Chronic systemic exposure to IL6 leads to deregulation of glycolysis and fat accumulation in the zebrafish liver.

Inflammation is a constant in Non-Alcoholic Fatty Liver Disease (NAFLD), although their relationship is unclear. In a transgenic zebrafish system with chronic systemic overexpression of human IL6 (IL6-OE) we show that inflammation can cause intra-hepatic accumulation of triglycerides. Transcriptomics and proteomics analysis of the IL6-OE liver revealed a deregulation of glycolysis/gluconeogenesis pathway, especially a striking down regulation of the glycolytic enzyme aldolase b. Metabolomics analysis by mass spectrometry showed accumulation of hexose monophosphates and their derivatives, which can act as precursors for triglyceride synthesis. Our results suggest that IL6-driven repression of glycolysis/gluconeogenesis, specifically aldolase b, may be a novel mechanism for fatty liver. This mechanism may be relevant for NAFLD in lean individuals, an emerging class of NAFLD prevalent more in Asian Indian populations.

Saliva microbiome in primary Sjögren's syndrome reveals distinct set of disease-associated microbes.

OBJECTIVE: This study systematically aims to evaluate the salivary microbiome in patients with primary Sjögren's syndrome (pSS) using 16S rRNA sequencing approach. METHODS: DNA isolation and 16S rRNA sequencing was performed on saliva of 37 pSS and 35 control (CC) samples on HiSeq 2500 platform. 16S rRNA sequence analysis was performed independently using two popular computational pipelines, QIIME and less operational taxonomic units scripts (LoTuS). RESULTS: There were no significant changes in the alpha diversity between saliva of patients and controls. However, four genera including Bifidobacterium, Lactobacillus, Dialister and Leptotrichia were found to be differential between the two sets, and common between both QIIME and LoTuS analysis pipelines (Fold change of 2 and p < .05). Bifidobacterium, Dialister and Lactobacillus were found to be enriched, while Leptotrichia was significantly depleted in pSS compared to the controls. Exploration of microbial diversity measures (Chao1, observed species and Shannon index) revealed a significant increase in the diversity in patients with renal tubular acidosis. An opposite trend was noted, with depletion of diversity in patients with steroids. CONCLUSION: Our analysis suggests that while no significant changes in the diversity of the salivary microbiome could be observed in Sjögren's syndrome compared to the controls, a set of four genera were significantly and consistently differential in the saliva of patients with pSS. Additionally, a difference in alpha diversity in patients with renal tubular acidosis and those on steroids was observed.

Investigating Coronary Artery Disease methylome through targeted bisulfite sequencing.

BACKGROUND: Gene environment interactions leading to epigenetic alterations play pivotal role in the pathogenesis of Coronary Artery Disease (CAD). Altered DNA methylation is one such epigenetic factor that could lead to altered disease etiology. In this study, we comprehensively identified methylation sites in several genes that have been previously associated with young CAD patients. METHODS: The study population consisted of 42 healthy controls and 33 young CAD patients (age group <50 years). We performed targeted bisulfite sequencing of promoter as well as gene body regions of several genes in various pathways like cholesterol synthesis and metabolism, endothelial dysfunction, apoptosis, which are implicated in the development of CAD. RESULTS: We observed that the genes like GALNT2, HMGCR were hypermethylated in the promoter whereas LDLR gene promoter was hypomethylated indicating that intracellular LDL uptake was higher in CAD patients. Although APOA1 did not show significant change in methylation but APOC3 and APOA5 showed variation in methylation in promoter and exonic regions. Glucokinase (GCK) and endothelial nitric oxide synthase 3 (NOS3) were hyper methylated in the promoter. Genes involved in apoptosis (BAX/BCL2/AKT2) and inflammation (PHACTR1/LCK) also showed differential methylation between controls and CAD patients. A combined analysis of the methylated CpG sites using machine learning tool revealed 14 CpGs in 11 genes that could discriminate CAD cases from controls with over 93% accuracy. CONCLUSIONS: This study is unique because it highlights important gene methylation alterations which might predict the risk of young CAD in Indian population. Large scale studies in different populations would be important for validating our findings and understanding the epigenetic events associated with CAD.

Loss of function mutation in the P2X7, a ligand-gated ion channel gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an inherited heart failure condition, mostly found to have genetic abnormalities, and is a leading cause of sudden death in young adults. Whole exome sequencing should be given consideration as a molecular diagnostic tool to identify disease-causing mutation/s. In this study, a HCM family with multiple affected members having history of sudden death were subjected to exome sequencing along with unaffected members. Quality passed variants obtained were filtered for rarity (MAF > 0.5%), evolutionary conservation, pathogenic prediction, and segregation in affected members after removing shared variants present in unaffected members. Only one non-synonymous mutation (p. Glu186Lys or E186K) in exon 6 of P2X7 gene segregated in HCM-affected individuals which was absent in unaffected family members and 100 clinically evaluated controls. The site of the mutation is highly conserved and led to complete loss of function which is in close vicinity to ATP-binding site-forming residues, affecting ATP binding, channel gating, or both. Mutations in candidate genes which were not segregated define clinical heterogeneity within affected members. P2X7 gene is highly expressed in the heart and shows direct interaction with major candidate genes for HCM. Our results reveal a significant putative HCM causative gene, P2X7, for the first time and show that germ-line mutations in P2X7 may cause a defective phenotype, suggesting purinergic receptor involvement in heart failure mediated through arrhythmias which need further investigations to be targeted for therapeutic interventions.

A genome-wide map of circular RNAs in adult zebrafish.

Circular RNAs (circRNAs) are transcript isoforms generated by back-splicing of exons and circularisation of the transcript. Recent genome-wide maps created for circular RNAs in humans and other model organisms have motivated us to explore the repertoire of circular RNAs in zebrafish, a popular model organism. We generated RNA-seq data for five major zebrafish tissues - Blood, Brain, Heart, Gills and Muscle. The repertoire RNA sequence reads left over after reference mapping to linear transcripts were used to identify unique back-spliced exons utilizing a split-mapping algorithm. Our analysis revealed 3,428 novel circRNAs in zebrafish. Further in-depth analysis suggested that majority of the circRNAs were derived from previously well-annotated protein-coding and long noncoding RNA gene loci. In addition, many of the circular RNAs showed extensive tissue specificity. We independently validated a subset of circRNAs using polymerase chain reaction (PCR) and divergent set of primers. Expression analysis using quantitative real time PCR recapitulate selected tissue specificity in the candidates studied. This study provides a comprehensive genome-wide map of circular RNAs in zebrafish tissues.

Organellar transcriptome sequencing reveals mitochondrial localization of nuclear encoded transcripts.

Mitochondria are organelles involved in a variety of biological functions in the cell, apart from their principal role in generation of ATP, the cellular currency of energy. The mitochondria, in spite of being compact organelles, are capable of performing complex biological functions largely because of the ability to exchange proteins, RNA, chemical metabolites and other biomolecules between cellular compartments. A close network of biomolecular interactions are known to modulate the crosstalk between the mitochondria and the nuclear genome. Apart from the small repertoire of genes encoded by the mitochondrial genome, it is now known that the functionality of the organelle is highly reliant on a number of proteins encoded by the nuclear genome, which localize to the mitochondria. With exceptions to a few anecdotal examples, the transcripts that have the potential to localize to the mitochondria have been poorly studied. We used a deep sequencing approach to identify transcripts encoded by the nuclear genome which localize to the mitoplast in a zebrafish model. We prioritized 292 candidate transcripts of nuclear origin that are potentially localized to the mitochondrial matrix. We experimentally demonstrated that the transcript encoding the nuclear encoded ribosomal protein 11 (Rpl11) localizes to the mitochondria. This study represents a comprehensive analysis of the mitochondrial localization of nuclear encoded transcripts. Our analysis has provided insights into a new layer of biomolecular pathways modulating mitochondrial-nuclear cross-talk. This provides a starting point towards understanding the role of nuclear encoded transcripts that localize to mitochondria and their influence on mitochondrial function.

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.

SAGE: a comprehensive resource of genetic variants integrating South Asian whole genomes and exomes.

South Asia is home to $\sim $20% of the world population and characterized by distinct ethnic, linguistic, cultural and genetic lineages. Only limited representative samples from the region have found its place in large population-scale international genome projects. The recent availability of genome scale data from multiple populations and datasets from South Asian countries in public domain motivated us to integrate the data into a comprehensive resource. In the present study, we have integrated a total of six datasets encompassing 1213 human exomes and genomes to create a compendium of 154 814 557 genetic variants and adding a total of 69 059 255 novel variants. The variants were systematically annotated using public resources and along with the allele frequencies are available as a browsable-online resource South Asian genomes and exomes. As a proof of principle application of the data and resource for genetic epidemiology, we have analyzed the pathogenic genetic variants causing retinitis pigmentosa. Our analysis reveals the genetic landscape of the disease and suggests subset of genetic variants to be highly prevalent in South Asia.

Genotype-Phenotype Correlations of Dystrophic Epidermolysis Bullosa in India: Experience from a Tertiary Care Centre.

Recent advances in the field of genomics have seen the successful implementation of whole exome sequencing as a rapid and efficient diagnostic strategy in several genodermatoses. The aim of this study was to explore the potential of molecular studies in dystrophic epidermolysis bullosa (DEB) in India. Whole exome sequencing was performed using genomic DNA from each case of epidermolysis bullosa, followed by massively parallel sequencing. Resulting reads were mapped to the human reference genome hg19. Sanger sequencing subsequently confirmed the potentially pathogenic mutations. Whole exome sequencing of 18 patients with DEB from 17 unrelated Indian families revealed 20 distinct sequence variants in the COL7A1 gene including 2 widely prevalent mutations. Dominant inheritance was seen in 7 patients, while 11 patients showed a highly variable recessive DEB. This preliminary study using exome sequencing is clearly encouraging and will serve as the basis for future large-scale molecular studies to actively identify and understand DEB in the Indian population.

Familial Hypertrophic Cardiomyopathy - Identification of cause and risk stratification through exome sequencing.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) with variable clinical presentations and heterogeneity is the common cause of sudden cardiac death. Genetic diagnosis is challenging in these complex diseases but exome sequencing as a genetic diagnostic tool provides explainable results. METHODS: In a familial Hypertrophic Cardiomyopathy with multigenerational inheritance with apparent phenotype, had a history of sudden death and severe arrhythmia followed by implantation of Implantable cardioverter defibrillator (ICD). Exome sequencing (100×) trailed by effective filtering steps for exome variants on the basis of different parameters, segregated variants are prioritized for the disease and further clinical relevance are evaluated for the variants. RESULTS: A rare causal variant in troponin-T gene (TNNT2, NM_000364.3;c.274C > T;p.Arg92Trp) is identified, shared by only affected members, absent in unaffected members and also in 200 unrelated control chromosomes. TNNT2 mutation act as a driver mutation but mutations in other disease-related genes, KCNMB1, LPL, APOE and other biochemical factors provides risk stratification within affected family members. CONCLUSION: This study contributes to the role of "rare variants" in complex disease phenotypes and heterogeneity within family and the necessity of whole exome targeted approaches in complex cardiomyopathy, which are known to harbor private mutations.

RNA secondary structure profiling in zebrafish reveals unique regulatory features.

BACKGROUND: RNA is known to play diverse roles in gene regulation. The clues for this regulatory function of RNA are embedded in its ability to fold into intricate secondary and tertiary structure. RESULTS: We report the transcriptome-wide RNA secondary structure in zebrafish at single nucleotide resolution using Parallel Analysis of RNA Structure (PARS). This study provides the secondary structure map of zebrafish coding and non-coding RNAs. The single nucleotide pairing probabilities of 54,083 distinct transcripts in the zebrafish genome were documented. We identified RNA secondary structural features embedded in functional units of zebrafish mRNAs. Translation start and stop sites were demarcated by weak structural signals. The coding regions were characterized by the three-nucleotide periodicity of secondary structure and display a codon base specific structural constrain. The splice sites of transcripts were also delineated by distinct signature signals. Relatively higher structural signals were observed at 3' Untranslated Regions (UTRs) compared to Coding DNA Sequence (CDS) and 5' UTRs. The 3' ends of transcripts were also marked by unique structure signals. Secondary structural signals in long non-coding RNAs were also explored to better understand their molecular function. CONCLUSIONS: Our study presents the first PARS-enabled transcriptome-wide secondary structure map of zebrafish, which documents pairing probability of RNA at single nucleotide precision. Our findings open avenues for exploring structural features in zebrafish RNAs and their influence on gene expression.

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.

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.

Egyptian tale from India: application of whole-exome sequencing in diagnosis of atypical familial Mediterranean fever.

Clinical diagnosis of autoinflammatory diseases requires a high degree of clinical suspicion and clinching molecular evidence to substantiate the diagnosis. This is more so in populations with low prevalence of these disorders. In this report, we describe the case of a young man from India with recurrent fever and persistent arthritis. The patient's forefathers were of Egyptian ancestry who practiced consanguinity. Molecular genetic analysis using whole-exome sequencing suggested the presence of variants c.443A>T:p.E148V and c.442G>C:p.E148Q in the MEFV gene, earlier independently shown to be associated with familial Mediterranean fever (FMF) in a compound heterozygous state. The variants were further confirmed by capillary sequencing. This report also highlights the application of whole exome sequencing to delineate the allelic differences in the variants apart from serving as a quick genetic screening approach for autoinflammatory diseases. To the best of our knowledge, this is the first report of a compound heterozygosity for the two well-characterized variants associated with atypical FMF in a patient.

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 lamininß3 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 reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa simplex (DEB) is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB) is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES). Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India.

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.

Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in the BTK gene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.