Stratification of rheumatoid arthritis cohort using Ayurveda based deep phenotyping approach identifies novel genes in a GWAS.

BACKGROUND AND AIM: Genome wide association studies have scaled up both in terms of sample size and range of complex disorders investigated, but these have explained relatively little phenotypic variance. Of the several reasons, phenotypic heterogeneity seems to be a likely contributor for missing out genetic associations of large effects. Ayurveda, the traditional Indian system of medicine is one such tool which adopts a holistic deep phenotyping approach and classifies individuals based on their body constitution/prakriti. We hypothesized that Ayurveda based phenotypic stratification of healthy and diseased individuals will allow us to achieve much desired homogeneous cohorts which would facilitate detection of genetic association of large effects. In this proof of concept study, we performed a genome wide association testing of clinically diagnosed rheumatoid arthritis patients and healthy controls, who were re-phenotyped into Vata, Pitta and Kapha predominant prakriti sub-groups. EXPERIMENTAL PROCEDURE: Genotypes of rheumatoid arthritis cases (Vata = 49; Pitta = 117; Kapha = 78) and controls (Vata = 33; Pitta = 175; Kapha = 85) were retrieved from the total genotype data, used in a recent genome-wide association study performed in our laboratory. A total of 528461 SNPs were included after quality control. Prakriti-wise genome-wide association analysis was employed. RESULTS AND CONCLUSION: This study identified (i) prakriti-specific novel disease risk genes of high effect sizes; (ii) putative candidates of novel therapeutic potential; and (iii) a good correlation between genetic findings and clinical knowledge in Ayurveda. Adopting Ayurveda based deep phenotyping may facilitate explaining hitherto undiscovered heritability in complex traits and may propel much needed progress in personalized medicine.

Compound heterozygous variants in Wiskott-Aldrich syndrome like (WASL) gene segregating in a family with early onset Parkinson's disease.

BACKGROUND: Knowledge of genetic determinants in Parkinson's disease is still limited. Familial forms of the disease continue to provide a rich resource to capture the genetic spectrum in disease pathogenesis, and this approach is exploited in this study. METHODS: Informative members from a three-generation family of Indian ethnicity manifesting a likely autosomal recessive mode of inheritance of Parkinson's disease were used for whole exome sequencing. Variant data analysis and in vitro functional characterisation of variant(s) segregating with the phenotype were carried out in HEK-293 and SH-SY5Y cells using gene constructs of interest. RESULTS: Two compound heterozygous variants, a rare missense (c.1139C > T:p.P380L) and a novel splice variant (c.1456 + 2 delTAGA, intron10) in Wiskott-Aldrich syndrome like gene (WASL, 7q31), both predicted to be deleterious were shared among the proband and two affected siblings. WASL, a gene not previously linked to a human Mendelian disorder is known to regulate actin polymerisation via Arp2/3 complex. Based on exon trapping assay using pSPL3 vector in HEK-293 cells, the splice variant showed skipping of exon10. Characterisation of the missense variant in SH-SY5Y cells demonstrated: i) significant alterations in neurite length and number; ii) decreased reactive oxygen species tolerance in mutation carrying cells on Tetrabutylphosphonium hydroxide induction and iii) increase in alpha-synuclein protein. Screening for WASL variants in two independent PD cohorts identified four individuals with heterozygous but none with biallelic variants. CONCLUSION: WASL, with demonstrated functional relevance in neurons may be yet another strong candidate gene for autosomal recessive PD encouraging assessment of its contribution across populations.

Early Onset Parkinson's disease due to DJ1 mutations: An Indian study.

INTRODUCTION: Early Onset Parkinson's Disease (EOPD) is genetically heterogeneous. PARK2 mutations are the commonest cause of autosomal recessive EOPD followed by PINK1.DJ1 mutations is rare and there is scarce literature on its phenotype and long term outcome. OBJECTIVES: We undertook a retrospective study to determine the prevalence of DJ1 mutation(s) in an Indian population and describe the clinical features and long term outcome of EOPD patients with these mutations. METHODS: One hundred EOPD patients and 114 controls were evaluated. All the seven coding exons of DJ1 gene were screened for novel and reported mutations by PCR- Sanger sequencing. RESULTS: A novel homozygous missense mutation (c.313 A > T, p. Ile105Phe) in exon 5 was seen in one patient and four unrelated patients had a homozygous missense single nucleotide variant rs71653619 (c.293 G > A, p.Arg98Gln). The clinical phenotype comprised of asymmetrical onset, slowly progressive Parkinsonism with levodopa induced motor restlessness in a patient with the novel mutation (c.313 A > T, p. Ile105Phe) while subjects with c.293 G > A, p.Arg98Gln had early onset levodopa responsive symmetrical Parkinsonism. CONCLUSION: DJ1 mutations account for ∼5% of EOPD patients from the Indian population. This study further adds to the clinical spectrum of EOPD with DJ1 mutations.

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.

Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations.

Ulcerative colitis and Crohn's disease are the two main forms of inflammatory bowel disease (IBD). Here we report the first trans-ancestry association study of IBD, with genome-wide or Immunochip genotype data from an extended cohort of 86,640 European individuals and Immunochip data from 9,846 individuals of East Asian, Indian or Iranian descent. We implicate 38 loci in IBD risk for the first time. For the majority of the IBD risk loci, the direction and magnitude of effect are consistent in European and non-European cohorts. Nevertheless, we observe genetic heterogeneity between divergent populations at several established risk loci driven by differences in allele frequency (NOD2) or effect size (TNFSF15 and ATG16L1) or a combination of these factors (IL23R and IRGM). Our results provide biological insights into the pathogenesis of IBD and demonstrate the usefulness of trans-ancestry association studies for mapping loci associated with complex diseases and understanding genetic architecture across diverse populations.

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.

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.

Role of immunodeficient animal models in the development of fructose induced NAFLD.

Cellular and humoral immunity had been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This study was designed to assess if T, B and natural killer (NK) cells are involved in the progress of NAFLD in mouse models after chronic fructose treatment. Mouse models that are deficient in either T cells, B cells or NK cells or lacking both T and B cells were fed with 30% fructose solution for 12 weeks. Typical features of NAFLD, including the relative body weight, food and water intake, biochemical analytes, liver histology, NAFLD activity score, and glucose tolerance and insulin tolerance test were characterized. Further, the percentage of CD3, B220 and NK cells in peripheral-blood mononuclear cell, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunodetection for hepatic apoptosis (p53) and for inflammation (TNFα) and quantitative real-time polymerase chain reaction for putative and inflammatory genes involved were determined. Our results conclude that mice deficient in T cells or NK cells fail to develop fructose induced NAFLD whereas the immunocompetent mice and mice with B-cell-specific defect developed NAFLD. Taken together, these data support that the onset of fructose-induced NAFLD is associated with involvement of T cells and NK cells in mice.

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.

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.

VPS35 and EIF4G1 mutations are rare in Parkinson's disease among Indians.

Mutations in 2 genes, vacuolar protein sorting homolog 35 (VPS35) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), have been recently reported as causal in autosomal dominant Parkinson's disease (PD) among Caucasians. Their contribution to PD in other ethnic groups remains limited with 1% of VPS35 mutations observed in Caucasian and Japanese populations, but none in Chinese, and 11.57% of EIF4G1 mutations in Caucasian families and 0.09% and 0.17% in Caucasian and Chinese sporadic cases, respectively. We investigated the contribution, if any, of these 2 genes to familial and sporadic PD among the ethnically distinct Indian population. Complete exonic regions of these 2 genes were resequenced in 15 well-characterized PD families; the reported p.Asp620Asn in VPS35 and p.Arg1205His in EIF4G1 mutations were screened in an additional 54 familial and 251 sporadic PD cases, and no mutations were observed. These results, together with our previous reports on the absence of mutations in SNCA and LRRK2, warrant a continuing search for novel causative genes for PD among Indians.

Antigen peptide transporter 1 is involved in the development of fructose-induced hepatic steatosis in mice.

BACKGROUND AND AIM: The purpose of this study is to assess whether the decrease in CD8 cells has any role in the development of non-alcoholic fatty liver disease (NAFLD). In this study, we therefore used antigen peptide transporter 1 (TAP1(-/-)) mice that cannot transport major histocompatibility complex class I antigens onto the cell surface resulting in failure of the generation of CD8 cells. METHODS: Wild-type C57Bl/6J and TAP1(-/-) mice were fed with 30% fructose solution for 8 weeks. The percentage of CD4, CD8 cells in peripheral blood mononuclear cells, and liver were sorted by fluorescence-activated cell sorting in both control and fructose-treated mice. Bodyweight, histopathological changes, oil red O staining, glucose tolerance test, intraperitoneal insulin tolerance test, serum levels of triglycerides, cholesterol, aspartate aminotransferase, and alanine aminotransferase were also evaluated. Quantitative real-time polymerase chain reaction was performed to determine the expression of specific genes involved in development of fatty changes in the liver. RESULTS: Chronic consumption of fructose in TAP1(-/-) mice did not develop NAFLD, insulin resistance, or change in level of CD8 cells. Moreover, there was delay in relative expression levels of genes involved in development of NAFLD in fructose-treated TAP1(-/-) mice. CONCLUSION: Taken together, the data suggest that TAP1(-/-) -deficient mice displayed reduced levels of CD8 cells that have a vital role in the initiation and propagation of liver inflammation and is a casual role in the beginning of fructose-induced liver damage as well as insulin resistance in mice.

Potential of ayurgenomics approach in complex trait research: leads from a pilot study on rheumatoid arthritis.

BACKGROUND: Inconsistent results across association studies including Genome-wide association, have posed a major challenge in complex disease genetics. Of the several factors which contribute to this, phenotypic heterogeneity is a serious limitation encountered in modern medicine. On the other hand, Ayurveda, a holistic Indian traditional system of medicine, enables subgrouping of individuals into three major categories namely Vata, Pitta and Kapha, based on their physical and mental constitution, referred to as Prakriti. We hypothesised that conditioning association studies on prior risk, predictable in Ayurveda, will uncover much more variance and potentially open up more predictive health. OBJECTIVES AND METHODS: Identification of genetic susceptibility markers by combining the prakriti based subgrouping of individuals with genetic analysis tools was attempted in a Rheumatoid arthritis (RA) cohort. Association of 21 markers from commonly implicated inflammatory and oxidative stress pathways was tested using a case-control approach in a total cohort comprising 325 cases and 356 controls and in the three subgroups separately. We also tested few postulates of Ayurveda on the disease characteristics in different prakriti groups using clinico-genetic data. RESULTS: Inflammatory genes like IL1ß (C-C-C haplotype, p=0.0005, OR=3.09) and CD40 (rs4810485 allelic, p=0.04, OR=2.27) seem to be the determinants in Vata subgroup whereas oxidative stress pathway genes are observed in Pitta (SOD3 rs699473, p=0.004, OR=1.83; rs2536512 p=0.005; OR=1.88 and PON1 rs662, p=0.04, OR=1.53) and Kapha (SOD3 rs2536512, genotypic, p=0.02, OR=2.39) subgroups. Fixed effect analysis of the associated markers from CD40, SOD3 and TNFα with genotype X prakriti interaction terms suggests heterogeneity of effects within the subgroups. Further, disease characteristics such as severity was most pronounced in Vata group. CONCLUSIONS: This exploratory study suggests discrete causal pathways for RA etiology in prakriti based subgroups, thereby, validating concepts of prakriti and personalized medicine in Ayurveda. Ayurgenomics approach holds promise for biomarker discovery in complex diseases.

Genetically modified mouse models for the study of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and type 2 diabetes. NAFLD represents a large spectrum of diseases ranging from (1) fatty liver (hepatic steatosis); (2) steatosis with inflammation and necrosis; to (3) cirrhosis. The animal models to study NAFLD/nonalcoholic steatohepatitis (NASH) are extremely useful, as there are still many events to be elucidated in the pathology of NASH. The study of the established animal models has provided many clues in the pathogenesis of steatosis and steatohepatitis, but these remain incompletely understood. The different mouse models can be classified in two large groups. The first one includes genetically modified (transgenic or knockout) mice that spontaneously develop liver disease, and the second one includes mice that acquire the disease after dietary or pharmacological manipulation. Although the molecular mechanism leading to the development of hepatic steatosis in the pathogenesis of NAFLD is complex, genetically modified animal models may be a key for the treatment of NAFLD. Ideal animal models for NASH should closely resemble the pathological characteristics observed in humans. To date, no single animal model has encompassed the full spectrum of human disease progression, but they can imitate particular characteristics of human disease. Therefore, it is important that the researchers choose the appropriate animal model. This review discusses various genetically modified animal models developed and used in research on NAFLD.

Caucasian and Asian specific rheumatoid arthritis risk loci reveal limited replication and apparent allelic heterogeneity in north Indians.

Genome-wide association studies and meta-analysis indicate that several genes/loci are consistently associated with rheumatoid arthritis (RA) in European and Asian populations. To evaluate the transferability status of these findings to an ethnically diverse north Indian population, we performed a replication analysis. We investigated the association of 47 single-nucleotide polymorphisms (SNPs) at 43 of these genes/loci with RA in a north Indian cohort comprising 983 RA cases and 1007 age and gender matched controls. Genotyping was done using Infinium human 660w-quad. Association analysis by chi-square test implemented in plink was carried out in two steps. Firstly, association of the index or surrogate SNP (r2>0.8, calculated from reference GIH Hap-Map population) was tested. In the second step, evidence for allelic/locus heterogeneity at aforementioned genes/loci was assessed for by testing additional flanking SNPs in linkage equilibrium with index/surrogate marker.Of the 44 European specific index SNPs, neither index nor surrogate SNPs were present for nine SNPs in the genotyping array. Of the remaining 35, associations were replicated at seven genes namely PTPN22 (rs1217407, p = 3×10(-3)); IL2-21 (rs13119723, p = 0.008); HLA-DRB1 (rs660895, p = 2.56×10(-5); rs6457617, p = 1.6×10(-09); rs13192471, p = 6.7×10(-16)); TNFA1P3 (rs9321637, p = 0.03); CCL21 (rs13293020, p = 0.01); IL2RA (rs2104286, p = 1.9×10(-4)) and ZEB1 (rs2793108, p = 0.006). Of the three Asian specific loci tested, rs2977227 in PADI4 showed modest association (p<0.02). Further, of the 140 SNPs (in LE with index/surrogate variant) tested, association was observed at 11 additional genes: PTPRC, AFF3, CD28, CTLA4, PXK, ANKRD55, TAGAP, CCR6, BLK, CD40 and IL2RB. This study indicates limited replication of European and Asian index SNPs and apparent allelic heterogeneity in RA etiology among north Indians warranting independent GWAS in this population. However, replicated associations of HLA-DRB1, PTPN22 (which confer ∼50% of the heritable risk to RA) and IL2RA suggest that cross-ethnicity fine mapping of such loci is apposite for identification of causal variants.

Leads from xenobiotic metabolism genes for Parkinson's disease among north Indians.

OBJECTIVE: Pesticide/neurotoxin/free radical-induced oxidative stress leading to dopaminergic neuronal vulnerability is known to promote sporadic Parkinson's disease (PD). This study investigated the contribution of polymorphisms in genes from drug-metabolizing enzymes (DMEs) and the oxidative stress pathway to PD susceptibility and severity among a north Indian cohort. METHODS: Three hundred and thirty-nine PD patients diagnosed using UK PD brain bank criteria and 344 age-, sex-, and ethnicity-matched controls were recruited. Univariate and multivariate analyses were carried out to test allelic, genotypic, and haplotypic associations, and gene-gene interactions were assessed for 18 polymorphisms from 13 genes. Disease severity was calculated on the basis of the Hoehn and Yahr (HY) scale and Unified Parkinson's Disease Rating Scale III scores and was compared among the genotypic categories of markers. RESULTS: An association of GSTO1-rs4925 (P=0.04) and NQO1-rs1800566 (P=0.02) in univariate and multivariate analysis (P=0.01 and P=0.03, respectively) with disease susceptibility was observed. Significant and novel association of PON2-rs7493 (P=0.00009 with UPDRS III, P=0.003 with HY) with disease severity was retained after Bonferroni correction. On categorizing the cohort into young-onset PD (YOPD, n=90 cases, 104 controls) and late-onset PD ( n=249 cases, 240 controls), the association of several single nucleotide polymorphisms (SNPs) in DMEs was observed with YOPD. CONCLUSIONS: The association of NQO1, PON2, and DME genes (this study) and NAT2 (previous study) with PD among Indians may point toward an inherent population-specific genetic predisposition. This, probably compounded by an increase in environmental toxins and the indiscriminate use of pesticides in our country in the last few decades, may suggest likely gene-environment interactions, which may explain the increasing incidence of YOPD among Indians.

Association of Cullin1 haplotype variants with rheumatoid arthritis and response to methotrexate.

Aberrations in ubiquitin pathway have been implicated in many diseases and drug response. In a previous study on rheumatoid arthritis (RA) in Japanese population, significant association of Cullin1 gene (CUL1), an ubiquitin E3 ligase, was observed. CUL1 also mediates degradation of IκBα and p27, levels of which has been associated with RA etiology and drug response, respectively. We carried out a replication study of association of CUL1 polymorphisms with RA in a north Indian population. Allelic, genotypic, and haplotypic associations of a promoter and two intronic polymorphisms of CUL1 with RA and with methotrexate response in patients with RA, were tested. A significant association (P=0.00056, adjusted) of a haplotype A-T-T with RA (odds ratio=3.68; 95% confidence interval=1.86-7.27) and in patients with RA poorly responding to methotrexate treatment (P=0.04, adjusted) was observed. Association with CUL1 haplotype indicates a possible role of CUL1 variation(s) in RA and its response to methotrexate.

An investigation of genome-wide studies reported susceptibility loci for ulcerative colitis shows limited replication in north Indians.

Genome-Wide Association studies (GWAS) of both Crohn's Disease (CD) and Ulcerative Colitis (UC) have unearthed over 40 risk conferring variants. Recently, a meta-analysis on UC revealed several loci, most of which were either previously associated with UC or CD susceptibility in populations of European origin. In this study, we attempted to replicate these findings in an ethnically distinct north Indian UC cohort. 648 UC cases and 850 controls were genotyped using Infinium Human 660W-quad. Out of 59 meta-analysis index SNPs, six were not in the SNP array used in the study. Of the remaining 53 SNPs, four were found monomorphic. Association (p<0.05) at 25 SNPs was observed, of which 15 were CD specific. Only five SNPs namely rs2395185 (HLA-DRA), rs3024505 (IL10), rs6426833 (RNF186), rs3763313 (BTNL2) and rs2066843 (NOD2) retained significance after Bonferroni correction. These results (i) reveal limited replication of Caucasian based meta-analysis results; (ii) reiterate overlapping molecular mechanism(s) in UC and CD; (iii) indicate differences in genetic architecture between populations; and (iv) suggest that resources such as HapMap need to be extended to cover diverse ethnic populations. They also suggest a systematic GWAS in this terrain may be insightful for identifying population specific IBD risk conferring loci and thus enable cross-ethnicity fine mapping of disease loci.

Role of polymorphisms in dopamine synthesis and metabolism genes and association of DBH haplotypes with Parkinson's disease among North Indians.

OBJECTIVES: Genetic and non-genetic components are believed to govern the etiology of common complex traits such as Parkinson's disease (PD). In view of the biochemical evidence of depleted dopamine levels in the affected brains and also the most common and effective therapeutic modality of administration of levodopa in PD, genes from the dopaminergic pathway emerge as major determinants. We have earlier shown the role of DRD4-120 bp duplication marker in PD susceptibility. In this study, contribution of six genes involved in dopamine synthesis and metabolism to PD susceptibility and disease severity was assessed in a North Indian PD cohort. METHODS: 339 patients diagnosed using UKPD brain bank criteria and 344 matched controls were recruited and disease severity was assessed using the Hoehn and Yahr scale and Unified Parkinson Disease Rating Scale III scores. Allelic, genotypic and haplotypic associations with PD were computed; severity was compared among the genotypic categories of markers; gene-gene interactions were assessed using multiple logistic regression. RESULTS: A highly significant association of dopamine beta-hydroxylase (DBH) haplotypes (rs1611115T>C - rs1108580A>G - rs5320A>G - rs129882C>T) with PD was observed; haplotypes C-A-G-C [P=0.000005, Odds ratio (95% confidence interval): OR (95% CI)=1.76 (1.38-2.25)] and C-A-G-T [P=0.000001, OR (95% CI)=0.49 (0.37-0.65)] retaining significance after Bonferroni correction. rs129882, a 3'UTR SNP in DBH showed significant association with disease severity [Hoehn and Yahr (P=0.005) and Unified Parkinson Disease Rating Scale (P=0.006)]. CONCLUSION: Observed association of DBH SNP/SNP haplotypes with PD susceptibility and its role in modulating disease severity reiterates the importance of dopamine pathway in sporadic PD etiology in general and potential therapeutic implications of DBH in particular.