Ankit Verma

RNA sequencing of db/db mice liver identifies lncRNA H19 as a key regulator of gluconeogenesis and hepatic glucose output

Liver plays a key role in maintaining glucose homeostasis and impaired hepatic glucose metabolism is associated with type 2 diabetes. In the present study, we used RNA sequencing to profile the transcriptome of the livers of diabetic db/db mice as compared to the normal db/+ mice and identified 218 differentially expressed genes. Amongst these, there were 3 lncRNAs that were significantly downregulated and H19 was the most altered lncRNA in the livers of db/db mice. H19 expression significantly correlated with the expression of genes of the glycolysis and gluconeogenesis pathways, which suggest that altered hepatic H19 levels can directly or indirectly modulate their expression. Inhibition of H19 using specific siRNA in HepG2 cells and primary mouse hepatocytes significantly increased the levels of gluconeogenic genes. This was subsequently accompanied by increased hepatic glucose output. Further,H19 depletion in HepG2 cells impaired insulin signaling and increased nuclear localization of FoxO1, an important transcriptional regulator of gluconeogenic gene expression. Our results reveal a novel link between decreased H19 levels and impaired gluconeogenesis via regulation of FoxO1 nuclear levels. These put forth interesting observations on the regulatory role of H19 in altering hepatic physiology during diabetes.

Chamber Specific Gene Expression Landscape of the Zebrafish Heart

The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6 tissue types of zebrafish. We hypothesize that clustering the differentially expressed genes with both known and unknown functions will deliver detailed insights on fundamental gene networks that are important for the development and specification of the cardiac chambers. It is also postulated that this transcriptome atlas will help utilize zebrafish in a better way as a model for studying cardiac development and to explore functional role of gene networks in cardiac disease pathogenesis.

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.

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

Application of whole exome sequencing in elucidating the phenotype and genotype spectrum of junctional epidermolysis bullosa: A preliminary experience of a tertiary care centre in India

BACKGROUND Junctional epidermolysis bullosa (JEB) is a diverse group of genodermatoses associated with extreme skin fragility. Despite several well-characterized genetic studies, molecular diagnosis of this heterogeneous group is still challenging. Recent advances in the field of genomics have seen the successful implementation of whole exome sequencing (WES) as a fast and efficient diagnostic strategy in several genodermatoses. OBJECTIVE In view of the scarcity and need of molecular studies for JEB in India, we sought to explore the potential of WES in understanding the mutational spectrum of this rare, in certain subtypes lethal, sub-group of EB. METHODS WES was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human reference genome hg19. Sanger sequencing subsequently confirmed the potentially pathogenic mutations. RESULTS Overall, four unrelated families (6 patients) of JEB with a highly variable clinical presentation including a rare case of LOC syndrome were studied. WES revealed 4 variations in 3 genes (LAMA3, LAMB3 and COL17A1) that are implicated in JEB. None of the variations were recurrent. In addition we proposed the probable molecular consequence of a missense mutation on the structure-function relationship of lamininβ3 protein through computational modeling studies. CONCLUSIONS Being the first report documenting the phenotype-genotype correlations of JEB patients from India, our preliminary experience with WES is clearly encouraging and serves as a nidus for future large-scale molecular studies to actively identify and understand JEB patients in Indian population.

Case Report: Whole exome sequencing identifies variation c.2308G>A p.E770K in RAG1 associated with B- T- NK+ severe combined immunodeficiency

Severe combined immunodeficiency is a large clinically heterogeneous group of disorders caused by a defect in the development of humoral or cellular immune responses. At least 13 genes are known to be involved in the pathophysiology of the disease and the mutation spectrum in SCID have been well documented. The widespread application of whole-exome sequencing based on next-generation sequencing has offered a new opportunity to systematically screen these genes in clinical scales. In this report, we describe the application of whole exome sequencing for arriving at a molecular diagnosis in a child suffering from B- T- NK+ severe combined immunodeficiency. Apart from making the accurate molecular diagnosis, we also add a genetic variation c.2308G>A p.E770K to the compendium of variations associated with the disease.

Draft Genome Sequence of the Extremely Halophilic Bacterium Halomonas salina Strain CIFRI1, Isolated from the East Coast of India

ABSTRACT Halomonas salina strain CIFRI1 is an extremely salt-stress-tolerant bacterium isolated from the salt crystals of the east coast of India. Here we report the annotated 3.45-Mb draft genome sequence of strain CIFRI1 having 86 contigs with 3,139 protein coding loci, including 62 RNA genes.

Case Report: Whole exome sequencing helps in accurate molecular diagnosis in siblings with a rare co-occurrence of paternally inherited 22q12 duplication and autosomal recessive non-syndromic ichthyosis.

Lamellar ichthyosis (LI), considered an autosomal recessive monogenic genodermatosis, has an incidence of approximately 1 in 250,000. Usually associated with mutations in the transglutaminase gene ( TGM1), mutations in six other genes have, less frequently, been shown to be causative. Two siblings, born in a collodion membrane, presented with fish like scales all over the body. Karyotyping revealed duplication of the chromosome arm on 22q12+ in the father and two siblings. Whole exome sequencing revealed a homozygous p.Gly218Ser variation in TGM1; a variation reported earlier in an isolated Finnish population in association with autosomal recessive non-syndromic ichthyosis. This concurrence of a potentially benign 22q12+ duplication and LI, both rare individually, is reported here likely for the first time.

Pharmacogenetic landscape of DPYD variants in south Asian populations by integration of genome-scale data.

AIM Adverse drug reactions to 5-Fluorouracil(5-FU) is frequent and largely attributable to genetic variations in the DPYD gene, a rate limiting enzyme that clears 5-FU. The study aims at understanding the pharmacogenetic landscape of DPYD variants in south Asian populations. MATERIALS & METHODS Systematic analysis of population scale genome wide datasets of over 3000 south Asians was performed. Independent evaluation was performed in a small cohort of patients. RESULTS Our analysis revealed significant differences in the the allelic distribution of variants in different ethnicities. CONCLUSIONS This is the first and largest genetic map the DPYD variants associated with adverse drug reaction to 5-FU in south Asian population. Our study highlights ethnic differences in allelic frequencies.

Large scale changes in the transcriptome of Eisenia fetida during regeneration

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.