A novel antisense lncRNA, ARBAG harboring an RNA destabilizing GWAS variant for C-peptide dictates the transcript isoforms of GABRA6 in cerebellum.

Human disease-associated genetic variations often map to long non-coding RNA (lncRNA) genes; however, elucidation of their functional impact is challenging. We previously identified a new genetic variant rs4454083 (A/G) residing in exon of an uncharacterized lncRNA ARBAG that strongly associates with plasma levels of C-peptide, a hormone that regulates insulin bioavailability. On the opposite strand, rs4454083 also corresponds to an intron of a cerebellum-specific GABA receptor subunit gene GABRA6 that mediates strengthening of inhibitory synapses by insulin. Here, we show that alleles of rs4454083 modulate transcript levels of the antisense gene, ARBAG, which then controls the expression of the sense gene, GABRA6. Predisposing to low C-peptide, GG (a minor allele genotype across ethnicities) stabilizes ARBAG lncRNA causing higher transcript levels in cerebellum. ARBAG lncRNA abundance leads to cleavage of GABRA6 mRNA at the complementary region, resulting in a dysfunctional GABRA6 protein that would not be recruited for synapse strengthening. Together, our findings in human cerebellar cell-line and induced Pluripotent Stem Cells (iPSCs) demonstrate biological role of a novel lncRNA in determining the ratio of mRNA isoforms of a protein-coding gene and the ability of an embedded variant in modulating lncRNA stability leading to inter-individual differences in protein expression.

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

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

Functional characterization of a novel p.Ser76Thr variant in IGFBP4 that associates with body mass index in American Indians.

Insulin-like growth factor binding protein 4 (IGFBP4) is involved in adipogenesis, and IGFBP4 null mice have decreased body fat through decreased PPAR-γ expression. In the current study, we assessed whether variation in the IGFBP4 coding region influences body mass index (BMI) in American Indians who are disproportionately affected by obesity. 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 the maximum BMI measured during adulthood (BMI 39.8 kg/m2 for Thr-allele homozygotes combined with heterozygotes vs. 36.2 kg/m2 for Ser-allele homozygotes, ß = 6.7% per Thr-allele, p = 8.0 × 10-5, adjusted for age, sex, birth-year and the first five genetic principal components) and the maximum age- and sex-adjusted BMI z-score measured during childhood/adolescence (z-score 0.70 SD for Thr-allele heterozygotes vs. 0.32 SD for Ser-allele homozygotes, ß = 0.37 SD per Thr-allele, p = 8.8 × 10-6). In vitro functional studies showed that IGFBP4 with the Thr-allele (BMI-increasing) had a 55% decrease (p = 0.0007) 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 PPARγ, CEBPα, AGPAT2 and SREBP1 expression. We propose that this American Indian specific variant in IGFBP4 affects obesity via an increase of IGF signaling.

Harnessing the Potential of Long Non-coding RNAs to Manage Metabolic Diseases.

Long non-coding RNAs (lncRNAs) are functionally versatile molecules that regulate gene expression at all levels of biological organization. RNA modulation, at the moment, has emerged as a powerful therapeutic technique to treat human diseases. Lately, lncRNAs have been acknowledged as key players in human metabolism and, indeed, implicated in the etiology of many common diseases other than cancers, where they can perhaps serve as reliable markers to determine disease status or assess outcomes of an intervention. Here, in this review, we cite examples of such lncRNAs, discuss their mechanistic role in human diseases and their genetic association, quote potential biomarkers found in human blood, summarize the methods for therapeutic targeting lncRNAs and examine the progress of lncRNA based drugs in clinical trials. Thus, we propose that lncRNAs serve as both a biomarker and an effective therapeutic target with promising clinical utility to manage human metabolic diseases.

Genetic variants entail type 2 diabetes as an innate immune disorder.

Type 2 Diabetes (T2D) is characterized by alteration in the circulatory levels of key inflammatory proteins, where our body strives to eliminate the perturbing factor through inflammation as a final resort to restore homeostasis. Plasma proteins play a crucial role to orchestrate this immune response. Over the past two decades, rigorous genetic efforts taken to comprehend T2D physiology have been partially successful and have left behind a dearth of knowledge of its causality. Here, we have investigated how the reported genetic variants of T2D are associated with circulatory levels of key plasma proteins. We identified 99 T2D genetic variants that serve as strong pQTL (protein Quantitative Trait Loci) for 72 plasma proteins, of which 4 proteins namely Small nuclear ribonucleoprotein F [SNRPF] (p = 2.99 × 10-14), Platelet endothelial cell adhesion molecule [PECAM1] (p = 1.9 × 10-45), Trypsin-2 [PRSS2] (p = 7.6 × 10-43) and Trypsin-3 [PRSS3] (p = 5.7 × 10-8) were previously not reported for association to T2D. The genes that encode these 72 proteins were observed to be highly expressed in at least one of the four T2D relevant tissues - liver, pancreas, adipose and whole blood. Comparative analysis of interactions of the studied proteins amongst these four tissues revealed distinct molecular connectivity. Assessment of biological function by gene-set enrichment highlighted innate immune system as the lead process enacted by the identified proteins (FDR q = 3.7 × 10-16). To validate the findings, we analyzed Coronary Artery Disease (CAD) and Rheumatoid Arthritis (RA) individually and as expected, we observed innate immune system as a top enriched pathway for RA but not for CAD. Our study illuminates strong regulation of plasma proteome by the established genetic variants of T2D.

Multifaceted genome-wide study identifies novel regulatory loci in SLC22A11 and ZNF45 for body mass index in Indians.

Obesity, a risk factor for multiple diseases (e.g. diabetes, hypertension, cancers) originates through complex interactions between genes and prevailing environment (food habit and lifestyle) that varies across populations. Indians exhibit a unique obesity phenotype with high abdominal adiposity for a given body weight compared to matched white populations suggesting presence of population-specific genetic and environmental factors influencing obesity. However, Indian population-specific genetic contributors for obesity have not been explored yet. Therefore, to identify potential genetic contributors, we performed a two-staged genome-wide association study (GWAS) for body mass index (BMI), a common measure to evaluate obesity in 5973 Indian adults and the lead findings were further replicated in 1286 Indian adolescents. Our study revealed novel association of variants-rs6913677 in BAI3 gene (p = 1.08 × 10-8) and rs2078267 in SLC22A11 gene (p = 4.62 × 10-8) at GWAS significance, and of rs8100011 in ZNF45 gene (p = 1.04 × 10-7) with near GWAS significance. As genetic loci may dictate the phenotype through modulation of epigenetic processes, we overlapped genetic data of identified signals with their DNA methylation patterns in 236 Indian individuals and performed methylation quantitative trait loci (meth-QTL) analysis. Further, functional roles of discovered variants and underlying genes were speculated using publicly available gene regulatory databases (ENCODE, JASPAR, GeneHancer, GTEx). The identified variants in BAI3 and SLC22A11 genes were found to dictate methylation patterns at unique CpGs harboring critical cis-regulatory elements. Further, BAI3, SLC22A11 and ZNF45 variants were located in repressive chromatin, active enhancer, and active chromatin regions, respectively, in human subcutaneous adipose tissue in ENCODE database. Additionally, these genomic regions represented potential binding sites for key transcription factors implicated in obesity and/or metabolic disorders. Interestingly, GTEx portal identify rs8100011 as a robust cis-expression quantitative trait locus (cis-eQTL) in subcutaneous adipose tissue (p = 1.6 × 10-7), and ZNF45 gene expression in skeletal muscle of Indian subjects showed an inverse correlation with BMI indicating its possible role in obesity. In conclusion, our study discovered 3 novel population-specific functional genetic variants (rs6913677, rs2078267, rs8100011) in 2 novel (SLC22A11 and ZNF45) and 1 earlier reported gene (BAI3) for BMI in Indians. Our study decodes key genomic loci underlying obesity phenotype in Indians that may serve as prospective drug targets in future.

Genome-Wide Association Study of Metabolic Syndrome Reveals Primary Genetic Variants at CETP Locus in Indians.

Indians, a rapidly growing population, constitute vast genetic heterogeneity to that of Western population; however they have become a sedentary population in past decades due to rapid urbanization ensuing in the amplified prevalence of metabolic syndrome (MetS). We performed a genome-wide association study (GWAS) of MetS in 10,093 Indian individuals (6,617 MetS and 3,476 controls) of Indo-European origin, that belong to our previous biorepository of The Indian Diabetes Consortium (INDICO). The study was conducted in two stages-discovery phase (N = 2,158) and replication phase (N = 7,935). We discovered two variants within/near the CETP gene-rs1800775 and rs3816117-associated with MetS at genome-wide significance level during replication phase in Indians. Additional CETP loci rs7205804, rs1532624, rs3764261, rs247617, and rs173539 also cropped up as modest signals in Indians. Haplotype association analysis revealed GCCCAGC as the strongest haplotype within the CETP locus constituting all seven CETP signals. In combined analysis, we perceived a novel and functionally relevant sub-GWAS significant locus-rs16890462 in the vicinity of SFRP1 gene. Overlaying gene regulatory data from ENCODE database revealed that single nucleotide polymorphism (SNP) rs16890462 resides in repressive chromatin in human subcutaneous adipose tissue as characterized by the enrichment of H3K27me3 and CTCF marks (repressive gene marks) and diminished H3K36me3 marks (activation gene marks). The variant displayed active DNA methylation marks in adipose tissue, suggesting its likely regulatory activity. Further, the variant also disrupts a potential binding site of a key transcription factor, NRF2, which is known for involvement in obesity and metabolic syndrome.

Genomewide association study of C-peptide surfaces key regulatory genes in Indians.

Insulin is a commonly used measure of pancreatic ß-cell function but exhibits a short half-life in the human body. During biosynthesis, insulin release is accompanied by C-peptide at an equimolar concentration which has a much higher plasma half-life and is therefore projected as a precise measure of ß-cell activity than insulin. Despite this, genetic studies of metabolic traits haveneglected the regulatory potential of C-peptide for therapeutic intervention of type-2 diabetes. The present study is aimed to search genomewide variants governing C-peptide levels in genetically diverse and high risk population for metabolic diseases-Indians. We performed whole genome genotyping in 877 healthy Indians of Indo-European origin followed by replication of variants with P ≤ 1 × 10-3 in an independent sample-set of 1829 Indians. Lead-associated signals were also tested in-silico in 773 Hispanics. To secure biological rationale for observed association, we further carried out DNA methylation quantitative trait loci analysis in 233 Indians and publicly available regulatory data was mined. We discovered novel lncRNA gene AC073333.8 with the strongest association with C-peptide levels in Indians that however missed genomewide significance. Also, noncoding genes, RP1-209A6.1 and RPS3AP5; protein gene regulators, ZNF831 and ETS2; and solute carrier protein gene SLC15A5 retained robust association with C-peptide after meta-analysis. Integration of methylation data revealed ETS2 and ZNF831 single-nucleotide polymorphisms as significant meth-QTLs in Indians. All genes showed reasonable expression in the human lung, signifying alternate important organs for C-peptide biology. Our findings mirror polygenic nature of C-peptide where multiple small-effect size variants in the regulatory genome principally govern the trait biology.

Normative range of blood biochemical parameters in urban Indian school-going adolescents.

Adolescence is the most critical phase of human growth that radically alters physiology of the body and wherein any inconsistency can lead to serious health consequences in adulthood. The timing and pace at which various developmental events occur during adolescence is highly diverse and thus results in a drastic change in blood biochemistry. Monitoring the physiological levels of various biochemical measures in ample number of individuals during adolescence can call up for an early intervention in managing metabolic diseases in adulthood. Today, only a couple of studies in different populations have investigated blood biochemistry in a small number of adolescents however, there is no comprehensive biochemical data available worldwide. In view, we performed a cross-sectional study in a sizeable group of 7,618 Indian adolescents (3,333 boys and 4,285 girls) aged between 11-17 years to inspect the distribution of values of common biochemical parameters that generally prevails during adolescence and we observed that various parameters considerably follow the reported values from different populations being 3.56-6.49mmol/L (fasting glucose), 10.60-199.48pmol/L (insulin), 0.21-3.22nmol/L (C-peptide), 3.85-6.25% (HbA1c), 2.49-5.54mmol/L (total cholesterol), 1.16-3.69mmol/L (LDL), 0.78-1.85mmol/L (HDL), 0.33-2.24mmol/L (triglycerides), 3.56-11.45mmol/L (urea), 130.01-440.15µmol/L (uric acid) and 22.99-74.28µmol/L (creatinine). Barring LDL and triglycerides, all parameters differed significantly between boys and girls (p< 0.001). Highest difference was seen for uric acid (p = 1.3 x10-187) followed by C-peptide (p = 6.6 x10-89). Across all ages during adolescence, glycemic and nitrogen metabolites parameters varied markedly with gender. Amongst lipid parameters, only HDL levels were found to be significantly associated with gender following puberty (p< 0.001). All parameters except urea, differed considerably in obese and lean adolescents (p< 0.0001). The present study asserts that age, sex and BMI are the essential contributors to variability in blood biochemistry during adolescence. Our composite data on common blood biochemical measures will benefit future endeavors to define reference intervals in adolescents especially when the global availability is scarce.

Genome-wide association study of blood lipids in Indians confirms universality of established variants.

Lipids foster energy production and their altered levels have been coupled with metabolic ailments. Indians feature high prevalence of metabolic diseases, yet uncharacterized for genes regulating lipid homeostasis. We performed first GWAS for quantitative lipids (total cholesterol, LDL, HDL, and triglycerides) exclusively in 5271 Indians. Further to corroborate our genetic findings, we investigated DNA methylation marks in peripheral blood in Indians at the identified loci (N = 233) and retrieved gene regulatory features from public domains. Recurrent GWAS loci-CELSR2, CETP, LPL, ZNF259, and BUD13 cropped up as lead signals in Indians, reflecting their universal applicability. Besides established variants, we found certain unreported variants at sub-genome-wide level-QKI, REEP3, TMCC2, FAM129C, FAM241B, and LOC100506207. These variants though failed to attain GWAS significance in Indians, but largely turned out to be active CpG sites in human subcutaneous adipose tissue and showed robust association to two or more lipid traits. Of which, QKI variants showed significant association to all four lipid traits and their designated region was observed to be a key gene regulatory segment denoting active transcription particularly in human subcutaneous adipose tissue. Both established and novel loci were observed to be significantly associated with altered DNA methylation in Indians for specific CpGs that resided in key regulatory elements. Further, gene-based association analysis pinpointed novel GWAS loci-LINC01340 and IQCJ-SCHIP1 for TC; IFT27, IFT88, and LINC02141 for HDL; and TEX26 for TG. Present study ascertains universality of selected known genes and also identifies certain novel loci for lipids in Indians by integrating data from various levels of gene regulation.

Common variants of ARID1A and KAT2B are associated with obesity in Indian adolescents.

Obesity involves alterations in transcriptional programs that can change in response to genetic and environmental signals through chromatin modifications. Since chromatin modifications involve different biochemical, neurological and molecular signaling pathways related to energy homeostasis, we hypothesize that genetic variations in chromatin modifier genes can predispose to obesity. Here, we assessed the associations between 179 variants in 35 chromatin modifier genes and overweight/obesity in 1283 adolescents (830 normal weight and 453 overweight/obese). This was followed up by the replication analysis of associated signals (18 variants in 8 genes) in 2247 adolescents (1709 normal weight and 538 overweight/obese). Our study revealed significant associations of two variants rs6598860 (OR = 1.27, P = 1.58 × 10-4) and rs4589135 (OR = 1.22, P = 3.72 × 10-4) in ARID1A with overweight/obesity. We also identified association of rs3804562 (ß = 0.11, P = 1.35 × 10-4) in KAT2B gene with BMI. In conclusion, our study suggests a potential role of ARID1A and KAT2B genes in the development of obesity in adolescents and provides leads for further investigations.

TNF-α (g.-308 G > A) and ADIPOQ (g. + 45 T > G) gene polymorphisms in type 2 diabetes and microvascular complications in the region of Punjab (North-West India).

AIMS: The present study aims to examine the association of tumor necrosis factor-α (TNF-α) g.-308 G > A and adiponectin (ADIPOQ) g. + 45 T > G gene polymorphisms in type 2 diabetes (T2D) and its microvascular complications diabetic retinopathy (DR) and diabetic nephropathy (DN). MATERIALS AND METHODS: A total of 672 individuals were analysed from the North-West population of Punjab. Genotyping was accomplished by a combination of allele specific amplification refractory mutation system and restriction digestion for TNF-α g. - 308 G > A and ADIPOQ g. + 45 T > G polymorphisms, respectively. Further, in silico modeling was done to predict secondary structure of mRNA for g. + 45 T > G polymorphism in the ADIPOQ gene by RNA fold. RESULTS: The minor allele frequency observed in the controls for the TNF-α G > A and ADIPOQ T > G polymorphisms were 0.07 and 0.10, respectively. The results show no significant association with TNF-α g. - 308 G > A polymorphism in T2D as well as in any of the microvascular complication. However, the ADIPOQ g. + 45 T > G polymorphism shows significant association in T2D (p = 0.048) and DR (p = 0.001) but in DN patients, no association was observed. Interactive analysis revealed that the two polymorphisms jointly conferred a 1.45-fold risk towards the occurrence of T2D [p = 0.031; OR = 1.45 (1.03-2.05)]. In the secondary structure of mRNA, slight free energy change was observed between the wild ( - 1370.28 kcal/mol) and variant allele (-1369.08 kcal/mol). CONCLUSIONS: Our results indicated a higher risk of T2D and DR in the background of ADIPOQ TT genotype. Further, the ADIPOQ g. + 45 T > G and TNF-α g. - 308 G > A polymorphisms jointly give 1.45-fold risk towards T2D.

Genome-wide association study for type 2 diabetes in Indians identifies a new susceptibility locus at 2q21.

Indians undergoing socioeconomic and lifestyle transitions will be maximally affected by epidemic of type 2 diabetes (T2D). We conducted a two-stage genome-wide association study of T2D in 12,535 Indians, a less explored but high-risk group. We identified a new type 2 diabetes-associated locus at 2q21, with the lead signal being rs6723108 (odds ratio 1.31; P = 3.32 × 10⁻9). Imputation analysis refined the signal to rs998451 (odds ratio 1.56; P = 6.3 × 10⁻¹²) within TMEM163 that encodes a probable vesicular transporter in nerve terminals. TMEM163 variants also showed association with decreased fasting plasma insulin and homeostatic model assessment of insulin resistance, indicating a plausible effect through impaired insulin secretion. The 2q21 region also harbors RAB3GAP1 and ACMSD; those are involved in neurologic disorders. Forty-nine of 56 previously reported signals showed consistency in direction with similar effect sizes in Indians and previous studies, and 25 of them were also associated (P < 0.05). Known loci and the newly identified 2q21 locus altogether explained 7.65% variance in the risk of T2D in Indians. Our study suggests that common susceptibility variants for T2D are largely the same across populations, but also reveals a population-specific locus and provides further insights into genetic architecture and etiology of T2D.