% and 87% lower, respectively, among South Asian men and women, but the risk differential narrowed at older ages (Table). In those with BMI Disclosure K. Narayan: None. D. Kondal: None. S. Kobes: None. M. Deepa: None. N.R. Daya: None. S.A. Patel: None. R. Anjana: None. L.R. Staimez: None. M.K. Ali: None. U. Gujral: None. D. Prabhakaran: None. R. Shivashankar: None. M. Kadir: None. E. Selvin: None. V. Mohan: None. R.L. Hanson: None. N. Tandon: None. Funding National Heart, Lung, and Blood Institute; Department of Health and Human Services (HHSN268200900026C); United Health Group, Minneapolis, MN; National Institute of Diabetes and Digestive and Kidney Diseases (P30DK111024)

Amerindians, particularly Pima Indians, are at high risk for type 2 diabetes (T2D). Our previous studies have shown that lipid levels (high triglycerides or TG, and low HDL-cholesterol, or HDL-C) are important predictors of T2D incidence. There are few data on the extent to which apolipoprotein levels, particularly apolipoprotein CIII (apoC-III), which is a potential therapeutic target to treat high TG, predict T2D. Thus, we examined whether circulating apolipoprotein levels predict T2D risk in addition to known risk factors. We measured serum TG, HDL-C and 5 apolipoprotein levels (apoA-I, apoA-V, apoB100, apoC-III, apoE), in 1,916 nondiabetic Pima Indians at baseline (mean age=31.2±10 years, 43% male). After mean follow-up of 6.26 years, ∼24% of subjects (n=454) developed T2D. BMI, HDL-C and TG predicted T2D, when adjusted for sex and age (hazard ratio [HR] per s.d. unit was 1.05, 0.74 and 1.18, respectively; all p Disclosure W. Hsueh: None. M. Walter: None. R. Nelson: None. W.C. Knowler: None. R.L. Hanson: None. Funding National Institutes of Health

Genome-wide association studies (GWAS) have identified many variants associated with type 2 diabetes (T2D), but there is little information on their effects in high risk populations such as American Indians. We analyzed 211 primary variants, associated with T2D (P Disclosure R.L. Hanson: None. L.E. Wedekind: None. W. Hsueh: None. S. Kobes: None. L.J. Baier: None. C. Bogardus: None. W.C. Knowler: None.

AIMS/HYPOTHESIS: The aim of this work was to estimate the impact of birthweight on early-onset (age

Type 2 diabetes is a common disease worldwide, but its prevalence varies widely by geographical region and by race/ethnicity. This review summarises differences in the frequencies of type 2 diabetes according to race, ethnicity, socioeconomic position, area of residence and environmental toxins. Type 2 diabetes susceptibility often begins early in life, starting with genetic susceptibility at conception and continuing in later life, via in utero, childhood and adult exposures. Early-life factors may lead to overt type 2 diabetes in childhood or in later life, supporting the concept of developmental origins of health and disease. The causes of the racial/ethnic differences in incidence of type 2 diabetes are not well understood. Specifically, the relative contributions of genetic and environmental factors to such differences are largely unknown. With a few exceptions in isolated populations, there is little evidence that differences in frequencies of known type 2 diabetes susceptibility genetic alleles account for racial/ethnic differences, although the search for genetic susceptibility has not been uniform among the world's racial/ethnic groups. In the USA, race/ethnicity is associated with many other risk factors for type 2 diabetes, including being overweight/obese, diet and socioeconomic status. Some studies suggest that some of these factors may account for the race/ethnic differences in prevalence of type 2 diabetes, although there is inadequate research in this area. A better understanding of the impact of these factors on type 2 diabetes risk should lead to more effective prevention and treatment of this disease. This has not yet been achieved but should be a goal for future research.

BackgroundGenetic factors that influence kidney traits have been understudied for low frequency and ancestry-specific variants.MethodsWe combined whole genome sequencing (WGS) data from 23,732 participants from 10 NHLBI Trans-Omics for Precision Medicine (TOPMed) Program multi-ethnic studies to identify novel loci for estimated glomerular filtration rate (eGFR). Participants included European, African, East Asian, and Hispanic ancestries. We applied linear mixed models using a genetic relationship matrix estimated from the WGS data and adjusted for age, sex, study, and ethnicity.FindingsWhen testing single variants, we identified three novel loci driven by low frequency variants more commonly observed in non-European ancestry (PRKAA2, rs180996919, minor allele frequency [MAF] 0.04%, P=6.1×10-11; METTL8, rs116951054, MAF 0.09%, P=4.5×10-9; and MATK, rs539182790, MAF 0.05%, P=3.4×10-9). We also replicated two known loci for common variants (rs2461702, MAF=0.49, P=1.2×10-9, nearest gene GATM, and rs71147340, MAF=0.34, P=3.3×10-9, CDK12). Testing aggregated variants within a gene identified the MAF gene. A statistical approach based on local ancestry helped to identify replication samples for ancestry-specific variants.InterpretationThis study highlights challenges in studying variants influencing kidney traits that are low frequency in populations and more common in non-European ancestry.

IntroductionSex hormone binding globulin (SHBG) levels are reported to be inversely associated with diabetes risk. It is unknown whether diabetes prevention interventions increase SHBG and whether resultant changes in SHBG affect diabetes risk. The purpose of this analysis was to determine whether intensive lifestyle intervention (ILS) or metformin changed circulating SHBG and if resultant changes influenced diabetes risk in the Diabetes Prevention Program (DPP).Research design and methodsThis is a secondary analysis from the DPP (1996–2001), a randomized trial of ILS or metformin versus placebo on diabetes risk over a mean follow-up of 3.2 years. The DPP was conducted across 27 academic study centers in the USA. Men, premenopausal and postmenopausal women without hormone use in the DPP were evaluated. The DPP included overweight/obese persons with elevated fasting glucose and impaired glucose tolerance. Main outcomes measures were changes in SHBG levels at 1 year and risk of diabetes over 3 years.ResultsILS resulted in significantly higher increases (postmenopausal women: pConclusionsLifestyle intervention may be associated with favorable changes in circulating SHBG, which is largely due to changes in adiposity. Changes in circulating SHBG do not independently predict reductions in diabetes incidence.

OBJECTIVE Long-term data validating glycated hemoglobin (HbA1c) in assessing the risk of type 2 diabetes in children are limited. HbA1c, fasting plasma glucose (FPG), and 2-h postload plasma glucose (2hPG) concentrations were measured in a longitudinal study of American Indians to determine their utility in predicting incident diabetes, all of which is thought to be type 2 in this population. RESEARCH DESIGN AND METHODS Incident diabetes (FPG ≥126 mg/dL [7.0 mmol/L], 2hPG ≥200 mg/dL [11.1 mmol/L], HbA1c ≥6.5% [8 mmol/mol], or clinical diagnosis) was determined in 2,095 children without diabetes ages 10–19 years monitored through age 39, and in 2,005 adults ages 20–39 monitored through age 59. Areas under the receiver operating characteristic (ROC) curve for HbA1c, FPG, and 2hPG in predicting diabetes within 10 years were compared. RESULTS During long-term follow-up of children and adolescents who did not initially have diabetes, the incidence rate of subsequent diabetes was fourfold (in boys) as high and more than sevenfold (in girls) as high in those with HbA1c ≥5.7% as in those with HbA1c ≤5.3%—greater rate ratios than experienced by adults in the same HbA1c categories. Analyses of ROCs revealed no significant differences between HbA1c, FPG, and 2hPG in sensitivity and specificity for identifying children and adolescents who later developed diabetes. CONCLUSIONS HbA1c is a useful predictor of diabetes risk in children and can be used to identify prediabetes in children with other type 2 diabetes risk factors with the same predictive value as FPG and 2hPG.

OBJECTIVE To determine whether early administration of losartan slows progression of diabetic kidney disease over an extended period. RESEARCH DESIGN AND METHODS We conducted a 6-year clinical trial in 169 American Indians with type 2 diabetes and urine albumin/creatinine ratio RESULTS After completion of the clinical trial, treatment with renin-angiotensin system inhibitors was equivalent in both groups. During a median of 13.5 years following randomization, 29 participants originally assigned to losartan and 35 to placebo reached the primary GFR outcome with an HR of 0.72 (95% CI 0.44–1.18). CONCLUSIONS Long-term risk of GFR decline was not significantly different between persons randomized to early treatment with losartan and those randomized to placebo. Accordingly, we found no evidence of an extended benefit of early losartan treatment on slowing GFR decline in persons with type 2 diabetes.

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.

SummaryBackground Childhood obesity is associated with increased cardiometabolic risk. Objective To study the relationship between body mass index (BMI) and cardiometabolic risk factors in American Indian children and adolescents. Methods Differences in metabolic variables by age and sex-specific BMI percentiles (2000 Centers for Disease Control and Prevention Growth Charts) were examined in a cross-sectional analysis of 2977 individuals across three age categories. Children with an exam in two consecutive age categories were included in a longitudinal analysis. Spearman's correlations were used to test the association of BMI percentile with anthropometric and biochemical variables. Results Body mass index percentile correlated with systolic (r = 0.24 to 0.38) and diastolic (r = 0.13 to 0.22) blood pressure, fasting plasma glucose (r = 0.20 to 0.33), 2-h plasma glucose (r = 0.30 to 0.46), total cholesterol (r = 0.12 to 0.23), serum triglycerides (r = 0.40 to 0.51) and HDL cholesterol (r = −0.36 to −0.43) in each age group (5–9, 10–13 and 14–17 years). Among participants examined in multiple age categories, BMI percentile increased over time. Change in BMI percentile from one age category to the next was associated with an increase in fasting glucose, 2-h glucose and triglycerides and a decrease in HDL cholesterol. Conclusion Higher BMI was associated with blood pressure elevation, hyperglycaemia and dyslipidaemia in American Indian children and adolescents.

Data are lacking on how metabolic risk factors during childhood affect the long-term risk of type 2 diabetes.Assess four metabolic risk factors as predictors of type 2 diabetes and determine whether the risk differs between younger and older children.In a prospective cohort study conducted between 1965 and 2007, participants were followed for development of diabetes. Baseline measurements included body mass index (BMI), blood pressure, serum cholesterol, and 2-hour plasma glucose after an oral glucose tolerance test. Additional analyses divided subjects into two groups according to baseline age, 5–11 and 12–19 years.Gila River Indian Community in Arizona.A total of 5532 nondiabetic Pima Indian children 5–19 years old.A total of 1281 children developed diabetes (median follow-up, 12.4 years). Diabetes incidence was higher in overweight children (BMI ≥ 85th percentile) than in nonoverweight children. Nonoverweight children had the lowest risk of diabetes (20-year cumulative incidence, 9.5%), whereas overweight children with impaired glucose tolerance (2-hour glucose ≥ 140 mg/dL) had the highest (79.0%). The relative risk for children with metabolic abnormalities compared with their healthy counterparts was higher in younger children than in older children early in follow-up. BMI and 2-hour glucose were related to incident diabetes in multivariable models (predicted 15-year cumulative incidence for the highest vs lowest quartile was 3.9 and 1.8 times as high for BMI and 2-hour glucose, respectively; P.001), whereas blood pressure and cholesterol were not.BMI and impaired glucose tolerance in children are strong predictors of type 2 diabetes. Other components of the “metabolic syndrome” are not.

Epidemiological studies in Pima Indians identified elevated levels of HDL-cholesterol (HDL-C) as a protective factor against type 2 diabetes risk in women. We assessed whether HDL-C-associated single-nucleotide polymorphisms (SNPs) also associate with type 2 diabetes in female Pima Indians.Twenty-one SNPs in established HDL-C loci were initially analysed in 2,675 full-heritage Pima Indians. SNPs shown to associate with HDL-C (12 SNPs) were assessed for association with type 2 diabetes in 7,710 Pima Indians (55.6% female sex). The CETP locus provided the strongest evidence for association with HDL-C and was further interrogated by analysing tag SNPs.Twelve of the 21 SNPs analysed had a significant association with HDL-C in Pima Indians; five SNPs representing four loci (CETP, DOCK6, PPP1R3B and ABCA1) reached genome-wide significance. Three SNPs, at CETP, KLF14 and HNF4A, associated with type 2 diabetes only in female participants with the HDL-C-lowering allele increasing diabetes risk (p values: 3.2 × 10(-4) to 7.7 × 10(-5)); the association remained significant even after adjustment for HDL-C. Additional analysis across CETP identified rs6499863 as having the strongest association with type 2 diabetes in female participants (p = 5.0 × 10(-6)) and this association remained independent of the HDL-C association.SNPs at the CETP, HNF4A and KLF14 locus are associated with HDL-C levels and type 2 diabetes (in female participants). However, since HNF4A and KLF14 are established loci for type 2 diabetes, it is unlikely that HDL-C solely mediates these associations.

AIMS: To assess peripheral neuropathy (PN) using graded Semmes Weinstein monofilaments (SWMs) and determine factors associated with PN among adult volunteers with and without diabetes. METHODS: Adult volunteers were assessed for distal sensory PN using three graded SWMs. Four PN levels were defined: 0 (no PN; felt all three filaments), 1 (subclinical PN; insensate to 1-gram filament), 2 (insensate to 10-gram), or 3 (insensate to 75-gram). Levels 2–3 were considered clinical PN. Associations with PN were determined using ordinal logistic regression. RESULTS: In 1,564 subjects (median age 41.9 years, 50.1% women), PN was subclinical or worse in 68.9% and clinical in 11.2%. Age-sex-race-adjusted prevalence of clinical PN was greater in people with diabetes (15.3%) than without (6.1%; P

OBJECTIVE Meta-analyses of genome-wide association studies in Europeans have identified > 98 loci for BMI. Transferability of these established associations in Pima Indians was analyzed. METHODS Among 98 lead single nucleotide polymorphisms (SNPs), 82 had minor allele frequency ≥ 0.01 in Pima Indians and were analyzed for association with the maximum BMI in adulthood (n = 3,491) and BMI z score in childhood (n = 1,958). Common tag SNPs across 98 loci were also analyzed for additional signals. RESULTS Among the lead SNPs, 13 (TMEM18, TCF7L2, MRPS33P4, PRKD1, ZFP64, FTO, TAL1, CALCR, GNPDA2, CREB1, LMX1B, ADCY9, NLRC3) were associated with BMI (P ≤ 0.05) in Pima adults. A multi-allelic genetic risk score (GRS), which summed the risk alleles for 82 lead SNPs, showed a significant trend for a positive relationship between GRS and BMI in adulthood (beta = 0.48% per risk allele; P = 1.6 × 10-9 ) and BMI z score in childhood (beta = 0.024 SD; P = 1.7 × 10-7 ). GRS was significantly associated with BMI across all age groups ≥ 5 years, except for those ≥ 50 years. The strongest association was seen in adolescence (age 14-16 years; P = 1.84 × 10-9 ). CONCLUSIONS In aggregate, European-derived lead SNPs had a notable effect on BMI in Pima Indians. Polygenic obesity in this population manifests strongly in childhood and adolescence and persists throughout much of adult life.

We recently completed a genome wide association study for type 2 diabetes (T2D) in 7659 American Indians who are predominantly of Pima Indian heritage using a custom designed Axiom array. A novel intronic variant (hg19 chr10:72276664 G>C) in PALD1 was identified that associated with T2D (risk allele frequency (RAF)=0.01, OR=2.19[1.49-3.22] per risk allele [C], P=6.6×10-5 adjusted for age, sex, birthyear, PCs 1-5 and accounting for genetic relatedness). Further analysis in a dataset of Urban American Indians (N=3029) representing several tribal groups living in the Phoenix area, identified a directionally consistent association (OR=2.32, P=0.12), but this variant was rare among the entire Urban American Indian sample (RAF=0.003). A meta-analysis of both datasets resulted in a summary OR of 2.15[1.49-3.13], P=4.9×10-5. In Pima Indians, this variant is highly concordant (r2=0.92) with a novel missense variant (hg19 chr10:72298980 C>T, p.[P568S]) in PALD1. The missense variant also had a significant association with T2D in Pima Indians (RAF=0.02, OR=1.80[1.25-2.60], P=0.002) and in Urban American Indians (RAF=0.008, OR=2.29[1.03-5.12], P=0.04). However, after conditional analysis, only the intronic variant remains significant in the Pima Indian dataset. PALD1 is a predicted phosphatase and acts as a negative regulator of insulin signaling. Overexpression of PALD1 leads to lower insulin stimulated AKT phosphorylation and insulin receptor abundance. For preliminary functional studies, a short fragment (100bp) containing each allele of the PALD1 intronic variant (C or G) was cloned upstream of a SV40 promoter luciferase vector and transfected into C2C12 myoblasts. The T2D risk allele (C) fragment had significantly (P=0.004) higher SV40 promoter mediated luciferase activity as compared to the non-risk (G) allele. This result is consistent with the possible role of PALD1 in T2D pathogenesis. Further mechanistic studies are currently ongoing. Disclosure A.K. Nair: None. J.R. Sutherland: None. G.R. Elson: None. P. Piaggi: None. S. Kobes: None. R.L. Hanson: None. C. Bogardus: None. L. Baier: None.

Background: Peripheral neuropathy (PN) is a diabetes complication but also occurs without diabetes. The effect of diabetes on PN is not well quantified. Methods: Subjects ≥ 18 years old had a health exam, including an OGTT for diabetes. Distal sensory PN, assessed using 3 graded Semmes Weinstein monofilaments, was classified as none (felt all filaments), latent (insensate to 1 g filament), clinical [mild (insensate to 10 g) or severe (75 g)]. Associations with PN were assessed using ordinal logistic regression assuming equal coefficients across classes of PN. Results: In 1564 subjects (median age 42 years, 50% women, 58% American Indians), PN was latent or worse in 68.9% (95% CI 66.7-71.2), clinical in 11.2% (CI 9.7-12.9), and associated with male sex, older age, and worse glucose regulation (Figure). Age-sex-race-adjusted prevalence was greater in people with than without diabetes (odds ratio; OR 2.2, CI 1.7-2.8), and in those with diabetes, with longer diabetes duration (OR 1.9 per decade, CI 1.5-2.4) or albuminuria, A/C ≥30 mg/g (OR 2.4, CI 1.6-3.6). In those without clinical PN, latent PN was associated with diabetes (OR 1.7, CI 1.2-2.2), longer diabetes duration (OR 2.2 per decade, CI 1.4-3.5) and albuminuria (OR 1.8, CI 1.0-3.2). Conclusions: Although PN is common in people with no diabetes, diabetes greatly increases its risk. Use of 3 monofilaments rather than 1 adds precision and allows detection of latent PN. Disclosure M.T. Olaiya: None. R.L. Hanson: None. K.G. Kavena: None. M. Sinha: None. R. Nelson: None. W.C. Knowler: None.

Background: Type 2 diabetes mellitus (T2DM) is increasingly diagnosed in adolescents and young adults. However, the impact of birth weight (BW) on this early onset T2DM is not well quantified. Methods: A longitudinal study of Southwestern American Indians, aged ≥ 5 years, was conducted from 1965-2007. Subjects who had a recorded BW and at least one research exam that included an OGTT were followed until they developed T2DM or their last exam before the age of 40 years, whichever came first. Age-sex-adjusted incidence rate of T2DM was computed and Poisson regression models were used to estimate the effect of BW on the incidence of T2DM, adjusted for sex, BMI z-score (fitted as time-varying), and maternal variables, such as the presence of diabetes during pregnancy. The effect of BW on T2DM was non-proportional over time, and in some regression models, the effect was non-linear (quadratic). Therefore, all analyses were stratified by decade of age at follow-up and BW was analyzed as quintiles. Results: Among 4,275 subjects (52% females, median BW 3.45 kg), there were 728 incident cases of T2DM over a median follow-up of 20.6 years (7.5 cases per 1000 person-years). Incidence rates, stratified by decade of age at follow-up, were greater in females and in older age groups. In multivariable regression analyses, the effect of BW on T2DM risk varied by age group, showing a quadratic (U-shaped) effect at 10-19 years (P Conclusions: Both low and high BWs increased the risk of T2DM in adolescence, but only the effect of low BW persisted into young adulthood. Effects of female sex, accelerated growth, or being the offspring of a diabetic pregnancy added to the effect of BW on the risk of early onset T2DM. Disclosure M.T. Olaiya: None. M. Sinha: None. R.L. Hanson: None. S. Kobes: None. R. Nelson: None. W.C. Knowler: None.

A recent genome-wide association study (GWAS) in Samoans (a Polynesian population) identified strong associations of CREBRF variants with BMI. The G allele at rs12513649 (frequency=25.8%) was associated with ∼1.3 kg/m2 higher BMI per copy, as was the Gln allele at rs373863828 (which codes for an ArgGln substitution). The Gln allele at rs373863828 has frequency of 25.9% in Samoans and is rare in most global populations, although it has been observed at low frequency in some other Oceanic populations. The alleles associated with higher BMI were suggestively associated with lower risk of type 2 diabetes [T2D, odds ratio (OR)=0.69 per copy, P=4.7×10-7]. We investigated the associations of rs12513649 and rs373863828 with BMI and T2D in 2029 Pacific Islanders from Guam and Saipan, who participated in a GWAS. Frequencies of the G allele at rs12513649 and the Gln allele at rs373863828 were 8.1 and 5.4% respectively in full-heritage Chamorros (N=928), 4.1 and 0.2% in Filipinos (N=249), 6.1 and 3.7% in Chuukis (N=153), 2.2 and 1.1% in Carolinians (N=43), and 20.7 and 2.2% in Palauans (n=45). Using a genomic control procedure with adjustment for age, sex, end-stage renal disease and the first four genetic principal components, the G allele at rs12513649 and the Gln allele at rs373863828 were associated with higher BMI (β=1.60 kg/m2, P=0.002 and β=1.36 kg/m2, P=0.058 respectively). They were also modestly associated with taller height (β=0.8 cm, P=0.11, and β=1.5 cm, P=0.019). The alleles conferring higher BMI were associated with lower prevalence of T2D (OR=0.62, P=0.0and OR=0.45, P=0.001). When combined with previously published results for Samoans via meta-analysis, the association of rs373863828 with T2D achieved genome-wide statistical significance (OR=0.67, P=2.9×10-8). These results suggest that CREBRF variants may be important contributors to obesity in Oceanic populations, and they confirm the associations of the allele conferring higher BMI with lower risk of T2D. Disclosure R.L. Hanson: None. S. Safabakhsh: None. J. Curtis: None. D.K. Mahkee: None. L. Baier: None. W.C. Knowler: None. R. Nelson: None. W. Hsueh: None.

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection.

Introduction: Using a population-based linkage analysis approach, we previously identified a major locus for serum triglyceride (TG) levels in Pima Indians, which largely reflects the effect of a functional SNP (p.(Ala43Thr), A43T, or rs147210663) in the APOC3 gene. This non-synonymous SNP is extremely rare in non-Amerindian populations and more common in the Pima Indians (frequency of the T43 allele≈3%). This SNP alters the structure of APO-CIII, which leads to loss of its function and to lower circulating levels of APO-CIII. Its effect size reduces TG level by approximately 1 SD unit (per allele), which is among the largest effects for any complex trait reported to date. Hypothesis: To further explore the functional mechanism underlying this association, we analyzed the relationships among A43T, TG and APO-CIII levels; we hypothesized that the APOC3 A43T SNP effect on TG is mediated through circulating APO-CIII in the blood. Methods: We measured serum APO-CIII level in 411 randomly selected Amerindian subjects from a larger cohort study with both the A43T genotype and TG levels measured; this included 22 carriers of a T43 allele (frequency=2.7%). All analyses were modeled using linear regressions based on cross-sectional data, with age, sex, diabetes status, and population admixture included as covariates. The natural logarithms of APO-CIII and TG levels were taken to reduce skewness. Results: The phenotypic correlation between APO-CIII and TG was 0.68. We found a strong association between the A43T SNP and TG levels (β=-0.78±0.21 SD unit per copy of the T43 allele, p=0.00026, explaining 3.2% variance) that was greatly attenuated with further adjustment for APO-CIII levels (β=-0.04±0.17 SD unit, p=0.81, explaining 0.01% variance). On the other hand, there was also a strong association between the A43T SNP and APO-CIII levels (β=-1.22±0.21 SD unit, p=1.2х10 -7 , explaining 6.5% variance), which remained significant even after adjustment for the TG effect (β=-0.61±0.16 SD unit, p=0.00016, explaining 3.4% variance). Formal mediation analysis, conducted by the Sobel method, suggested significant and virtually complete mediation of the A43T effect on TG through APO-CIII levels (p=5.2х10 -7 , percent mediation=95%). Conclusion: Our results suggest that the APOC3 A43T SNP effect on TG may be primarily mediated through APO-CIII, and lend further strength to the possibility of making APO-CIII a therapeutic target for CVD interventions. We are in the process of expanding our analysis to a much larger sample.

Background: Type 2 diabetes mellitus (T2DM) is increasingly diagnosed in adolescents and young adults. However, the impact of birth weight on early onset (aged

Aims Insulin-like growth factor 1 receptor (IGF1R) is involved in cell growth and glucose homeostasis. In the current study, the IGF1R locus was analysed as a candidate gene for type 2 diabetes (T2D) in American Indians. Materials and methods Whole genome sequence data from 335 American Indians identified 3 novel missense variants in IGF1R. The associations of IGF1R variants with T2D, age of T2D onset and birth weight were analysed in a population-based sample of 7701 American Indians. Results A novel glycine-to-aspartic acid substitution (G310D) in IGF1R was identified, which associated with T2D in a sex-specific manner (Psex interaction = 0.02). In women, the aspartic acid (D) allele (frequency = 0.034) was associated with increased risk for T2D (n = 4292, P = 2.0 × 10-5 adjusted for age, birth year, and the first 5 genetic principal components; odds ratio [OR] = 2.23 [1.54-3.23] per risk allele) and an earlier age of T2D onset (n = 4292, P = 2 × 10-4 , hazard rate ratio = 1.45 [1.20-1.75], Psex interaction = 0.05). Female carriers of the D-allele also had lower birth weight (n = 1313, β = -163 g, P = .006, Psex interaction = 0.008). Among 85 siblings discordant for G310D, carriers of the D-allele had shorter stature as compared with carriers of the G-allele (β = -1.6 cm, P = .001, within family model). The G310D variant was functionally studied in vitro, where the D-allele had a 22% increase (P = .0005) in FOXO1-induced transcriptional activity, due to decreased activation of the PI3K/AKT pathway mediated through reduced IGF1R activity. Conclusion A unique G310D variant in IGF1R, which occurs in 6% American Indians, may impair IGF1R signalling pathways, thereby increasing the risk of T2D.

OBJECTIVES: To illustrate the difficulties in optimal growth monitoring of children with severe obesity or underweight by using the Centers for Disease Control and Prevention (CDC) 2000 age- and sex-specific BMI percentile growth charts. We also aimed to examine the utility of a new modified CDC BMI z score chart to monitor growth in children with normal and extreme BMI percentiles by using real-life clinical scenarios. METHODS: Modified BMI z score charts were created by using the 2000 CDC algorithm. Three cases of children with extreme BMI values and abnormal growth patterns were plotted by using the standard CDC 2000 clinical growth chart, the modified BMI z score chart, and the CDC BMI percentile chart, modified to include the percentage of the 95th percentile (%BMIp95) curves. RESULTS: Children with severe obesity could not be plotted on the standard CDC BMI percentile chart because their BMI points lay above the chart cutoff. Children with a low BMI ( CONCLUSIONS: The modified CDC z score chart is suitable for growth tracking of children with normal and extreme growth patterns; the measures correlate well with the %BMIp95, and the chart can be incorporated easily into existing electronic health record systems for clinical use.

Background— Identity-by-descent mapping using empirical estimates of identity-by-descent allele sharing may be useful for studies of complex traits in founder populations, where hidden relationships may augment the inherent genetic information that can be used for localization. Methods and Results— Through identity-by-descent mapping, using ≈400 000 single-nucleotide polymorphisms (SNPs), of serum lipid profiles, we identified a major linkage signal for triglycerides in 1007 Pima Indians (LOD=9.23; P =3.5×10 −11 on chromosome 11q). In subsequent fine-mapping and replication association studies in ≈7500 Amerindians, we determined that this signal reflects effects of a loss-of-function Ala43Thr substitution in APOC3 (rs147210663) and 3 established functional SNPs in APOA5 . The association with rs147210663 was particularly strong; each copy of the Thr allele conferred 42% lower triglycerides (β=−0.92±0.059 SD unit; P =9.6×10 −55 in 4668 Pimas and 2793 Southwest Amerindians combined). The Thr allele is extremely rare in most global populations but has a frequency of 2.5% in Pimas. We further demonstrated that 3 APOA5 SNPs with established functional impact could explain the association with the most well-replicated SNP (rs964184) for triglycerides identified by genome-wide association studies. Collectively, these 4 SNPs account for 6.9% of variation in triglycerides in Pimas (and 4.1% in Southwest Amerindians), and their inclusion in the original linkage model reduced the linkage signal to virtually null. Conclusions— APOC3/APOA5 constitutes a major locus for serum triglycerides in Amerindians, especially the Pimas, and these results provide an empirical example for the concept that population-based linkage analysis is a useful strategy to identify complex trait variants.

Objective A prior genome-wide association study (GWAS) in Pima Indians identified variation within FOXO1A that modestly associated with early-onset (onset age

Objective Identifying groups of individuals with similar patterns of body mass index (BMI) change during childhood may increase understanding of the relationship between childhood BMI and adult health. Methods Discrete classes of BMI z-score change were determined in 1,920 American Indian children with at least four non diabetic health examinations between the ages of 2 and 18 years using latent class trajectory analysis. In subsets of subjects, data were available for melanocortin-4 receptor (MC4R) sequencing; in utero exposure to type 2 diabetes (T2D); or, as adults, oral glucose tolerance tests, onset of T2D, or body composition. Results Six separate groups were identified. Individuals with a more modern birth year, an MC4R mutation, or in utero exposure to T2D were clustered in the two groups with high increasing and chronic overweight z-scores (P

OBJECTIVE The global epidemics of type 2 diabetes and obesity have been attributed to the interaction between lifestyle changes and genetic predisposition to these diseases. We compared the prevalences of type 2 diabetes and obesity in Mexican Pima Indians, presumed to have a high genetic predisposition to these diseases, to those in their non-Pima neighbors, both of whom over a 15-year period experienced a transition from a traditional to a more modern lifestyle. RESEARCH DESIGN AND METHODS Prevalence of diabetes, impaired fasting glucose, impaired glucose tolerance, and obesity in Mexican Pimas (n = 359) and non-Pima Mexicans (n = 251) were determined in 2010 using methods identical to those used in 1995. RESULTS During this 15-year period, age-adjusted diabetes prevalence was unchanged in Pima men (5.8% in 1995 vs. 6.1% in 2010) yet increased in non-Pima men from 0.0 to 8.6% (P < 0.05). Diabetes prevalence tended to increase in both Pima women (9.4 vs. 13.4%) and non-Pima women (4.8 vs. 9.5%). Age-adjusted prevalence of obesity increased significantly in all groups (6.6 vs. 15.7% in Pima men; 8.5 vs. 20.5% in non-Pima men; 18.9. vs 36.3% in Pima women; 29.5 vs. 42.9% in non-Pima women). CONCLUSIONS Type 2 diabetes prevalence increased between 1995 and 2010 in non-Pima men, and to a lesser degree in women of both groups, but it did not increase in Pima men. Prevalence of obesity increased among Pimas and non-Pimas of both sexes. These changes occurred concomitantly with an environmental transition from a traditional to a more modernized lifestyle.

Background In Caucasians, lower triglycerides (TG), total or LDL cholesterol and high HDL cholesterol are generally associated with lower mortality. However, low cholesterol is associated with higher mortality in some Asian populations. This study examines the relationship between serum lipids and mortality in American Indians. Methods 2125 American Indians aged ≥ 40 years were examined biennially between 1993 and 2007. Vital status was determined through 2011. Mortality rates, adjusted for age, sex and diabetes, were calculated using Poisson regression. Results The median baseline age was 46 years and 61% were women. Over a median follow-up of 10.1 years, 522 deaths occurred. Relationships between baseline lipids, except for HDL cholesterol, and all-cause mortality were negative and linear in persons without diabetes and U-shaped in persons with diabetes. For HDL cholesterol, the relationship was U-shaped in the total cohort. Cardiovascular mortality was positively associated with total, LDL and non-HDL cholesterol whereas lower lipid concentrations were adversely associated with mortality from liver disease or external causes, except for HDL cholesterol, where associations were positive. Conclusion The common belief that low cholesterol and TG are beneficial for health is not universally observed; evidence suggests increased mortality at both ends of the cholesterol and TG distributions.

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.

Identity-by-descent mapping using empirical estimates of identity-by-descent allele sharing may be useful for studies of complex traits in founder populations, where hidden relationships may augment the inherent genetic information that can be used for localization.Through identity-by-descent mapping, using ≈400 000 single-nucleotide polymorphisms (SNPs), of serum lipid profiles, we identified a major linkage signal for triglycerides in 1007 Pima Indians (LOD=9.23

Objective Relationships were examined between persistent organic pollutants (POPs) and incident type 2 diabetes, end-stage renal disease (ESRD) and mortality. Methods In a nested case–control study, 300 persons without diabetes had baseline examinations between 1969 and 1974; 149 developed diabetes (cases) and 151 remained non-diabetic (controls) during 8.0 and 23.1 years of follow-up, respectively. POPs were measured at baseline. ORs for diabetes were computed by logistic regression analysis. The cases were followed from diabetes onset to ESRD, death or 2013. HRs for ESRD and mortality were computed by cause-specific hazard models. Patterns of association were explored using principal components analysis. Results PCB151 increased the odds for incident diabetes, whereas hexachlorobenzene (HCB) was protective after adjusting for age, sex, body mass index, sample storage characteristics, glucose and lipid levels. Associations between incident diabetes and polychlorinatedbiphenyl (PCB) or persistent pesticide (PST) components were mostly positive but non-significant. Among the cases, 29 developed ESRD and 48 died without ESRD. PCB28, PCB49 and PCB44 increased the risk of ESRD after adjusting for baseline demographic and clinical characteristics. Several PCBs and PSTs increased the risk of death without ESRD. The principal components analysis identified PCBs with low-chlorine load positively associated with ESRD and death without ESRD, and several PSTs associated with death without ESRD. Conclusions Most POPs were positively but not significantly associated with incident diabetes. PCB151 was significantly predictive and HCB was significantly protective for diabetes. Among participants with diabetes, low-chlorine PCBs increase the risk of ESRD and death without ESRD, whereas several PSTs predict death without ESRD.

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

Understanding the physiological mechanisms by which common variants predispose to type 2 diabetes requires large studies with detailed measures of insulin secretion and sensitivity. Here we performed the largest genome-wide association study of first-phase insulin secretion, as measured by intravenous glucose tolerance tests, using up to 5,567 individuals without diabetes from 10 studies. We aimed to refine the mechanisms of 178 known associations between common variants and glycemic traits and identify new loci. Thirty type 2 diabetes or fasting glucose–raising alleles were associated with a measure of first-phase insulin secretion at P < 0.05 and provided new evidence, or the strongest evidence yet, that insulin secretion, intrinsic to the islet cells, is a key mechanism underlying the associations at the HNF1A, IGF2BP2, KCNQ1, HNF1B, VPS13C/C2CD4A, FAF1, PTPRD, AP3S2, KCNK16, MAEA, LPP, WFS1, and TMPRSS6 loci. The fasting glucose–raising allele near PDX1, a known key insulin transcription factor, was strongly associated with lower first-phase insulin secretion but has no evidence for an effect on type 2 diabetes risk. The diabetes risk allele at TCF7L2 was associated with a stronger effect on peak insulin response than on C-peptide–based insulin secretion rate, suggesting a possible additional role in hepatic insulin clearance or insulin processing. In summary, our study provides further insight into the mechanisms by which common genetic variation influences type 2 diabetes risk and glycemic traits.