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The aim of this study is to investigate the potential role of DNA methylation in mediating the increased risk of developing type 2 diabetes in offspring of mothers who had diabetes during pregnancy.
Materials and Methods
Peripheral blood leukocytes were collected from non-diabetic Pima Indians who were either offspring of diabetic mothers (ODM; n = 14) or offspring of nondiabetic mothers (ONDM; n = 14). The two groups were matched for age, sex, age of mother, and fraction of Pima ethnicity. Differentially methylated regions were determined using a MeDIP-chip assay on an Affymetrix Human Tiling 2.0R Array. Data were analyzed using the model based analysis of tiling arrays (MAT) algorithm, and 4883 regions overlapping with putative promoters, were identified as differentially methylated, having met an empirically derived threshold (nominal p < 0.0077). The list of genes with differentially methylated promoters were subjected to KEGG pathway analysis to determine canonical metabolic pathways enriched by these genes.
Pathway analysis of genes with differentially methylated promoters identified the top 3 enriched pathways as maturity onset diabetes of the young (MODY), type 2 diabetes, and Notch signaling. Several genes in these pathways are known to affect pancreatic development and insulin secretion
These findings support the hypothesis that epigenetic changes may increase the risk of type 2 diabetes via an effect on β-cell function in the offspring of mothers with diabetes during pregnancy.
PMCID: PMC3995826  PMID: 24582139
in utero; maternal diabetes; immunoprecipitation; microarray
2.  A Genome-Wide Association Study in American Indians Implicates DNER as a Susceptibility Locus for Type 2 Diabetes 
Diabetes  2013;63(1):369-376.
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 β-cells suggested that DNER regulates expression of notch signaling pathway genes. These studies implicate DNER as a susceptibility gene for T2DM in American Indians.
PMCID: PMC3868048  PMID: 24101674
3.  Strong Parent-of-Origin Effects in the Association of KCNQ1 Variants With Type 2 Diabetes in American Indians 
Diabetes  2013;62(8):2984-2991.
Parent-of-origin effects were observed in an Icelandic population for several genetic variants associated with type 2 diabetes, including those in KLF14 (rs4731702), MOB2 (rs2334499), and KCNQ1 (rs2237892, rs231362). We analyzed parent-of-origin effects for these variants, along with two others in KCNQ1 identified in previous genome-wide association studies (rs2237895, rs2299620), in 7,351 Pima Indians from 4,549 nuclear families; 34% of participants had diabetes. In a subset of 287 normoglycemic individuals, acute insulin secretion was measured by an intravenous glucose tolerance test. Statistically significant (P < 0.05) parent-of-origin effects were seen for association with type 2 diabetes for all variants. The strongest effect was seen at rs2299620 in KCNQ1; the C allele was associated with increased diabetes when maternally derived (odds ratio [OR], 1.92; P = 4.1 × 10−12), but not when paternally derived (OR, 0.93; P = 0.47; P = 9.9 × 10−6 for difference in maternal and paternal effects). A maternally derived C allele also was associated with a 28% decrease in insulin secretion (P = 0.002). This study confirms parent-of-origin effects in the association with type 2 diabetes for variants in KLF14, MOB2, and KCNQ1. In Pima Indians, the effect of maternally derived KCNQ1 variants appears to be mediated through decreased insulin secretion and is particularly strong, accounting for 4% of the variance in liability to diabetes.
PMCID: PMC3717865  PMID: 23630301
4.  Whole Exome Sequencing Identifies Variation in CYB5A and RNF10 Associated with Adiposity and Type 2 Diabetes 
Obesity (Silver Spring, Md.)  2013;22(4):984-988.
Few coding variants in genes associated with type 2 diabetes (T2D) have been identified, and the underlying physiologic mechanisms whereby susceptibility genes influence T2D risk are often unknown. The objective of this study was to identify coding variation that increases risk for T2D via an effect on a pre-diabetic trait.
Design and Methods
Whole exome sequencing was done in 177 Pima Indians. Selected variants (N=345) were genotyped in 555 subjects characterized for body fatness, glucose disposal rates during a clamp, acute insulin response to glucose, and 2-hour plasma glucose concentrations during an OGTT, and were also genotyped in up to 5,880 subjects with longitudinal measures of BMI. Variants associated with quantitative traits were assessed for association with T2D in 7,667 subjects.
rs7238987 in CYB5A associated with body fatness (p=7.0×10−6). This SNP and a novel SNP in RNF10 also associated with maximum recorded BMI (p=6.2×10−7 and p=7.2×10−4) and maximum childhood BMI z-score (p=5.9×10−4 and p=8.5×10−7). The BMI increasing alleles increased risk for T2D (p= 0.01; OR=1.13 [1.03–1.24] and 9.5×10−3, OR=1.49 [1.10–2.02]).
CYB5A, which has a role in stearyl-CoA-desaturase activity, and RNF10, with an unknown role in weight regulating pathways, associated with adiposity and nominally increased risk for T2D in American Indians.
PMCID: PMC3968243  PMID: 24151200
BMI; Exome sequencing; RNF10; CYB5A; SCD1
5.  Effect of Losartan on Prevention and Progression of Early Diabetic Nephropathy in American Indians With Type 2 Diabetes 
Diabetes  2013;62(9):3224-3231.
Angiotensin receptor blockers are renoprotective in hypertensive azotemic patients with type 2 diabetes, but their efficacy in early diabetic kidney disease is uncertain. We performed a 6-year randomized clinical trial in 169 American Indians with type 2 diabetes and normoalbuminuria (albumin/creatinine ratio [ACR] <30 mg/g; n = 91) or microalbuminuria (ACR 30–299 mg/g; n = 78) at baseline. The primary outcome was decline in glomerular filtration rate (GFR) to ≤60 mL/min or to half the baseline value in subjects who entered with GFR <120 mL/min. Another outcome was differences in glomerular structure at end of treatment. Subjects received 100 mg losartan or placebo daily. GFR was measured annually; 111 subjects underwent kidney biopsies. Only nine subjects reached the GFR outcome, and the unadjusted hazard ratio (losartan vs. placebo) was 0.50 (95% CI, 0.12–1.99). Differences in mesangial fractional volume were not estimated in the combined albuminuria groups because of an interaction with treatment assignment. In separate analyses, mesangial fractional volume was lower in subjects treated with losartan in the microalbuminuria group (18.8 vs. 25.6%; P = 0.02), but not in the normoalbuminuria group (19.6 vs. 17.8%; P = 0.86). Treatment with losartan may preserve some features of kidney structure in American Indians with type 2 diabetes and microalbuminuria.
PMCID: PMC3749332  PMID: 23545707
6.  Variants associated with type 2 diabetes identified by the transethnic meta-analysis study: assessment in American Indians and evidence for a new signal in LPP 
Diabetologia  2014;57(11):2334-2338.
A recent genome-wide trans-ancestry meta-analysis identified seven new loci associated with type 2 diabetes. We assessed the replication of the seven lead single nucleotide polymorphisms (SNPs) and evaluated these loci for additional signals in American Indians.
Seven SNPs were genotyped in 7,710 individuals from a longitudinally studied American Indian population, and associations with type 2 diabetes, BMI and related phenotypes were assessed. Previous genome-wide association study (GWAS) data from these individuals were used to screen for additional type 2 diabetes signals at these loci. A variant independent of the trans-ancestry meta-analysis was identified within LPP, and its replication was assessed in an additional 3,106 urban American Indians.
SNP rs6813195 near to TMEM154 was nominally associated with type 2 diabetes (p = 0.01, OR 1.12 [95% CI 1.03, 1.22]) and adiposity: the type 2 diabetes risk allele was associated with a lower percentage body fat (β = −1.451%, p = 4.8 × 10−4). Another SNP, rs3130501 near to POU5F1–TCF19, was associated with BMI (β = −0.012, p = 0.004), type 2 diabetes adjusted for BMI (p = 0.02, OR 1.11 [95% CI 1.02, 1.22]), 2 h glucose concentrations (β = 0.080 mmol/l, p = 0.02) and insulin resistance estimated by homeostatic model (β = 0.039, p = 0.009). The independent variant identified at the LPP locus in our American Indian GWAS for type 2 diabetes was replicated in the additional samples (all American Indian meta-analysis, p = 8.9 × 10−6, OR 1.29 [95% CI 1.15, 1.45]).
For two of the seven newly identified variants, there was nominal evidence for association with type 2 diabetes and related traits in American Indians. Identification of an independent variant at the LPP locus suggests the existence of more than one type 2 diabetes signal at this locus.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3351-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC4180905  PMID: 25112377
American Indians; ARL15; FAF1; GWAS; LPP; MPHOSPH9; POU5F1–TCF19; SSR1–RREB1; Trans-ancestry meta-analysis; Type 2 diabetes
7.  Common genetic variation in and near the melanocortin 4 receptor gene (MC4R) is associated with body mass index in American Indian adults and children 
Human Genetics  2014;133(11):1431-1441.
Six rare functional coding mutations were previously identified in melanocortin 4 receptor (MC4R) in 6,760 American Indians. Individuals heterozygous for one of these mutations become obese while young. We now investigate whether common non-coding variation near MC4R also contributes to obesity. Fifty-six tag single-nucleotide polymorphisms (SNPs) were genotyped in 3,229 full-heritage Pima Indians, and nine of these SNPs which showed evidence for association were genotyped in additional 3,852 mixed-heritage American Indians. Associations of SNPs with maximum body mass index (BMI) in adulthood (n = 5,918), BMI z score in childhood (n = 5,350), percent body fat (n = 864), energy expenditure (n = 358) and ad libitum food intake (n = 178) were assessed. Conditional analyses demonstrated that SNPs, rs74861148 and rs483125, were independently associated with BMI in adulthood (β = 0.68 kg/m2 per risk allele, p = 5 × 10−5; β = 0.58 kg/m2, p = 0.002, respectively) and BMI z score in childhood (β = 0.05, p = 0.02; β = 0.07, p = 0.01, respectively). One haplotype (frequency = 0.35) of the G allele at rs74861148 and the A allele at rs483125 provided the strongest evidence for association with adult BMI (β = 0.89 kg/m2, p = 5.5 × 10−7), and was also associated with childhood BMI z score (β = 0.08, p = 0.001). In addition, a promoter SNP rs11872992 was nominally associated with adult BMI (β = 0.61 kg/m2, p = 0.05) and childhood BMI z score (β = 0.11, p = 0.01), where the risk allele also modestly decreased transcription in vitro by 12 % (p = 0.005). This risk allele was further associated with increased percent body fat (β = 2.2 %, p = 0.002), increased food intake (β = 676 kcal/day, p = 0.007) and decreased energy expenditure (β = −53.4 kcal/day, p = 0.054). Common and rare variation in MC4R contributes to obesity in American Indians.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-014-1477-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4185108  PMID: 25103139
8.  Assessing Accuracy of Genotype Imputation in American Indians 
PLoS ONE  2014;9(7):e102544.
Genotype imputation is commonly used in genetic association studies to test untyped variants using information on linkage disequilibrium (LD) with typed markers. Imputing genotypes requires a suitable reference population in which the LD pattern is known, most often one selected from HapMap. However, some populations, such as American Indians, are not represented in HapMap. In the present study, we assessed accuracy of imputation using HapMap reference populations in a genome-wide association study in Pima Indians.
Data from six randomly selected chromosomes were used. Genotypes in the study population were masked (either 1% or 20% of SNPs available for a given chromosome). The masked genotypes were then imputed using the software Markov Chain Haplotyping Algorithm. Using four HapMap reference populations, average genotype error rates ranged from 7.86% for Mexican Americans to 22.30% for Yoruba. In contrast, use of the original Pima Indian data as a reference resulted in an average error rate of 1.73%.
Our results suggest that the use of HapMap reference populations results in substantial inaccuracy in the imputation of genotypes in American Indians. A possible solution would be to densely genotype or sequence a reference American Indian population.
PMCID: PMC4094523  PMID: 25014012
9.  Comparison of Serum Cystatin C, Serum Creatinine, Measured GFR, and Estimated GFR to Assess the Risk of Kidney Failure in American Indians With Diabetic Nephropathy 
We compared values of baseline serum cystatin C (SCysC), serum creatinine (SCr), and measured GFR (mGFR) for predicting end-stage renal disease (ESRD) in patients with type 2 diabetes and elevated albuminuria.
Study Design
Observational longitudinal study.
Setting & Participants
Pima Indians with type 2 diabetes and elevated albumin/creatinine ratio (ACR ≥ 30 mg/g).
Baseline SCysC, SCr, and mGFR.
Outcomes & Measurements
Individuals were followed from their first diabetic examination with ACR ≥ 30 mg/g until December 2010, onset of ESRD, or death, whichever came first. Incidence rates adjusted for age, and sex were computed by Mantel-Haenszel stratification. The abilities of SCysC, SCr, and mGFR to predict ESRD were compared with receiver operating characteristic curves.
Of 234 Pima Indians with a mean age of 42.8 years who were followed for a median of 10.7 (range, 0.6–21.3) years, 68 (29%) developed ESRD. The incidence of ESRD was significantly higher among patients in the lowest vs. highest tertile of 1/SCysC (incidence rate ratio, 2.43; 95%CI, 1.31–4.50). By contrast, mGFR and 1/SCr had J-shaped associations with ESRD. In unadjusted analyses, 1/SCysC had the highest area under the receiver operating characteristic curve (AUROC; 0.719±0.035) and mGFR the lowest (0.585±0.042; P<0.001); the AUROC for 1/SCr was intermediate (0.672±0.040; P of 0.1 and 0.03 vs 1/SCysC and mGFR, respectively). In analyses adjusted for age, sex, diabetes duration, height, weight, hemoglobin A1c, and ACR, 1/SCysC had the highest AUROC (0.845±0.026). Models with mGFR or 1/SCr alone had similar AUROCs (0.815±0.028) and both were lower than the model with 1/SCysC alone (P=0.02 and P=0.03, respectively).
The predictive values of the filtration markers are limited to the extent that their precision is based on a single measurement.
SCysC was a better predictor of ESRD than mGFR or SCr in Pima Indians with type 2 diabetes and elevated albuminuria.
PMCID: PMC3664248  PMID: 23347458
11.  Secular Trends in Treatment and Control of Type 2 Diabetes in an American Indian Population: A 30-Year Longitudinal Study 
Diabetes Care  2010;33(11):2383-2389.
Treatment guidelines for diabetes have become increasingly stringent as most research shows that more aggressive intervention reduces the risks for complications. Community data on the effect of these interventions are lacking.
Changes in the pharmacologic treatment of diabetes, blood pressure, and cholesterol in adults with diabetes were analyzed in a longitudinal population-based study of American Indians from 10 independent 3-year time intervals between 1975 and 2004. Trends in drug use were assessed by logistic regression models and trends in glycemia, blood pressure, and cholesterol were assessed by linear models.
Among the study participants, the use of any medicine for the treatment of diabetes increased from 53% in 1975–1978 to 67% in 2002–2004, Ptrend < 0.0001. The use of insulin as a single agent declined, and the use of combinations of insulin and oral agents increased. In 1990–1992, 23% of subjects had an A1C <7% and by 2002–2004, the proportion had increased to 33%, Ptrend < 0.0001. The use of anti-hypertensive medicine increased from 21% in 1975–1977 to 58% in 2002–2004, Ptrend < 0.0001, coincident with a decline in mean systolic blood pressure from 137 mmHg in 1975–1977 to 123 mmHg in 2002–2004, Ptrend < 0.0001. The use of lipid-lowering medicine also increased with an accompanying increase in HDL and a decrease in non-HDL cholesterol concentration.
Major changes in community treatment patterns for diabetes and related conditions coincided with improvements in glycemia, blood pressure, and cholesterol.
PMCID: PMC2963499  PMID: 20855550
12.  Common genetic variation in the glucokinase gene (GCK) is associated with type 2 diabetes and rates of carbohydrate oxidation and energy expenditure 
Diabetologia  2014;57(7):1382-1390.
Glucokinase (GCK) plays a role in glucose metabolism and glucose-stimulated insulin secretion. Rare mutations in GCK cause MODY. We investigated whether common variation (minor allele frequency ≥0.01) in GCK is associated with metabolic traits and type 2 diabetes.
Four exonic single-nucleotide polymorphisms (SNPs) and three SNPs predicted to cause loss of promoter function were identified in whole-genome sequence data from 234 Pima Indians. These seven tag SNPs and rs4607517, a type 2 diabetes variant established in other studies, were analysed in 415 full-heritage non-diabetic Pima Indians characterised for metabolic traits, and 7,667 American Indians who had data on type 2 diabetes and BMI.
A novel 3′ untranslated region (3′UTR) SNP, chr7:44184184-G/A, was associated with the rate of carbohydrate oxidation post-absorptively (β = 0.22 mg [kg estimated metabolic body size (EMBS)]−1 min−1, p = 0.005) and during a hyperinsulinaemic–euglycaemic clamp (β = 0.24 mg [kg EMBS]−1 min−1, p = 0.0002), the rate of carbohydrate oxidation in a respiratory chamber (β = 311 kJ/day, p = 0.03) and 24 h energy expenditure, which was attributable to the thermic effect of food (β = 520 kJ/day, p = 3.39 × 10−6). This 3′UTR SNP was also associated with diabetes (OR 1.36, 95% CI 1.11, 1.65, p = 0.002), where the A allele (allele frequency 0.05) was associated with a lower rate of carbohydrate oxidation, lower 24 h energy expenditure and higher risk for diabetes. In a Cox proportional hazards model, a rate of insulin-stimulated carbohydrate oxidation lower than the mean rate at baseline predicted a higher risk for developing diabetes than for those above the mean (hazard rate ratio 2.2, 95% CI 1.3, 3.6, p = 0.002).
Common variation in GCK influences the rate of carbohydrate oxidation, 24 h energy expenditure and diabetes risk in Pima Indians.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3234-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC4052004  PMID: 24728127
Carbohydrate oxidation; Energy expenditure; GCK; Thermic effect of food; Type 2 diabetes
13.  A Genome-Wide Search for Linkage of Estimated Glomerular Filtration Rate (eGFR) in the Family Investigation of Nephropathy and Diabetes (FIND) 
PLoS ONE  2013;8(12):e81888.
Estimated glomerular filtration rate (eGFR), a measure of kidney function, is heritable, suggesting that genes influence renal function. Genes that influence eGFR have been identified through genome-wide association studies. However, family-based linkage approaches may identify loci that explain a larger proportion of the heritability. This study used genome-wide linkage and association scans to identify quantitative trait loci (QTL) that influence eGFR.
Genome-wide linkage and sparse association scans of eGFR were performed in families ascertained by probands with advanced diabetic nephropathy (DN) from the multi-ethnic Family Investigation of Nephropathy and Diabetes (FIND) study. This study included 954 African Americans (AA), 781 American Indians (AI), 614 European Americans (EA) and 1,611 Mexican Americans (MA). A total of 3,960 FIND participants were genotyped for 6,000 single nucleotide polymorphisms (SNPs) using the Illumina Linkage IVb panel. GFR was estimated by the Modification of Diet in Renal Disease (MDRD) formula.
The non-parametric linkage analysis, accounting for the effects of diabetes duration and BMI, identified the strongest evidence for linkage of eGFR on chromosome 20q11 (log of the odds [LOD] = 3.34; P = 4.4×10−5) in MA and chromosome 15q12 (LOD = 2.84; P = 1.5×10−4) in EA. In all subjects, the strongest linkage signal for eGFR was detected on chromosome 10p12 (P = 5.5×10−4) at 44 cM near marker rs1339048. A subsequent association scan in both ancestry-specific groups and the entire population identified several SNPs significantly associated with eGFR across the genome.
The present study describes the localization of QTL influencing eGFR on 20q11 in MA, 15q21 in EA and 10p12 in the combined ethnic groups participating in the FIND study. Identification of causal genes/variants influencing eGFR, within these linkage and association loci, will open new avenues for functional analyses and development of novel diagnostic markers for DN.
PMCID: PMC3866106  PMID: 24358131
14.  MAP2K3 is associated with body mass index in American Indians and Caucasians and may mediate hypothalamic inflammation 
Human Molecular Genetics  2013;22(21):4438-4449.
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.
PMCID: PMC3792696  PMID: 23825110
15.  Variants in the LEPR gene are nominally associated with higher BMI and lower 24 hour energy expenditure in Pima Indians 
Obesity (Silver Spring, Md.)  2012;20(12):2426-2430.
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 high a prevalence of both diseases. In these studies, a variant (rs2025804) in the LEPR gene was nominally associated with BMI in 1082 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=2842) 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=2969) and 3 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 hour energy expenditure 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 non-coding variation in the LEPR gene is associated with higher BMI and lower energy expenditure in Native Americans.
PMCID: PMC3479320  PMID: 22810975
16.  Identification of genetic variation that determines human trehalase activity and its association with type 2 diabetes 
Human Genetics  2013;132(6):697-707.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-013-1278-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3654185  PMID: 23468175
17.  An ACACB Variant Implicated in Diabetic Nephropathy Associates with Body Mass Index and Gene Expression in Obese Subjects 
PLoS ONE  2013;8(2):e56193.
Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m2 (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m2 (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m2; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m2 for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.
PMCID: PMC3584087  PMID: 23460794
18.  Genetic Association and Gene-Gene Interaction Analyses in African American Dialysis Patients With Nondiabetic Nephropathy 
African Americans (AAs) have increased susceptibility to non-diabetic nephropathy relative to European Americans.
Study Design
Follow-up of a pooled genome-wide association study (GWAS) in AA dialysis patients with nondiabetic nephropathy; novel gene-gene interaction analyses.
Setting & Participants
Wake Forest sample: 962 AA nondiabetic nephropathy cases; 931 non-nephropathy controls. Replication sample: 668 Family Investigation of Nephropathy and Diabetes (FIND) AA nondiabetic nephropathy cases; 804 non-nephropathy controls.
Individual genotyping of top 1420 pooled GWAS-associated single nucleotide polymorphisms (SNPs) and 54 SNPs in six nephropathy susceptibility genes.
APOL1 genetic association and additional candidate susceptibility loci interacting with, or independently from, APOL1.
The strongest GWAS associations included two non-coding APOL1 SNPs, rs2239785 (odds ratio [OR], 0.33; dominant; p = 5.9 × 10−24) and rs136148 (OR, 0.54; additive; p = 1.1 × 10−7) with replication in FIND (p = 5.0 × 10−21 and 1.9 × 10−05, respectively). Rs2239785 remained significantly associated after controlling for the APOL1 G1 and G2 coding variants. Additional top hits included a CFH SNP(OR from meta-analysis in above 3367 AA cases and controls, 0.81; additive; p = 6.8 × 10−4). The 1420 SNPs were tested for interaction with APOL1 G1 and G2 variants. Several interactive SNPs were detected, the most significant was rs16854341 in the podocin gene (NPHS2) (p = 0.0001).
Non-pooled GWAS have not been performed in AA nondiabetic nephropathy.
This follow-up of a pooled GWAS provides additional and independent evidence that APOL1 variants contribute to nondiabetic nephropathy in AAs and identified additional associated and interactive non-diabetic nephropathy susceptibility genes.
PMCID: PMC3259209  PMID: 22119407
African American; APOL1; CFH; end-stage renal disease; FIND; FSGS; hypertension
19.  Greater Impact of Melanocortin-4 Receptor Deficiency on Rates of Growth and Risk of Type 2 Diabetes During Childhood Compared With Adulthood in Pima Indians 
Diabetes  2011;61(1):250-257.
Features of melanocortin-4 receptor (MC4R) deficiency have been observed to be more pronounced in childhood. Longitudinal data from a population-based study were used to separate the phenotypic effects of MC4R deficiency during childhood and adulthood. The MC4R exon was sequenced in 6,760 individuals of predominantly Pima Indian heritage, and discovered mutations were functionally assessed in vitro. Effects on BMI, height, and slope of BMI change were assessed during childhood (ages 5–20 years) and adulthood (ages 20–45 years). Six mutations affecting MC4R function, including three that may be private to Pima Indians, were found in 159 individuals (2.4%). The slope of BMI increase was greater in individuals carrying an MC4R mutation compared with noncarriers during childhood but not during adulthood. The final adult height obtained was higher in individuals with MC4R deficiency. There was an increased risk for developing type 2 diabetes in individuals with a defective MC4R during childhood and adulthood, but this was only independent of BMI in childhood. The greater rates of body mass accumulation and risk of type 2 diabetes before the age of 20 years in individuals with MC4R deficiency indicate that the effects of these mutations are more apparent during the active growth of childhood.
PMCID: PMC3237672  PMID: 22106157
20.  SIRT1 is Associated with a Decrease in Acute Insulin Secretion and a Sex Specific Increase in Risk for Type 2 Diabetes in Pima Indians 
Molecular genetics and metabolism  2011;104(4):661-665.
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.
PMCID: PMC3224181  PMID: 21871827
SIRT1; type 2 diabetes; insulin secretion; genetic associations
21.  Predictive value of albuminuria in American Indian youth with or without type 2 diabetes 
Pediatrics  2010;125(4):e844-e851.
We examined the prognostic significance of elevated albuminuria in youth with type 2 diabetes.
Patients and Methods
Cross-sectional and prospective studies were conducted in Pima Indian youth aged 5-19 years at baseline who were examined between July 1, 1982 and December 31, 2007. Prevalence and sequential changes in the level of microalbuminuria (30≤ albumin-to-creatinine ratio <300 mg/g) and macroalbuminuria (albumin-to-creatinine ratio ≥300 mg/g) and incidence of macroalbuminuria were computed according to the presence or absence of type 2 diabetes.
The prevalence of micro- and macroalbuminuria was 6.5% and 0.6% in the 3,856 nondiabetic youth and 18.5% and 2.9% in the 103 youth with diabetes. One-hundred-forty-one (75.4%) of 187 nondiabetic youth, but only one (7.1%) of 14 diabetic youth with elevated albumin-to-creatinine ratio (≥30 mg/g) regressed to undetectable or normal albumin-to-creatinine ratio (<30 mg/g) on subsequent examination. In a subset of 2,666 youth with a median follow-up of 8.1 years, 36 nondiabetic and 30 diabetic youth with baseline albumin-to-creatinine ratio <300 mg/g developed macroalbuminuria. For a given albumin-to-creatinine ratio level, the incidence of macroalbuminuria was 15.9-fold (95% CI = 11.1 to 22.6) higher in the diabetic than in the nondiabetic youth.
Elevated albuminuria is infrequent and largely transient in nondiabetic youth, but is relatively frequent and largely persistent in those with diabetes. Microalbuminuria in youth with type 2 diabetes strongly predicts progression to macroalbuminuria, supporting annual screening for albuminuria.
PMCID: PMC3481836  PMID: 20194283
diabetic nephropathy; epidemiology; incidence; longitudinal; prevalence; risk factors
22.  Diabetic Nephropathy in American Indians, with a Special Emphasis on the Pima Indians 
Current diabetes reports  2008;8(6):486-493.
Diabetes affects American Indians disproportionately compared with other racial/ethnic groups in the United States and is almost exclusively type 2 diabetes. Much of our knowledge about diabetes in American Indians comes from studies in a few tribes. The most extensively studied American Indians are the Pima Indians from the Gila River Indian Community in Arizona, who participated in a longitudinal study of diabetes and its complications between 1965 and 2007. They have one of the highest reported incidence and prevalence of type 2 diabetes in the world, and kidney disease attributable to diabetes is a major cause of morbidity and mortality. In this article, we examine the course, determinants, and trends of diabetic kidney disease in American Indians, with special emphasis on studies conducted in the Pima Indians. We also review therapeutic strategies for managing diabetic kidney disease.
PMCID: PMC3480511  PMID: 18990306
23.  New Susceptibility Loci Associated with Kidney Disease in Type 1 Diabetes 
Sandholm, Niina | Salem, Rany M. | McKnight, Amy Jayne | Brennan, Eoin P. | Forsblom, Carol | Isakova, Tamara | McKay, Gareth J. | Williams, Winfred W. | Sadlier, Denise M. | Mäkinen, Ville-Petteri | Swan, Elizabeth J. | Palmer, Cameron | Boright, Andrew P. | Ahlqvist, Emma | Deshmukh, Harshal A. | Keller, Benjamin J. | Huang, Huateng | Ahola, Aila J. | Fagerholm, Emma | Gordin, Daniel | Harjutsalo, Valma | He, Bing | Heikkilä, Outi | Hietala, Kustaa | Kytö, Janne | Lahermo, Päivi | Lehto, Markku | Lithovius, Raija | Österholm, Anne-May | Parkkonen, Maija | Pitkäniemi, Janne | Rosengård-Bärlund, Milla | Saraheimo, Markku | Sarti, Cinzia | Söderlund, Jenny | Soro-Paavonen, Aino | Syreeni, Anna | Thorn, Lena M. | Tikkanen, Heikki | Tolonen, Nina | Tryggvason, Karl | Tuomilehto, Jaakko | Wadén, Johan | Gill, Geoffrey V. | Prior, Sarah | Guiducci, Candace | Mirel, Daniel B. | Taylor, Andrew | Hosseini, S. Mohsen | Parving, Hans-Henrik | Rossing, Peter | Tarnow, Lise | Ladenvall, Claes | Alhenc-Gelas, François | Lefebvre, Pierre | Rigalleau, Vincent | Roussel, Ronan | Tregouet, David-Alexandre | Maestroni, Anna | Maestroni, Silvia | Falhammar, Henrik | Gu, Tianwei | Möllsten, Anna | Cimponeriu, Danut | Ioana, Mihai | Mota, Maria | Mota, Eugen | Serafinceanu, Cristian | Stavarachi, Monica | Hanson, Robert L. | Nelson, Robert G. | Kretzler, Matthias | Colhoun, Helen M. | Panduru, Nicolae Mircea | Gu, Harvest F. | Brismar, Kerstin | Zerbini, Gianpaolo | Hadjadj, Samy | Marre, Michel | Groop, Leif | Lajer, Maria | Bull, Shelley B. | Waggott, Daryl | Paterson, Andrew D. | Savage, David A. | Bain, Stephen C. | Martin, Finian | Hirschhorn, Joel N. | Godson, Catherine | Florez, Jose C. | Groop, Per-Henrik | Maxwell, Alexander P.
PLoS Genetics  2012;8(9):e1002921.
Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.
Author Summary
The global prevalence of diabetes has reached epidemic proportions, constituting a major health care problem worldwide. Diabetic kidney disease, or diabetic nephropathy (DN)—the major long term microvascular complication of diabetes—is associated with excess mortality among patients with type 1 diabetes. Even though DN has been shown to cluster in families, the underlying genetic and molecular pathways remain poorly defined. We have undertaken the largest genome-wide association study and meta-analysis to date on DN and on its most severe form of kidney disease, end-stage renal disease (ESRD). We identified new loci significantly associated with diabetic ESRD: AFF3 and an intergenic locus on chromosome 15q26 residing between RGMA and MCTP2. Our functional analyses suggest that AFF3 influences renal tubule fibrosis, a pathological hallmark of severe DN. Another locus in ERBB4 was suggestively associated with DN and resides in the same intronic region as a variant affecting the expression of ERBB4. Subsequent pathway analysis of the genes co-expressed with ERBB4 indicated involvement of fibrosis.
PMCID: PMC3447939  PMID: 23028342
24.  Association of variants in the carnosine peptidase 1 gene (CNDP1) with diabetic nephropathy in American Indians 
Molecular genetics and metabolism  2011;103(2):185-190.
CNDP1 is located on 18q22.3, where linkage with diabetic nephropathy has been observed in several populations, including Pima Indians. However, evidence for association between CNDP1 alleles and diabetic nephropathy is equivocal and population-dependent. This study investigated CNDP1 as a candidate for diabetic kidney disease in Pima Indians. Nineteen tag single nucleotide polymorphisms spanning the CNDP1 locus were selected using genotype data from Chinese individuals in the HapMap resource along with 2 variants previously associated with diabetic nephropathy. All variants were genotyped in 3 different samples including a diabetic end-stage renal disease (ESRD) case-control study, a family-based study of diabetic individuals who participated in the linkage study for nephropathy, and a cohort of diabetic individuals in whom longitudinal measures of glomerular filtration rates (GFR) were performed. There was no statistically significant evidence for association with diabetic ESRD. However, nominal evidence for association was found in the family study, where markers rs12957330 (Odds ratio [OR]=0.29 per copy of G allele; p=0.04) and rs17817077 (OR=0.46 per copy of G allele; p=0.05) were associated with diabetic nephropathy. In addition, markers rs12964454, rs7244647, and rs7229005 were associated with changes in GFR (−8.5 ml/min per copy of the G allele; p=0.04; 18.8 ml/min per copy of the C allele; p=0.03; and −13.4 ml/min per copy of the C allele; p=0.001, respectively). These findings provide nominal evidence supporting a role between CNDP1 variants and diabetic kidney disease.
PMCID: PMC3101283  PMID: 21393041
kidney disease; single nucleotide polymorphism; proteinuria
25.  Genomewide Linkage Scan for Diabetic Renal Failure and Albuminuria: The FIND Study 
American Journal of Nephrology  2011;33(5):381-389.
Diabetic nephropathy (DN) is a leading cause of mortality and morbidity in patients with type 1 and type 2 diabetes. The multicenter FIND consortium aims to identify genes for DN and its associated quantitative traits, e.g. the urine albumin:creatinine ratio (ACR). Herein, the results of whole-genome linkage analysis and a sparse association scan for ACR and a dichotomous DN phenotype are reported in diabetic individuals.
A genomewide scan comprising more than 5,500 autosomal single nucleotide polymorphism markers (average spacing of 0.6 cM) was performed on 1,235 nuclear and extended pedigrees (3,972 diabetic participants) ascertained for DN from African-American (AA), American-Indian (AI), European-American (EA) and Mexican-American (MA) populations.
Strong evidence for linkage to DN was detected on chromosome 6p (p = 8.0 × 10−5, LOD = 3.09) in EA families as well as suggestive evidence for linkage to chromosome 7p in AI families. Regions on chromosomes 3p in AA, 7q in EA, 16q in AA and 22q in MA displayed suggestive evidence of linkage for urine ACR. The linkage peak on chromosome 22q overlaps the MYH9/APOL1 gene region, previously implicated in AA diabetic and nondiabetic nephropathies.
These results strengthen the evidence for previously identified genomic regions and implicate several novel loci potentially involved in the pathogenesis of DN.
PMCID: PMC3078269  PMID: 21454968
Albuminuria; Diabetes mellitus; Renal failure; End-stage renal disease; Linkage; Allelic association

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