Shamsudheen Karuthedath Vellarikkal

Human 45,X fibroblast transcriptome reveals distinct differentially expressed genes including long noncoding RNAs potentially associated with the pathophysiology of Turner syndrome.

Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes.

Comparative whole-genome analysis of clinical isolates reveals characteristic architecture of Mycobacterium tuberculosis pangenome.

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

mit‐o‐matic: A Comprehensive Computational Pipeline for Clinical Evaluation of Mitochondrial Variations from Next‐Generation Sequencing Datasets

The human mitochondrial genome has been reported to have a very high mutation rate as compared with the nuclear genome. A large number of mitochondrial mutations show significant phenotypic association and are involved in a broad spectrum of diseases. In recent years, there has been a remarkable progress in the understanding of mitochondrial genetics. The availability of next‐generation sequencing (NGS) technologies have not only reduced sequencing cost by orders of magnitude but has also provided us good quality mitochondrial genome sequences with high coverage, thereby enabling decoding of a number of human mitochondrial diseases. In this study, we report a computational and experimental pipeline to decipher the human mitochondrial DNA variations and examine them for their clinical correlation. As a proof of principle, we also present a clinical study of a patient with Leigh disease and confirmed maternal inheritance of the causative allele. The pipeline is made available as a user‐friendly online tool to annotate variants and find haplogroup, disease association, and heteroplasmic sites. The “mit‐o‐matic” computational pipeline represents a comprehensive cloud‐based tool for clinical evaluation of mitochondrial genomic variations from NGS datasets. The tool is freely available at http://genome.igib.res.in/mitomatic/.

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×108 M-1; ΔH = 4.32×104±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a ‘wiki’-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a ‘structured wiki’ or rather a ‘semantic wiki’. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki

Draft Genome Sequence of a Clinical Isolate of Multidrug-Resistant Mycobacterium tuberculosis East African Indian Strain OSDD271.

ABSTRACT We describe the genome sequencing and analysis of a clinical isolate of Mycobacterium tuberculosis East African Indian (EAI) strain OSDD271 from India.

Whole‐exome sequencing solves diagnostic dilemma in a rare case of sporadic acrokeratosis verruciformis

riasis and in some patients the increasing severity of psoriasis may have a more negative impact for the patient than the actual malignancy diagnosis. In these circumstances, both the physician and the patient need to weigh the risks against the benefits of selecting alternative treatment methods. It has to be acknowledged that all six patients were exposed to systemic treatments before starting biologics and it may be the case that malignancies while taking biologics are more common because of the preceding immunosuppressive burden. In five of our six patients, systemic therapy was introduced following malignancy diagnosis but despite this approach, they had recalcitrant psoriasis with suboptimal control. Until more data including information on the cancer risk profile of biologic and systemic therapies are available, through the ongoing dermatology intervention registries, it is difficult to postulate if the solid tumours observed in our cohort are related or not, to biologic therapy. Our experience, also confirmed that there are limited treatment options for psoriasis patients who develop malignancy.

Exome sequencing reveals a novel mutation, p.L325H, in the KRT5 gene associated with autosomal dominant Epidermolysis Bullosa Simplex Koebner type in a large family from western India.

We report a large, non-consanguineous family comprising five generations of individuals residing in Gujarat, India affected with localized Epidermolysis Bullosa Simplex (EBS) Koebner type. We analyzed 14 individuals including 9 affected individuals from this family. Exome sequencing in two cases suggested a novel non-synonymous variation, p.L325H, in the KRT5 gene. The present analysis also reports the first causative mutation of EBS Koebner type from India.

Draft Genome Sequence of an Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolate of the Ural Strain OSDD493

ABSTRACT We describe the genome sequencing and analysis of a clinical isolate of Mycobacterium tuberculosis belonging to the Ural strain OSDD493 from India.

AB0188 Systematic Analysis of the Oral Microbiome in Primary SjÖgren's Syndrome Suggest Enrichment of Distinct Microbes

Background Dysbiosis has been hypothesized to play a role in the pathogenesis of autoimmune disease. Primary Sjögrens syndrome (pSS) is an autoimmune disease characterized by sicca symptoms resulting from salivary and lacrimal gland dysfunction. This could result in dysbiosis of oral cavity. At the same time, dysbiosis could also be hypothesized to have a causative role. Previous studies using culture dependent approaches have provided evidence for altered oral microflora in pSS. However culture dependent approaches have caveats which have been abrogated with the advent of culture independent methodologies. Objectives To systematically evaluate the microbiome in the oral cavity in patients with pSS using a culture independent shotgun metagenome sequencing Methods Cases included adult patients fulfilling criteria for pSS by American-European Consensus Group (AECG) 2002 or American College of Rheumatology (ACR) 2012 classification criteria, while controls included healthy volunteers. Neither cases nor controls had oral disease or other obvious co morbidities, recent antibiotic intake. Individuals who had chronic intake of alcohol and tobacco were excluded. Saliva was collected in sterile tubes with buffer. DNA was extracted using Qiagen DNA isolation kit. Libraries were prepared and sequenced on Illumina Hiseq 2500 (Illumina Inc, USA). Reads mapping to the Human reference genome (hg19) were tagged. Reads were further reference mapped to the core oral microbiome sequences available from the Human Oral Microbiome Database. The read counts were normalized for the input reads as well as the genome size of the organism. A fold change (FC) of >2 and p value <0.05 by Students t-test was considered significant. Results Oral microbiome of 13 pSS patients and 12 healthy controls were analysed. Organisms significantly enriched in pSS included the following (FC; p-value) Capnocytophaga (2.09; 0.01), Dialister (2.13; 0.02), Fusobacterium (2.84; 0.04), Helicobacter (4.83; 0.03), Streptococcus (3.33; 0.01) and Veilonella (3.82; 0.006) spp. A paucity of Pseudomonas (8.9;0.03) spp. was noted compared to controls. Conclusions A distinct subset of organisms was enriched in the oral cavity of patients with pSS. This subset includes Capnocytophaga previously shown to be associated with the pathogenesis and T cell activation in pSS. References Almståhl A, Kroneld U, Tarkowski A, Wikström M. Oral microbial flora in Sjögrens syndrome. J Rheumatol. 1999;26:110-4. Almståhl A, Wikström M, Kroneld U. Microflora in oral ecosystems in primary Sjögrens syndrome. J Rheumatol. 2001;28:1007-13. Szymula A, Rosenthal J, Szczerba BM, Bagavant H, Fu SM, Deshmukh US.T cell epitope mimicry between Sjögrens syndrome Antigen A (SSA)/Ro60 and oral, gut, skin and vaginal bacteria. Clin Immunol. 2014;152:1-9. Acknowledgements Authors acknowledge colleagues at Department of Clinical Immunology and Rheumatology, CMC Vellore for help in recruiting volunteers for the study. Authors VS and SS acknowledge funding from CSIR, India through Grant OLP1105 (EMPOWER). Disclosure of Interest None declared