Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

Transethnic analysis of the human leukocyte antigen region for ulcerative colitis reveals not only shared but also ethnicity-specific disease associations.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gut. Genetic association studies have identified the highly variable human leukocyte antigen (HLA) region as the strongest susceptibility locus for IBD and specifically DRB1*01:03 as a determining factor for ulcerative colitis (UC). However, for most of the association signal such as delineation could not be made because of tight structures of linkage disequilibrium within the HLA. The aim of this study was therefore to further characterize the HLA signal using a transethnic approach. We performed a comprehensive fine mapping of single HLA alleles in UC in a cohort of 9272 individuals with African American, East Asian, Puerto Rican, Indian and Iranian descent and 40 691 previously analyzed Caucasians, additionally analyzing whole HLA haplotypes. We computationally characterized the binding of associated HLA alleles to human self-peptides and analyzed the physicochemical properties of the HLA proteins and predicted self-peptidomes. Highlighting alleles of the HLA-DRB1*15 group and their correlated HLA-DQ-DR haplotypes, we not only identified consistent associations (regarding effects directions/magnitudes) across different ethnicities but also identified population-specific signals (regarding differences in allele frequencies). We observed that DRB1*01:03 is mostly present in individuals of Western European descent and hardly present in non-Caucasian individuals. We found peptides predicted to bind to risk HLA alleles to be rich in positively charged amino acids. We conclude that the HLA plays an important role for UC susceptibility across different ethnicities. This research further implicates specific features of peptides that are predicted to bind risk and protective HLA proteins.

Significance of an altered lncRNA landscape in schizophrenia and cognition: clues from a case-control association study.

Genetic etiology of schizophrenia is poorly understood despite large genome-wide association data. Long non-coding RNAs (lncRNAs) with a probable regulatory role are emerging as important players in neuro-psychiatric disorders including schizophrenia. Prioritising important lncRNAs and analyses of their holistic interaction with their target genes may provide insights into disease biology/etiology. Of the 3843 lncRNA SNPs reported in schizophrenia GWASs extracted using lincSNP 2.0, we prioritised n = 247 based on association strength, minor allele frequency and regulatory potential and mapped them to lncRNAs. lncRNAs were then prioritised based on their expression in brain using lncRBase, epigenetic role using 3D SNP and functional relevance to schizophrenia etiology. 18 SNPs were finally tested for association with schizophrenia (n = 930) and its endophenotypes-tardive dyskinesia (n = 176) and cognition (n = 565) using a case-control approach. Associated SNPs were characterised by ChIP seq, eQTL, and transcription factor binding site (TFBS) data using FeatSNP. Of the eight SNPs significantly associated, rs2072806 in lncRNA hsaLB_IO39983 with regulatory effect on BTN3A2 was associated with schizophrenia (p = 0.006); rs2710323 in hsaLB_IO_2331 with role in dysregulation of ITIH1 with tardive dyskinesia (p < 0.05); and four SNPs with significant cognition score reduction (p < 0.05) in cases. Two of these with two additional variants in eQTL were observed among controls (p < 0.05), acting likely as enhancer SNPs and/or altering TFBS of eQTL mapped downstream genes. This study highlights important lncRNAs in schizophrenia and provides a proof of concept of novel interactions of lncRNAs with protein-coding genes to elicit alterations in immune/inflammatory pathways of schizophrenia.

Construction and benchmarking of a multi-ethnic reference panel for the imputation of HLA class I and II alleles.

Genotype imputation of the human leukocyte antigen (HLA) region is a cost-effective means to infer classical HLA alleles from inexpensive and dense SNP array data. In the research setting, imputation helps avoid costs for wet lab-based HLA typing and thus renders association analyses of the HLA in large cohorts feasible. Yet, most HLA imputation reference panels target Caucasian ethnicities and multi-ethnic panels are scarce. We compiled a high-quality multi-ethnic reference panel based on genotypes measured with Illumina's Immunochip genotyping array and HLA types established using a high-resolution next generation sequencing approach. Our reference panel includes more than 1,300 samples from Germany, Malta, China, India, Iran, Japan and Korea and samples of African American ancestry for all classical HLA class I and II alleles including HLA-DRB3/4/5. Applying extensive cross-validation, we benchmarked the imputation using the HLA imputation tool HIBAG, our multi-ethnic reference and an independent, previously published data set compiled of subpopulations of the 1000 Genomes project. We achieved average imputation accuracies higher than 0.924 for the commonly studied HLA-A, -B, -C, -DQB1 and -DRB1 genes across all ethnicities. We investigated allele-specific imputation challenges in regard to geographic origin of the samples using sensitivity and specificity measurements as well as allele frequencies and identified HLA alleles that are challenging to impute for each of the populations separately. In conclusion, our new multi-ethnic reference data set allows for high resolution HLA imputation of genotypes at all classical HLA class I and II genes including the HLA-DRB3/4/5 loci based on diverse ancestry populations.

Determination of Dopamine-ß-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection.

Dopamine-ß-hydroxylase (DBH, EC 1.14.17.1) is an enzyme with implications in various neuropsychiatric and cardiovascular diseases and is a known drug target. There is a dearth of cost effective and fast method for estimation of activity of this enzyme. A sensitive UHPLC based method for the estimation of DBH activity in human sera samples based on separation of substrate tyramine from the product octopamine in 3 min is described here. In this newly developed protocol, a Solid Phase Extraction (SPE) sample purification step prior to LC separation, selectively removes interferences from the reaction cocktail with almost no additional burden on analyte recovery. The response was found to be linear with an r2 = 0.999. The coefficient of variation for assay precision was < 10% and recovery > 90%. As a proof of concept, DBH activity in sera from healthy human volunteers (n = 60) and schizophrenia subjects (n = 60) were successfully determined using this method. There was a significant decrease in sera DBH activity in subjects affected by schizophrenia (p < 0.05) as compared to healthy volunteers. This novel assay employing SPE to separate octopamine and tyramine from the cocktail matrix may have implications for categorising subjects into various risk groups for Schizophrenia, Parkinson's disease as well as in high throughput screening of inhibitors.

Functional characterisation of ADP ribosylation factor-like protein 15 in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: ARL15 is a novel susceptibility gene identified in a recent GWAS in a north Indian rheumatoid arthritis (RA) cohort. However, the role of ARL15 or ARF family genes in RA aetiology remains unknown. Therefore, we aimed to i) establish the expression of ARL15 in rheumatoid arthritis synovial fibroblasts (RASF) and ii) its functional characterisation by assessing its effects on major inflammatory cytokines and interacting partners using a knockdown approach. METHODS: RASF were cultured from synovial tissue obtained from RA patients (n=5) and osteoarthritis (OA) patients (n=3) serving as controls. Expression of ARL15, ARF1 and ARF6 in RASF was checked by semi-quantitative PCR and western blots; and altered expression of ARL15, if any, by induction of RASF with TNF using real-time PCR. The effect of ARL15 on the expression of adiponectin, adiponectin receptor I, IL6 and GAPDH and on cell mobility by invasion and migration assays were assessed by siRNA mediated gene knockdown. RESULTS: Expression of ARL15, ARF1 and ARF6 was confirmed in RASF and OASF samples but ARL15 expression remained unaltered on TNF induction. Notably, ARL15 knockdown resulted in downregulation of IL6 and GAPDH, upregulation of adiponectin and adiponectin receptor I genes; and significant reduction in migration and invasion of RASF. Genemania showed significant interactions of ARL15 with genes responsible for insulin resistance and phospholipase D. CONCLUSIONS: This first report on ARL15 expression in RASF and its likely role in inflammation and metabolic syndromes through a TNF independent pathway, encourages hypothesis-free studies to identify additional pathways underlying RA disease biology.

Characterization of SNPs in the dopamine-ß-hydroxylase gene providing new insights into its structure-function relationship.

Dopamine-ß-hydroxylase (DBH, EC 1.14.17.1), an oxido-reductase that catalyses the conversion of dopamine to norepinephrine, is largely expressed in sympathetic neurons and adrenal medulla. Several regulatory and structural variants in DBH associated with various neuropsychiatric, cardiovascular diseases and a few that may determine enzyme activity have also been identified. Due to paucity of studies on functional characterization of DBH variants, its structure-function relationship is poorly understood. The purpose of the study was to characterize five non-synonymous (ns) variants that were prioritized either based on previous association studies or Sorting Tolerant From Intolerant (SIFT) algorithm. The DBH ORF with wild type (WT) and site-directed mutagenized variants were transfected into HEK293 cells to generate transient and stable lines expressing these variant enzymes. Activity was determined by UPLC-PDA and corresponding quantity by MRMHR on a TripleTOF 5600 MS respectively of spent media from stable cell lines. Homospecific activity computed for the WT and variant proteins showed a marginal decrease in A318S, W544S and R549C variants. In transient cell lines, differential secretion was observed in the case of L317P, W544S and R549C. Secretory defect in L317P was confirmed by localization in ER. R549C exhibited both decreased homospecific activity and differential secretion. Of note, all the variants were seen to be destabilizing based on in silico folding analysis and molecular dynamics (MD) simulation, lending support to our experimental observations. These novel genotype-phenotype correlations in this gene of considerable pharmacological relevance have implications for dopamine-related disorders.

Evidence of mutations in RIC3 acetylcholine receptor chaperone as a novel cause of autosomal-dominant Parkinson's disease with non-motor phenotypes.

BACKGROUND: The known genetic determinants of Parkinson's disease (PD) do not explain all cases investigated to date. Contemporary sequencing technologies hold promise for enhanced causal variant discovery. We attempted to identify the putative causal variant in an Indian PD family by whole exome sequencing (WES). METHODS: WES data generated for two affected cousins from a 14-member PD family with some non-motor phenotypes were analysed. Variants prioritised were checked for segregation with disease by targeted sequencing. An independent PD cohort (n=280) was screened for additional mutations in the prioritised gene. Variants were functionally validated in PC12 cells differentiated into neurons. RESULTS: A heterozygous mutation c.169C>A, p.P57T in RIC3 acetylcholine receptor chaperone (11p15) segregated with disease in the family confirming an autosomal-dominant mode of inheritance. Another heterozygous mutation c.502G>C, p.V168L was detected in an unrelated PD case. Both mutations were absent in 144 healthy control and in 74 non-PD WES data available in-house and in 186 age and sex-matched controls screened by PCR sequencing. RIC3 is a known chaperone of neuronal nicotinic acetylcholine receptor subunit α-7 (CHRNA7). Dominant negative effect of RIC3 mutants in transfected PC12 cells was reflected by the reduced levels of endogenous CHRNA7 in the membrane fractions in western blots and lower colocalisation profiles in confocal micrographs. CONCLUSION: The novel demonstration of a chaperone-mediated receptor density alteration due to RIC3 mutants provides strong evidence for the role of cholinergic pathway for the first time in PD aetiology. This may also be insightful for some non-motor symptoms and personalised treatment.

Discovery of a frameshift mutation in podocalyxin-like (PODXL) gene, coding for a neural adhesion molecule, as causal for autosomal-recessive juvenile Parkinsonism.

BACKGROUND: Mutations in known genes for inherited forms of Parkinson's disease (PD) account for <30% of familial PD (FPD) implying that more causal gene(s) remain to be identified. We attempted to discover the putative causal variant in an Indian family with autosomal-recessive juvenile Parkinsonism (ARJP), tested negative for mutations in PARK2, PINK1 and DJ1. METHODS: Whole exomes of two affected siblings were sequenced. Variants prioritised were screened for segregation with disease in the family by targeted sequencing. Gene thus identified was screened for index/additional exonic mutations, if any, in an independent PD cohort by PCR sequencing. Variants observed were functionally validated in differentiated PC12 cells. RESULTS: A novel homozygous frameshift mutation, c.89_90insGTCGCCCC in exon 1 of podocalyxin-like gene (PODXL, 7q32-33), resulting in loss of protein, segregated with disease in the family. Mutant allele was absent in 186 healthy controls screened by PCR sequencing and in control exomes available in the laboratory and public databases. Screening of additional 212 sporadic and 68 FPD cases identified three novel heterozygous missense variants namely c.1285C>A, c.1118G>A and c.881G>A in three unrelated cases. Significant differences in neurite branching and length (p<0.0001) were observed in PC12 cells with wild-type and mutant constructs. CONCLUSIONS: Based on the genetic and functional evidence in this study and literature support on the role of PODXL in neural development, a novel frameshift mutation in PODXL seems to be the likely cause of ARJP in this family. This is the first report suggesting the possible role of a neurodevelopmental pathway in PD aetiology.

Convergent evolution in European and Rroma populations reveals pressure exerted by plague on Toll-like receptors.

Recent historical periods in Europe have been characterized by severe epidemic events such as plague, smallpox, or influenza that shaped the immune system of modern populations. This study aims to identify signals of convergent evolution of the immune system, based on the peculiar demographic history in which two populations with different genetic ancestry, Europeans and Rroma (Gypsies), have lived in the same geographic area and have been exposed to similar environments, including infections, during the last millennium. We identified several genes under evolutionary pressure in European/Romanian and Rroma/Gipsy populations, but not in a Northwest Indian population, the geographic origin of the Rroma. Genes in the immune system were highly represented among those under strong evolutionary pressures in Europeans, and infections are likely to have played an important role. For example, Toll-like receptor 1 (TLR1)/TLR6/TLR10 gene cluster showed a strong signal of adaptive selection. Their gene products are functional receptors for Yersinia pestis, the agent of plague, as shown by overexpression studies showing induction of proinflammatory cytokines such as TNF, IL-1ß, and IL-6 as one possible infection that may have exerted evolutionary pressures. Immunogenetic analysis showed that TLR1, TLR6, and TLR10 single-nucleotide polymorphisms modulate Y. pestis-induced cytokine responses. Other infections may also have played an important role. Thus, reconstruction of evolutionary history of European populations has identified several immune pathways, among them TLR1/TLR6/TLR10, as being shaped by convergent evolution in two human populations with different origins under the same infectious environment.

Identification of critical variants within SLC44A4, an ulcerative colitis susceptibility gene identified in a GWAS in north Indians.

SLC44A4 is one of the seven novel susceptibility genes that were discovered in the first ever genome-wide association study (GWAS) on ulcerative colitis (UC) in the genetically distinct north Indians. This gene seems to be functionally relevant to disease biology as it may contribute to the associated phenotype of Vitamin B1 deficiency among UC patients, hence playing a role in disease pathogenesis. A large number of single-nucleotide polymorphisms (SNPs) are known to be distributed throughout this gene, but the functional status of most are not known. Thus, an extensive investigation of structural and regulatory variants within this gene was undertaken in this study to identify the critical variants amongst them using a combination of fine mapping, in silico and in vitro approaches. A few intronic SNPs were predicted to have regulatory roles on the basis of in silico analysis, suggesting that they may be the critical variants within SLC44A4. This highlights the importance of this gene in UC biology, thus confirming the finding of the GWAS and also warranting additional studies.

Deep sequencing identifies novel regulatory variants in the distal promoter region of the dopamine-ß-hydroxylase gene.

OBJECTIVE: Dopamine-ß-hydroxylase (DBH), an enzyme that converts dopamine into norepinephrine, is a drug target in cardiovascular and neuropsychiatric disorders. We aimed to identify functional variants in this gene by deep sequencing and enzyme phenotyping in an Indian cohort. MATERIALS AND METHODS: Targeted resequencing of 12 exons and 10 kb upstream sequences of DBH in healthy volunteers (n=50) was performed using the Ion Personal Genome Machine System. Enzyme quantity and activity in their sera samples were determined by ELISA and ultra performance liquid chromatography, respectively. The association of markers with phenotypes was determined using Matrix eQTL. Global P-values for haplotypes generated using UNPHASED 3.1.5 were graphed using GrASP v.082 beta. RESULTS: Of the 49 variants identified, nine were novel (minor allele frequency≥0.01). Though individual markers associated with enzyme quantity did not withstand multiple corrections, a novel distal promoter block driven by rs113249250 (global P=1.5×10) was associated. Of the nine single nucleotide polymorphisms (SNPs) associated with enzyme activity, rs3025369, rs1076151 and rs1611115, all from the upstream region, withstood false discovery rate correction (false discovery rate=0.03, 0.03 and 2.9×10, respectively). Conditioning for rs1611115 identified rs1989787 also to affect activity. Importantly, we report an association of a novel haplotype block distal to rs1076151 driven by rs3025369 (global P=8.9×10) with enzyme activity. This regulatory SNP explained 4.9% of the total 46.1% of variance in DBH activity caused by associated SNPs. CONCLUSION: This first study combining deep sequencing and enzyme phenotyping identified yet another regulatory SNP suggesting that regulatory variants may be central in the physiological or metabolic role of this gene of therapeutic and pharmacological relevance.

Genome-wide association scan in north Indians reveals three novel HLA-independent risk loci for ulcerative colitis.

OBJECTIVE: Over 100 ulcerative colitis (UC) loci have been identified by genome-wide association studies (GWASs) primarily in Caucasians (CEUs). Many of them have weak effects on disease susceptibility, and the bulk of the heritability cannot be ascribed to these loci. Very little is known about the genetic background of UC in non-CEU groups. Here we report the first GWAS on UC in a genetically distinct north Indian (NI) population. DESIGN: A genome-wide scan was performed on 700 cases and 761 controls. 18 single-nucleotide polymorphisms (SNPs) (p<5×10(-5)) were genotyped in an independent cohort of 733 cases and 1148 controls. A linear mixed model was used for case-control association tests. RESULTS: Seven novel human leucocyte antigen (HLA)-independent SNPs from chromosome 6, located in 3.8-1, BAT2, MSH5, HSPA1L, SLC44A4, CFB and NOTCH4, exceeded p<5×10(-8) in the combined analysis. To assess the independent biological contribution of such genes from the extended HLA region, we determined the percentage alternative pathway activity of complement factor B (CFB), the top novel hit. The activity was significantly different (p=0.01) between the different genotypes at rs12614 in UC cases. Transethnic comparisons revealed a shared contribution of a fraction of UC risk genes between NI and CEU populations, in addition to genetic heterogeneity. CONCLUSIONS: This study shows varying contribution of the HLA region to UC in different populations. Different environmental exposures and the characteristic genetic structure of the HLA locus across ethnic groups collectively make it amenable to the discovery of causative alleles by transethnic resequencing. This may lead to an improved understanding of the molecular mechanisms underlying UC.

Targeted deep resequencing identifies MID2 mutation for X-linked intellectual disability with varied disease severity in a large kindred from India.

We report a novel missense mutation (c.1040G>A, p.Arg347Gln) in MID2, which encodes ubiquitin ligase E3, as the likely cause of X-linked mental retardation in a large kindred. The mutation was observed in all affected and obligate carriers but not in any unaffected males of the family or in population controls (n = 200). When transiently expressed in HEK293T cell line, the mutation was found to abolish the function of the COS domain in the protein. The GFP-tagged mutant protein accumulated in the cytoplasm instead of binding to the cytoskeleton resulting in its altered subcellular localization. Screening of coding exons of this gene in additional 480 unrelated individuals with idiopathic intellectual disability identified another novel variation p.Asn343Ser. This study highlights the growing role of the ubiquitin pathway in intellectual disability and also, the difference in MID2 determined phenotype observed in this study compared with that of its paralogue MID1 reported in literature.

Genome-wide analysis of methotrexate pharmacogenomics in rheumatoid arthritis shows multiple novel risk variants and leads for TYMS regulation.

OBJECTIVE: Methotrexate (MTX) is the drug of first choice for the treatment of rheumatoid arthritis (RA), but is effective only in around 60% of the patients. Identification of genetic markers to predict response is essential for effective treatment within a critical window period of 6 months after diagnosis, but have been hitherto elusive. In this study, we used genome-wide genotype data to identify the potential risk variants associated with MTX (poor)response in a north Indian RA cohort. MATERIALS AND METHODS: Genome-wide genotyping data for a total of 457 RA patients [297 good (DAS28-3≤3.2) and 160 poor (DAS28-3≥5.1) responders] on MTX monotherapy were tested for association using an additive model. Support vector machine and genome-wide pathway analysis were used to identify additional risk variants and pathways. All risk loci were imputed to fine-map the association signals and identify causal variant(s) of therapeutic/diagnostic relevance. RESULTS: Seven novel suggestive loci from genome-wide (P≤5×10(-5)) and three from support vector machine analysis were associated with MTX (poor)response. The associations of published candidate genes namely DHFR (P=0.014), FPGS (P=0.035), and TYMS (P=0.005) and purine and nucleotide metabolism pathways were reconfirmed. Imputation, followed by bioinformatic analysis indicated possible interaction between two reversely oriented overlapping genes namely ENOSF1 and TYMS at the post-transcriptional level. CONCLUSION: In this first ever genome-wide analysis on MTX treatment response in RA patients, 10 new risk loci were identified. These preliminary findings warrant replication in independent studies. Further, TYMS expression at the post-transcriptional level seems to be probably regulated through an antisense-RNA involving the 6-bp ins/del marker in the overlapping segment at 3'UTR of TYMS-ENOSF1, a finding with impending pharmacogenetic applications.

Population and genomic lessons from genetic analysis of two Indian populations.

Indian demographic history includes special features such as founder effects, interpopulation segregation, complex social structure with a caste system and elevated frequency of consanguineous marriages. It also presents a higher frequency for some rare mendelian disorders and in the last two decades increased prevalence of some complex disorders. Despite the fact that India represents about one-sixth of the human population, deep genetic studies from this terrain have been scarce. In this study, we analyzed high-density genotyping and whole-exome sequencing data of a North and a South Indian population. Indian populations show higher differentiation levels than those reported between populations of other continents. In this work, we have analyzed its consequences, by specifically assessing the transferability of genetic markers from or to Indian populations. We show that there is limited genetic marker portability from available genetic resources such as HapMap or the 1,000 Genomes Project to Indian populations, which also present an excess of private rare variants. Conversely, tagSNPs show a high level of portability between the two Indian populations, in contrast to the common belief that North and South Indian populations are genetically very different. By estimating kinship from mates and consanguinity in our data from trios, we also describe different patterns of assortative mating and inbreeding in the two populations, in agreement with distinct mating preferences and social structures. In addition, this analysis has allowed us to describe genomic regions under recent adaptive selection, indicating differential adaptive histories for North and South Indian populations. Our findings highlight the importance of considering demography for design and analysis of genetic studies, as well as the need for extending human genetic variation catalogs to new populations and particularly to those with particular demographic histories.

A genome-wide association study reveals ARL15, a novel non-HLA susceptibility gene for rheumatoid arthritis in North Indians.

OBJECTIVE: Genome-wide association studies (GWAS) and their subsequent meta-analyses have changed the landscape of genetics in rheumatoid arthritis (RA) by uncovering several novel genes. Such studies are heavily weighted by samples from Caucasian populations, but they explain only a small proportion of total heritability. Our previous studies in genetically distinct North Indian RA cohorts have demonstrated apparent allelic/genetic heterogeneity between North Indian and Western populations, warranting GWAS in non-European populations. We undertook this study to detect additional disease-associated loci that may be collectively important in the presence or absence of genes with a major effect. METHODS: High-quality genotypes for >600,000 single-nucleotide polymorphisms (SNPs) in 706 RA patients and 761 controls from North India were generated in the discovery stage. Twelve SNPs showing suggestive association (P < 5 × 10(-5)) were then tested in an independent cohort of 927 RA patients and 1,148 controls. Additional disease-associated loci were determined using support vector machine (SVM) analyses. Fine-mapping of novel loci was performed by using imputation. RESULTS: In addition to the expected association of the HLA locus with RA, we identified association with a novel intronic SNP of ARL15 (rs255758) on chromosome 5 (Pcombined = 6.57 × 10(-6); odds ratio 1.42). Genotype-phenotype correlation by assaying adiponectin levels demonstrated the functional significance of this novel gene in disease pathogenesis. SVM analysis confirmed this association along with that of a few more replication stage genes. CONCLUSION: In this first GWAS of RA among North Indians, ARL15 emerged as a novel genetic risk factor in addition to the classic HLA locus, which suggests that population-specific genetic loci as well as those shared between Asian and European populations contribute to RA etiology. Furthermore, our study reveals the potential of machine learning methods in unraveling gene-gene interactions using GWAS data.

Association of polymorphisms of CYP2C9, CYP2C19, and ABCB1, and activity of P-glycoprotein with response to anti-epileptic drugs.

BACKGROUND AND OBJECTIVE: Epilepsy, the most common neurological disorder, has treatment failure rate of 20 to 25%. Inter-individual variability in drug response can be attributed to genetic polymorphism in genes encoding different drug metabolizing enzymes, drug transporters (P-gp), and enzymes involved in sodium channel biosynthesis. The present study attempted to evaluate association of polymorphisms of CYP2C9, CYP2C19, and ABCB1, and P-gp activity with treatment response in patients with epilepsy. MATERIALS AND METHODS: Patients with epilepsy on phenytoin and/or phenobarbital and/or carbamazepine were categorized into responders and non-responders as per the International League Against Epilepsy. Plasma drug concentration was estimated by high-performance liquid chromatography. P-gp activity was measured by flow cytometry using rhodamine efflux. The polymerase chain reaction (PCR-RFLP) was used to study polymorphisms of ABCB1 (C3435T), CYP2C9 (416 C > T, and 1061 A > T), and CYP2C19 (681 G > A and 636 G > A). RESULTS: Of total 117 patients enrolled in this study, genotype data was available for 115 patients. P-gp activity was higher in non-responders (n = 68) compared to responders (n = 47) (P<0.001). No association of 416 C > T and 1061 A > T in CYP2C9 or 681 G > A and 636 G > A in CYP2C19 was observed with response phenotype in genotypic analysis. Significant genotypic (odds ratio, OR = 4.5; 95% CI, 1.04 to 20.99) and allelic association (OR = 1.73; 95% CI, 1.02 to 2.95) was observed with ABCB1 C3435T and response phenotype. CONCLUSIONS: The response to antiepileptics seems to be modulated by C3435T in ABCB1 or P-gp activity. At present, role of other genetic factors in treatment responsiveness in epilepsy appears limited, warranting analysis in a larger cohort.

ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

Building polarization into protein-inhibitor binding dynamics in rational drug design for rheumatoid arthritis.

Standard force field-based simulations to accomplish structure-based evaluations of lead molecules is a powerful tool. Combining protein fragmentation into tractable sub-systems with continuum solvation method is envisaged to enable quantum mechanics-based electronic structure calculations of macromolecules in their realistic environment. This along with incorporation of many-body polarization effect in molecular dynamics simulations may augment an accurate description of electrostatics of protein-inhibitor systems for effective drug design. Rheumatoid arthritis (RA) is a complex autoimmune disorder plagued by the ceiling effect of current targeted therapies, encouraging identification of new druggable targets and corresponding drug design to tackle the refractory form of disease. In this study, polarization-inclusive force field approach has been used to model protein solvation and ligand binding for 'Mitogen-activated protein kinase' (MAP3K8), a regulatory node of notable pharmacological relevance in RA synovial biology. For MAP3K8 inhibitors belonging to different scaffold series, the calculations illustrated differential electrostatic contribution to their relative binding affinities and successfully explained examples from available structure-activity relationship studies. Results from this study exemplified i) the advantage of this approach in reliably ranking inhibitors having close nanomolar range activities for the same target; and ii) its prospective application in lead molecule identification aiding drug discovery efforts in RA.Communicated by Ramaswamy H. Sarma.