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.

The Spectrum of Non-Parkinsonian Tremor: A Registry at a Tertiary Care Teaching Institute.

Background: Tremors other than those associated with Parkinson's disease (non-parkinsonian tremor) are commonly observed in clinical settings. However, their frequency and clinical characteristics have rarely been reported. Objectives: To classify non-parkinsonian tremors based on the consensus statement on the classification of tremors, from the task force of the International Parkinson and Movement Disorder Society published in 2018. Methods: A prospective registry at a tertiary care teaching institute. Results: A total of 475 patients with non-parkinsonian tremors were recruited for the study. 67.57% (n = 321) of our patients were male and a family history of tremor was present in 20.84% (n = 99) of patients. Dystonic tremor (DT) was the most common non-parkinsonian tremor (33.26%). 27.78% of patients fulfilled the new classification criteria for essential tremor, with 13.47% classified as pure ET (ET) and 14.31% exhibiting neurological soft signs, leading to the classification of ET plus (ETP). Patients with ETP had more family history (57.35%) [vs DT (26.48%, p = 0.00004) and ET (10.93%, p = 0.00003], longer duration of disease [mean ± standard deviation (SD) = 9.53 ± 8.64 years] [vs DT (5.60 ± 5.93, p = 0.0003) and ET (6.38 ± 5.97, p = 0.01) years], and more severe tremor as measured by the essential tremor rating assessment scale total score [mean ± SD = 27.42 ± 11.70] [vs DT (23.50 ± 8.62, p = 0.007) and ET (22.12 ± 8.19, p = 0.007)] compared with patients with DT and ET. Conclusions: DT was the most common cause of non-parkinsonian tremor in our registry followed by essential tremor syndrome. ETP was more common than ET.

Mutation spectrum and enzyme profiling of G6PD deficiency in neonates of north India: a prospective study.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked disorder with well-established clinical and allelic heterogeneity and ethnic disparity. With ~390,000 annual births with G6PD deficiency in India, it emerges as the most predictable and preventable inbornmetabolic error. Disease prevalence and mutation spectrum have been reasonably reported fromcentral, western and southern parts of India and are mostly retrospective studies.Although prevalence data fromnorth India is available, there is paucity of data on the mutation spectrum and genotype-phenotype correlation (GxP). Thus, we aimed at establishing the clinical and mutation profiles for G6PD, as a part of a large prospective newborn screening study conducted between 2014 and 2016 across hospitals in Delhi, India. G6PD activity levels were measured at 24-48 h of life for ~200,000 neonates using Victor 2D and/or Genomic Screening Processor followed by confirmatory spectrophotometric analysis usingRBClysates of the respective neonates based on clinical symptoms.Asubset of 570 enzyme deficient neonates were screened formutations by polymerase chain reaction-restriction fragment length polymorphismand/or Sanger sequencing.Mediterraneanwas the most common mutation (n=318; 55.8%) with the lowest enzyme activity and most severe phenotype, followed by G6PD Orissa (n=187;32.8%); Kerala-Kalyan (n=25); Jammu (n=24);Mahidol (n=14); Chattam(n=1) andNilgiri/Coimbra (n=1).Of the 163 intramural neonates followed up, 68 developed clinical jaundice. However, no correlation was observed between jaundice and enzyme level. Notable outcome of this first ever prospective screening approach for G6PD deficiency in neonates may help in prediction of disease severity and appropriate timely management.

Effect of rs1108580 of DBH and rs1006737 of CACNA1C on Cognition and Tardive Dyskinesia in a North Indian Schizophrenia Cohort.

Genetic perturbations in dopamine neurotransmission and calcium signaling pathways are implicated in the etiology of schizophrenia. We aimed to test the association of a functional splice variant each in Dopamine ß-Hydroxylase (DBH; rs1108580) and Calcium voltage-gated channel subunit alpha1 C (CACNA1C; rs1006737) genes in these pathways with schizophrenia (506 cases, 443 controls); Abnormal Involuntary Movement Scale (AIMS) scores in subjects assessed for tardive dyskinesia (76 TD-positive, 95 TD-negative) and Penn Computerized Neurocognitive Battery (PennCNB) scores (334 cases, 234 controls). The effect of smoking status and SNP genotypes on AIMS scores were assessed using ANOVA; health status and SNP genotypes on three performance functions of PennCNB cognitive domains were assessed by ANCOVA with age and sex as covariates. Association with Positive and Negative Syndrome Scale (PANSS) scores in the TD cohort and cognitive scores in healthy controls of the cognition cohort were tested by linear regression. None of the markers were associated with schizophrenia. Smoking status [F(2, 139) = 10.6; p = 5 × 10-5], rs1006737 [F(2, 139) = 7.1; p = 0.001], TD status*smoking [F(2, 139) = 8.0; p = 5.0 × 10-4] and smoking status*rs1006737 [F(4, 139) = 2.7; p = 0.03] had an effect on AIMS score. Furthermore, rs1006737 was associated with orofacial [F(2, 139) = 4.6; p = 0.01] and limb-truncal TD [(F(2, 139) = 3.8; p = 0.02]. Main effect of rs1108580 on working memoryprocessing speed [F(2, 544) = 3.8; p = 0.03] and rs1006737 on spatial abilityefficiency [F(1, 550) = 9.4; p = 0.02] was identified. Health status*rs1006737 interaction had an effect on spatial memoryprocessing speed [F(1, 550) = 6.9; p = 0.01]. Allelic/genotypic association (p = 0.01/0.03) of rs1006737 with disorganized/concrete factor and allelic association of rs1108580 (p = 0.04) with a depressive factor of PANSS was observed in the TD-negative subcohort. Allelic association of rs1006737 with sensorimotor dexterityaccuracy (p = 0.03), attentionefficiency (p = 0.05), and spatial abilityefficiency (p = 0.02); allelic association of rs1108580 with face memoryaccuracy (p = 0.05) and emotionefficiency (p = 0.05); and allelic/genotypic association with emotionaccuracy (p = 0.003/0.009) were observed in healthy controls of the cognition cohort. These association findings may have direct implications for personalized medicine and cognitive remediation.

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.

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.

Celiac disease-associated loci show considerable genetic overlap with neuropsychiatric diseases but with limited transethnic applicability.

Clinical and public health research has revealed the co-occurrence of several neuropsychiatric diseases among patients with celiac disease (CD). The significant presence of CD-specific autoantibodies in patients with neuropsychiatric diseases and vice versa are often reported. To explain the genetic basis of such frequent disease co-occurrence and investigate the underlying common pathways/processes, we performed an extensive cross-disease association study followed by supporting in silico functional validation of the leads. Genomewide association study (GWAS) data for CD and eight commonly co-occurring neuropsychiatric diseases from Caucasian populations were analysed, and the shared loci were determined.We performed Immunochip-based fine mapping of these overlapping association signals in an independent European CD data and tested their cross-ethnic transferability using CD association data from the genetically distinct north Indian population. This study identified 12 shared loci between the two diseases with genomewide significance (P = 5e-8). Of these five loci, namely NFIA, KIA1109, NOTCH4-TSBP1-PBX2, HLA-DQA1 and CSK replicated in an independent Dutch cohort representing European ancestry. Three of these loci, namely NFIA, NOTCH4-TSBP1-PBX2 and HLA-DQA1 that are common between CD, anxiety, migraine and schizophrenia respectively withstood locus transferability test in north Indians. Tissue-specific eQTL analysis of SNPs from transferable loci revealed expression QTL effects in brain tissue besides the small intestine and whole blood. Pathway analysis and evidence of epigenetic regulation highlighted the potential contribution of these SNPs to disease pathology. The replicable and transferable association of genetic variants from MHC locus and their functional implications suggest the process of antigen presentation and adaptive/innate immune response regulated by non-HLA genes in the locus may dominate the shared pathogenesis of CD and neuropsychiatric diseases. Functional validation of the shared candidate genes is warranted to unravel the molecular mechanism for the co-occurrence of CD and specific neuropsychiatric diseases.

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.

Microhomology-mediated endjoining repair mechanism enables rapid and effective indel generations in stem cells.

Functional characterization of gene(s) using a transgene approach in a human cell line or in an animal model generally poses limitations due to persistent transgene overexpression. Conversely, the CRISPR/Cas9 geneediting technology enables precise variant(s) introduction in a gene, thus facilitating accurate characterization in human iPSC-derived target cell/tissue. Such editing is generally mediated by non-homologous end joining, the predominant and error-prone double-strand break repair mechanism which mostly results in gene knockout due to indel(s) generation. However, in most cases the best in silico predicted sgRNAs fail to generate indels especially in iPSCs, encouraging a revisit of DNA damage repair principles. Microhomology-mediated end joining (MMEJ) is another error-prone repair mechanism which relies on exposed microhomologous sequences flanking the broken ends to fix double-strand breaks. Therefore, sgRNAs targeting the exonic region encompassing di- or tri-nucleotide repeats along with non-repeat exonic region as control, in RIC3, a gene of our interest, were designed to generate effective indel(s) exploiting the MMEJ DSB repair mechanism. iPSCs were co-transfected with eCas9+EGFP and sgRNA+puromycin plasmids and positive clones enriched by transient puromycin selection. Multiple deletion lines adjacent to microhomologous (repeat) region and several lines heterozygous with only 1 bp insertion for the non-repeat region were obtained, and confirmed by Sanger sequencing. These findings suggest that (i) designing sgRNAs from different exonic regions with microrepeats and (ii) MMEJ combined with a rapid and less expensive method of using antibiotic screening of edited lines without cumbersome cell sorting may be effective strategies for indel(s) generation in stem cells.

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.

Genetic variations in evolutionary accelerated regions disrupt cognition in schizophrenia.

Cognition is believed to be a product of human evolution, while schizophrenia is ascribed as the by-product with cognitive impairment as it's genetically mediated endophenotype. Genomic loci associated with these traits are enriched with recent evolutionary markers such as Human accelerated regions (HARs). HARs are markedly different in humans since their divergence with chimpanzees and mostly regulate gene expression by binding to transcription factors and/or modulating chromatin interactions. We hypothesize that variants within HARs may alter such functions and thus contribute to disease pathogenesis. 49 systematically prioritized variants from 2737 genome-wide HARs were genotyped in a north-Indian schizophrenia cohort (331 cases, 235 controls). Six variants were significantly associated with cognitive impairment in schizophrenia, thirteen with general cognition in healthy individuals. These variants were mapped to 122 genes; predicted to alter 79 transcription factors binding sites and overlapped with promoters, enhancers and/or repressors. These genes and TFs are implicated in neurocognitive phenotypes, autism, schizophrenia and bipolar disorders; a few are targets of common or repurposable antipsychotics suggesting their draggability; and enriched for immune response and brain developmental pathways. Immune response has been more strongly targeted by natural selection during human evolution and has a prominent role in neurodevelopment. Thus, its disruption may have deleterious consequences for neuronal and cognitive functions. Importantly, among the 15 associated SNPs, 12 showed association in several independent GWASs of different neurocognitive functions. Further analysis of HARs may be valuable to understand their role in cognition biology and identify improved therapeutics for schizophrenia.

New Druggable Targets for Rheumatoid Arthritis Based on Insights From Synovial Biology.

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease characterized by chronic inflammation and destruction of multiple small joints which may lead to systemic complications. Altered immunity via pathogenic autoantibodies pre-date clinical symptom development by several years. Incompletely understood range of mechanisms trigger joint-homing, leading to clinically evident articular disease. Advances in therapeutic approaches and understanding pathogenesis have improved prognosis and likely remission. However, partial/non-response to conventional and biologic therapies witnessed in a subset of patients highlights the need for new therapeutics. It is now evident that joint disease chronicity stems from recalcitrant inflammatory synovial environment, majorly maintained by epigenetically and metabolically reprogrammed synoviocytes. Therefore, interference with effector functions of activated cell types seems a rational strategy to reinstate synovial homeostasis and complement existing anti-inflammatory interventions to mitigate chronic RA. Presenting this newer aspect of fibroblast-like synoviocytes and myeloid cells underlying the altered synovial biology in RA and its potential for identification of new druggable targets is attempted in this review. Major leads from i) molecular insights of pathogenic cell types from hypothesis free OMICS approaches; ii) hierarchy of their dysregulated signaling pathways; and iii) knowledge of druggability of molecular nodes in these pathways are highlighted. Development of such synovial biology-directed therapeutics hold promise for an enriched drug repertoire for RA.

Computational insight into the three-dimensional structure of ADP ribosylation factor like protein 15, a novel susceptibility gene for rheumatoid arthritis.

The ARL15 gene (ADP ribosylation factor like protein 15) encodes for an uncharacterized small GTP-binding protein. Its exact role in human physiology remains unknown, but a number of genetic association studies have recognised different variants in this gene to be statistically associated with numerous traits and complex diseases. We have previously reported a novel association of ARL15 with rheumatoid arthritis (RA) based on a genome-wide association study in a north Indian cohort. Subsequent investigations have provided leads for its involvement in RA pathophysiology, especially its potential as a novel therapeutic target. However, the absence of an experimentally determined tertiary structure for ARL15 significantly hinders the understanding of its biochemical and physiological functions, as well as development of potential lead molecules. We, therefore, aimed to derive a high quality, refined model of the three dimensional structure of human ARL15 protein using two different computational protein structure prediction methods - template-based threading and ab initio modelling. The best model each from among the five each derived from both the approaches was selected based on stringent quality assessment and refinement. Molecular dynamics simulations over long timescales revealed the ab initio model to be relatively more stable, and it marginally outperformed the template-based model in the quality assessment as well. A putative GTP-binding site was also predicted using homology for the ARL15 protein, where potential competitive inhibitors can be targeted. This high quality predicted model may provide insights to the biological role(s) of ARL15 and inform and guide further experimental, structural and biochemical characterization efforts.Communicated by Ramaswamy H. Sarma.

Transethnic analysis of psoriasis susceptibility in South Asians and Europeans enhances fine-mapping in the MHC and genomewide.

Because transethnic analysis may facilitate prioritization of causal genetic variants, we performed a genomewide association study (GWAS) of psoriasis in South Asians (SAS), consisting of 2,590 cases and 1,720 controls. Comparison with our existing European-origin (EUR) GWAS showed that effect sizes of known psoriasis signals were highly correlated in SAS and EUR (Spearman ρ = 0.78; p < 2 × 10-14). Transethnic meta-analysis identified two non-MHC psoriasis loci (1p36.22 and 1q24.2) not previously identified in EUR, which may have regulatory roles. For these two loci, the transethnic GWAS provided higher genetic resolution and reduced the number of potential causal variants compared to using the EUR sample alone. We then explored multiple strategies to develop reference panels for accurately imputing MHC genotypes in both SAS and EUR populations and conducted a fine-mapping of MHC psoriasis associations in SAS and the largest such effort for EUR. HLA-C*06 was the top-ranking MHC locus in both populations but was even more prominent in SAS based on odds ratio, disease liability, model fit and predictive power. Transethnic modeling also substantially boosted the probability that the HLA-C*06 protein variant is causal. Secondary MHC signals included coding variants of HLA-C and HLA-B, but also potential regulatory variants of these two genes as well as HLA-A and several HLA class II genes, with effects on both chromatin accessibility and gene expression. This study highlights the shared genetic basis of psoriasis in SAS and EUR populations and the value of transethnic meta-analysis for discovery and fine-mapping of susceptibility loci.

Genome wide study of tardive dyskinesia in schizophrenia.

Tardive dyskinesia (TD) is a severe condition characterized by repetitive involuntary movement of orofacial regions and extremities. Patients treated with antipsychotics typically present with TD symptomatology. Here, we conducted the largest GWAS of TD to date, by meta-analyzing samples of East-Asian, European, and African American ancestry, followed by analyses of biological pathways and polygenic risk with related phenotypes. We identified a novel locus and three suggestive loci, implicating immune-related pathways. Through integrating trans-ethnic fine mapping, we identified putative credible causal variants for three of the loci. Post-hoc analysis revealed that SNPs harbored in TNFRSF1B and CALCOCO1 independently conferred three-fold increase in TD risk, beyond clinical risk factors like Age of onset and Duration of illness to schizophrenia. Further work is necessary to replicate loci that are reported in the study and evaluate the polygenic architecture underlying TD.

Correlation between an intronic SNP genotype and ARL15 level in rheumatoid arthritis.

ADP ribosylation factor like protein 15 (ARL15) was identified as a novel susceptibility gene for rheumatoid arthritis (RA) based on a genomewide association study in a north Indian cohort. Mechanism of its action and functional relevance in RA biology remain largely unknown. In this study, we aimed to establish (i) ARL15 protein level in sera samples of RA patients; and (ii) its correlation, if any, with the RA associated ARL15 intronic single-nucleotide polymorphism (SNP) rs255758 (A>C). DNA, RNA and sera were isolated from blood samples of 117 RA patients and 25 age-matched healthy controls recruited at All India Institute of Medical Sciences, New Delhi with institutional ethical committee clearance. SNP rs255758 (A>C) was genotyped by Sanger sequencing; ARL15 RNA and protein levels were estimated by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively; and genotype-phenotype correlation established using Mann-Whitney nonparametric test. Very low level of ARL15 expression in human blood was confirmed at both RNA and protein levels. Genotype-wise distribution showed increased levels (P = 0.05) of ARL15 protein in RA patients with the homozygous variant (CC) as compared to AA + AC genotypes of rs255758. This first-ever correlation between higher ARL15 protein levels and the intronic susceptibility genotype (CC; rs255758) in RA patients may be of diagnostic and therapeutic relevance encouraging additional investigations.

Multiple allelic associations from genes involved in energy metabolism were identified in celiac disease.

Energy metabolism is a critical factor that influences disease pathogenesis. Recent high-throughput genomic studies have enabled us to look into disease biology with greater details. Celiac disease (CD) is an inflammatory autoimmune disease where ~60 non-HLA genes were identified which in conjunction with HLA genes explain ~55% of the disease heritability. In this study we aimed to identify susceptibility energy metabolism genes and investigate their role in CD. We re-analysed published Immunochip genotyping data, which were originally analysed for CD association studies in north Indian and Dutch population. 269 energy metabolism genes were tested. Meta-analysis was done for the identified SNPs. To validate the functional implications of identified markers and/or genes, in silico functional annotation was performed. Six SNPs were identified in north Indians, of which three markers from two loci were replicated in Dutch. rs2071592 (PMeta=5.01e-75) and rs2251824 (PMeta=1.87e-14) from ATP6V1G2-NFKBIL1-DDX39B locus and rs4947331 (PMeta= 9.85e-13) from NEU1 locus were found significantly associated. Identified genes are key regulators of cellular energy metabolism and associated with several immune mediated diseases. In silico functional annotation showed significant biological relevance of these novel markers and genes. FDI approved therapeutics against ATP6V1G2 and NEU1 are currently in use to treat chronic and inflammatory diseases. This study identified two pathogenic loci, originally involved in energy metabolism. Extensive investigation showed their synergistic role in CD pathogenesis by promoting immune mediated enteric inflammation. Proposed CD pathogenesis model in this study needs to be tested through tissue-on-chip and in vivo methods to ensure its translational application.

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.

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.