Structural Analysis Of Candidate Genes For Type 2 Diabetes and Obesity

一个基因被认为是皮马印第安人肥胖或2型糖尿病（T2D）的候选基因，如果1)它在与T2D/肥胖相关的途径中具有已知的生理功能，或2)它与另一个人群或动物模型中的糖尿病/肥胖相关。在过去的一年里，我们对几个生理候选基因的所有标记变异进行了直接测序和基因分型，用于与BMI或T2D的关联分析。对从吉拉河印第安社区收集的两个基于人群的大型个体样本进行了基因分型。

A gene is considered a candidate gene for obesity or type 2 diabetes (T2D) in Pima Indians if 1) it has a known physiological function in a pathway relevant to T2D/obesity or 2) it is associated with diabetes/obesity in another human population or in an animal model. In the past year we have directly sequenced and genotyped all tagging variants in several physiologic candidate genes for association analysis with BMI or T2D. Genotyping was performed in two large population-based samples of individuals collected from the Gila River Indian Community. Missense variants in KCNJ11 and ABCC8, which encode the KIR6.2 and SUR1 subunits of the -cell KATP channel, have previously been implicated in type 2 diabetes, neonatal diabetes, and hyperinsulinemic hypoglycemia of infancy (HHI). To determine whether variation in these genes affect risk for type 2 diabetes, or increased birth weight as a consequence of fetal hyperinsulinemia in Pima Indians, missense and common non-coding variants were analyzed in individuals living in the Gila River Indian Community. A R1420H variant in SUR1 (ABCC8) was identified in 3.3% of the population (N = 7,710). R1420H carriers had higher mean birth weights, and a twofold increased risk for type 2 diabetes with a 7 year earlier onset age despite being leaner than non-carriers. One individual homozygous for R1420H was identified; retrospective review of his medical records was consistent with HHI and a diagnosis of diabetes at age 3.5 years. In vitro studies show that the R1420H substitution decreases KATP channel activity. Identification of this loss-of-function variant in ABCC8 with a carrier frequency of 3.3% impacts clinical care since homozygous inheritance and potential HHI will occur in 1/3,600 births in this American Indian population. The PTF1 complex is critical for pancreatic development and maintenance of adult exocrine pancreas. Therefore, we analyzed variation in genes that form this complex, namely PTF1A, RBPJ, and its paralogue RBPJL. A Thr280Met variant (rs200998587, risk allele frequency = 0.03) in RBPJL, identified only in Amerindian-derived populations, had a nominal association with T2D (OR = 1.601.21-2.13 per Met allele, P = 0.001) and age of diabetes onset (HR = 1.401.14-1.72, P = 0.001). Knockdown of Rbpjl in mouse acinar cells resulted in a significant decrease in the expression of Cel and Ctrb. Interestingly, CTRB1/2 is a previously reported T2D locus where the protective allele associates with increased GLP-1 stimulated insulin secretion and higher expression of CTRB1/2. Further in-vitro studies showed 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 protein is less stable. Luciferase assays in HEK293 cells which examined two different RBPJL responsive promoters, including the promoter for CTRB1, identified reduced transactivation by the Met280 RBPJL. Similarly, overexpression of both Met280 and Thr280 RBPJL in mouse acinar cell identified a significant impairment in the expression of Cel when transactivated by the Met280 RBPJL. Therefore, we believe that this functional Thr280Met variant in RBPJL modifies T2D risk by regulating exocrine enzyme expression. We have also identified a novel missense variant (G310D) in the candidate gene IGF1R; this variant occurred in 6% of Pima Indians but did not occur in 2,687 American Indians who also live in Arizona but are from non-Pima tribes. Therefore, we believe this variant, which is not reported in any public databases, may be private to Pima Indians of Arizona. Genotyping of the G310D in a population-based sample of Pima Indians showed that it associates with a 2.3-fold increased risk for T2D, and an earlier age of T2D onset in women, but not men. Consistent with the previously defined role of IGF1R in prenatal and postnatal growth, subjects carrying the T2D risk allele D at G310D also had lower birth weights and shorter stature in adulthood as compared to subjects homozygous for the non-risk allele. Recently we collaborated with A. Bartolomucci who conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human and mouse molecules. Mouse TLQP-21 showed enhanced binding affinity and potency for human C3aR1 compared with human TLQP-21. Consistently, mouse TLQP-21, but not human TLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs. In an ongoing effort to identify genetic variation that contributes to obesity in American Indians, we analyzed known Bardet-Biedl syndrome (BBS) genes for an effect on BMI and leptin signaling. Potentially deleterious variants (CADD score >20) in BBS genes were identified in whole exome sequence data from 6,851 American Indians informative for BMI. Common variants (detected in 10 individuals) were analyzed for association with BMI; rare variants (detected in <10 individuals) were analyzed for mean BMI of carriers. Functional assessment of variants effect on STAT3 activity was performed in vitro. We identified one common variant, rs59252892 (Thr549Ile) in BBS9 that associated with BMI (P=0.0008, =25% increase per risk allele). Among rare variants where carriers were severely obese (mean BMI >40 kg/m2), 4 were in BBS9. In vitro analysis of BBS9 found the Ile-allele at Thr549Ile had a 20% increase in STAT3 activity compared to the Thr-allele (P=0.01). Western blot analysis showed the Ile-allele had a 15% increase in STAT3 phosphorylation (P=0.006). Comparable functional results were observed with Ser545Gly and Val209Leu, but not Leu665Phe and Lys810Glu. Therefore we conclude that functional variants in BBS genes may contribute to polygenic obesity in American Indians. Since insulin-like growth factor binding protein 4 (IGFBP4) is involved in adipogenesis, and IGFBP4 null mice have decreased body fat through decreased PPAR- expression, we recently assessed whether variation in the IGFBP4 coding region influences BMI in humans. Whole exome sequence data from a population-based sample of 6779 American Indians with longitudinal measures of BMI were used to identify variation in IGFBP4 that associated with BMI. A novel variant that predicts a p.Ser76Thr in IGFBP4 (Thr-allele frequency = 0.02) was identified which associated with BMI measurements during childhood and adulthood In vitro functional studies showed that IGFBP4 with the Thr-allele (BMI-increasing) had a decrease in FOXO-induced transcriptional activity, reflecting increased activation of the PI3K/AKT pathway mediated through increased IGF signaling. Over-expression and knock-down of IGFBP4 in OP9 cells during differentiation showed that IGFBP4 upregulates adipogenesis through PPAR, CEBP, AGPAT2 and SREBP1 expression. We propose that this American Indian specific variant in IGFBP4 affects obesity via an increase of IGF signaling.