Effect of calcium supplementation on reversing metformin-based inhibition of vitamin B12 bioavailability in healthy adults using a [13C] cyanocobalamin tracer - A pilot study.

BACKGROUND & AIMS: Metformin is a widely prescribed first line drug for the treatment of type 2 diabetes mellitus (DM). Studies have shown that the use of metformin is often associated with a decrease in vitamin B12 (B12) levels in patients with DM. Few studies have shown that this effect could be mitigated with calcium supplementation. In the present study, we quantified the effect of metformin, and metformin co-administered with calcium on B12 absorption using a novel stable isotope [13C] cyanocobalamin tracer. METHODS: A pilot crossover study was conducted to estimate the bioavailability of B12 in healthy subjects, using [13C] cyanocobalamin as a tracer. In the study, [13C] cyanocobalamin was administered orally to the participants followed by hourly venous sampling to measure the concentration of the tracer and estimate bioavailability. This protocol was followed for three experiment days, each separated by a one month wash out period. As part of the study, all participants received the tracer alone for the control day (C), metformin 850 mg along with the tracer for the metformin day (M) and metformin 850 mg with calcium 500 mg and the tracer for the metformin calcium day (MC). RESULTS: Seven participants completed all three experiment days. The mean B12 bioavailability (±SD, n = 7) was 42.6 ± 10.2% for the control day (C), 30.8 ± 15.3% for the metformin day (M) and 46.4 ± 8.6% for the metformin-calcium day (MC). Repeated measures ANOVA was done and the pairwise comparison showed a significant difference in the B12 bioavailability between control and metformin day (C vs M p = 0.010), and between the metformin and metformin with calcium day (M vs MC p = 0.003). CONCLUSION: B12 bioavailability reduced significantly from baseline (C) when metformin (M) was administered and this reduction was reversed when calcium was co-administered (MC) in healthy participants. In patients using metformin, calcium supplementation as a strategy to prevent B12 deficiency needs to be further studied.

The genomic landscape of CYP2D6 variation in the Indian population.

Aim: The CYP2D6 gene is highly polymorphic, causing large interindividual variability in the metabolism of several clinically important drugs. Materials & methods: The authors investigated the diversity and distribution of CYP2D6 alleles in Indians using whole genome sequences (N = 1518). Functional consequences were assessed using pathogenicity scores and molecular dynamics simulations. Results: The analysis revealed population-specific CYP2D6 alleles (*86, *7, *111, *112, *113, *99) and remarkable differences in variant and phenotype frequencies with global populations. The authors observed that one in three Indians could benefit from a dose alteration for psychiatric drugs with accurate CYP2D6 phenotyping. Molecular dynamics simulations revealed large conformational fluctuations, confirming the predicted reduced function of *86 and *113 alleles. Conclusion: The findings emphasize the utility of comprehensive CYP2D6 profiling for aiding precision public health.

Landscape of pharmacogenetic variants associated with non-insulin antidiabetic drugs in the Indian population.

INTRODUCTION: Genetic variants contribute to differential responses to non-insulin antidiabetic drugs (NIADs), and consequently to variable plasma glucose control. Optimal control of plasma glucose is paramount to minimizing type 2 diabetes-related long-term complications. India's distinct genetic architecture and its exploding burden of type 2 diabetes warrants a population-specific survey of NIAD-associated pharmacogenetic (PGx) variants. The recent availability of large-scale whole genomes from the Indian population provides a unique opportunity to generate a population-specific map of NIAD-associated PGx variants. RESEARCH DESIGN AND METHODS: We mined 1029 Indian whole genomes for PGx variants, drug-drug interaction (DDI) and drug-drug-gene interactions (DDGI) associated with 44 NIADs. Population-wise allele frequencies were estimated and compared using Fisher's exact test. RESULTS: Overall, we found 76 known and 52 predicted deleterious common PGx variants associated with response to type 2 diabetes therapy among Indians. We report remarkable interethnic differences in the relative cumulative counts of decreased and increased response-associated alleles across NIAD classes. Indians and South Asians showed a significant excess of decreased metformin response-associated alleles compared with other global populations. Network analysis of shared PGx genes predicts high DDI risk during coadministration of NIADs with other metabolic disease drugs. We also predict an increased CYP2C19-mediated DDGI risk for CYP3A4/3A5-metabolized NIADs, saxagliptin, linagliptin and glyburide when coadministered with proton-pump inhibitors (PPIs). CONCLUSIONS: Indians and South Asians have a distinct PGx profile for antidiabetes drugs, marked by an excess of poor treatment response-associated alleles for various NIAD classes. This suggests the possibility of a population-specific reduced drug response in atleast some NIADs. In addition, our findings provide an actionable resource for accelerating future diabetes PGx studies in Indians and South Asians and reconsidering NIAD dosing guidelines to ensure maximum efficacy and safety in the population.

Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function.

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

Whole genome sequencing followed by functional analysis of genomic deletion encompassing ERCC8 and NDUFAF2 genes in a non-consanguineous Indian family reveals dysfunctional mitochondrial bioenergetics leading to infant mortality.

Genomic investigations on an infant who presented with a putative mitochondrial disorder led to identification of compound heterozygous deletion with an overlapping region of ∼142 kb encompassing two nuclear encoded genes namely ERCC8 and NDUFAF2. Investigations on fetal-derived fibroblast culture demonstrated impaired bioenergetics and mitochondrial dysfunction, which explains the phenotype and observed infant mortality in the present study. The genetic findings from this study extended the utility of whole-genome sequencing as it led to development of a MLPA-based assay for carrier screening in the extended family and the prenatal testing aiding in the birth of two healthy children.

Bioavailability and daily requirement of vitamin B12 in adult humans: an observational study of its colonic absorption and daily excretion as measured by [13C]-cyanocobalamin kinetics.

BACKGROUND: Clinical and biochemical vitamin B12 (B12) deficiency is lower than anticipated in vegetarians. Extraileal absorption, such as from the colon, as well as reduced daily excretion, may be adaptive mechanisms to maintain B12 homeostasis with marginal intakes. OBJECTIVE: To measure the absorption of B12 from the small and large intestine, and its daily rate of excretion from the body, using a [13C]-cyanocobalamin tracer. METHODS: Oral B12 bioavailability was measured over 12 h after administration of [13C]-cyanocobalamin tracer (2.5 µg) in normal participants. The colonic B12 bioavailability was evaluated by direct instillation of [13C]-cyanocobalamin (5 µg) into the ascending colon. Bioavailability was calculated from 2-compartmental modeling of the tracer appearance in plasma. The excretion rate of B12 was measured from [13C]-cyanocobalamin elimination from the body over 4 wk after oral dosing (5 µg). RESULTS: The oral B12 bioavailability (n = 11) was 63% ± 10% measured over 12 h. A late absorption peak, accounting for 12% of the absorption, was observed after an average lag time of 8.7 h from dosing. The colonic B12 bioavailability (n = 10) was 7% ± 5% over 4 h. The daily B12 excretion rate (n = 4) was 0.7 ± 0.2 µg/d. The minimum daily requirement of B12 in these participants was derived at 1 µg /d. CONCLUSIONS: B12 is absorbed in the human colon. This observation confirms the potential contribution of the colon in daily B12 nutriture, and along with a possible lower requirement, could explain the absence of clinical deficiency in populations with marginal B12 intakes. TRIAL REGISTRATION NUMBER: This study was registered in Clinical Trials Registry of India (CTRI) with the registration number CTRI/2018/04/012957, available from https://ctri.nic.in/Clinicaltrials/showallp.php?mid1=49319&EncHid=&userName=029108.

Maternal Vitamin A Status as a Risk Factor of Hirschsprung Disease in the Child.

INTRODUCTION: The gene-environment interaction of the REarranged during Transfection ( RET ) gene with vitamin A in the etiopathogenesis of Hirschsprung disease (HSCR) has been suggested in rodents. The aim of this study was to evaluate vitamin A status in mothers of children with HSCR and to assess its association with pathogenic variants of the RET gene in affected children. METHODS: This was a case-control study of stable isotope-based vitamin A measurement stores of mothers of children diagnosed with HSCR (within 8 months from birth, n = 7) and age-matched mothers of normal children (n = 6). Next-generation sequencing of RET exons, along with their upstream promoter region, was performed in the 7 HSCR proband-parent triads to evaluate pathogenic variants. RESULTS: Maternal vitamin A stores in the HSCR group was almost 50% that of those in controls, tending toward significance (0.50 ± 0.17 vs 0.89 ± 0.51 µmol/g respectively, P = 0.079). Two novel pathogenic de novo mutations were identified in 2 cases, and a rare single-nucleotide deletion was detected in the 3.5-kb RET upstream region, in a heterozygous state, in all 7 proband-parent triads. Low-penetrance RET haplotypes associated with HSCR were detected in 5 cases. DISCUSSION: Mothers with children with HSCR had lower vitamin A liver stores than mothers with normal children, and the children who were affected had HSCR despite having no established pathogenic RET variants. Lower maternal vitamin A status may increase the penetrance of genetic mutations in RET , and vitamin-A mediated gene-environment interactions may underpin some of the etiology of HSCR.

Multi-omics Analysis Classifies Colorectal Cancer into Distinct Methylated Immunogenic and Angiogenic Subtypes Based on Anatomical Laterality.

We employed supervised machine learning algorithms to a cohort of colorectal cancer patients from the NCI to differentiate and classify the heterogenous disease based on anatomical laterality and multi-omics stratification, in a first of its kind. Multi-omics integrative analysis shows distinct clustering of left and right colorectal cancer with disentangled representation of methylome and delineation of transcriptome and genome. We present novel multi-omics findings consistent with augmented hypermethylation of genes in right CRC, epigenomic biomarkers on the right in conjunction with immune-mediated pathway signatures, and lymphocytic invasion which unlocks unique therapeutic avenues. Contrarily, left CRC multi-omics signature is found to be marked by angiogenesis, cadherins, and epithelial-mesenchymal transition (EMT). An integrated multi-omics molecular signature of RNF217-AS1, hsa-miR-10b, and panel of FBX02, FBX06, FBX044, MAD2L2, and MIIP copy number altered genes have been found by the study. Overall survival analysis reveals genomic biomarkers ABCA13 and TTN in 852 LCRC cases, and SOX11 in 170 RCRC cases that predicts a significant survival benefit. Our study exemplifies the translational competence and robustness of machine learning in effective translational bridging of research and clinic. Supplementary Information: The online version contains supplementary material available at 10.1007/s13193-023-01760-6.

Multi-omics analyses provide novel biological insights to distinguish lobular ductal types of invasive breast cancers.

BACKGROUND: Invasive lobular carcinoma (ILC) treatment is similar to invasive ductal carcinoma (IDC; now invasive carcinoma-no special type, IBC-NST), based on its intrinsic subtype. However, further investigation is required for an integrative understanding of differentially perturbed molecular patterns and pathways in these histotypes. METHODS: A dataset of 780 IDC and 201 ILC samples from the TCGA-BRCA project for cross-platform multi-omics was analyzed. We leveraged a consensus approach integrating different bioinformatic algorithms to analyze mutations, CNAs, mRNA, miRNA abundance, methylation, and protein abundance to understand the complex crosstalks that distinguish ILC and IDC samples. A histotype-matched comparison was performed. We performed Cox survival analyses for prognosis based on our identified 53 histotype-specific and four discordant genes. RESULTS: Approximately 90% of ILC cases were of the luminal subtype. Somatic mutations in CDH1 were higher in ILC than in IDC (FDR-adjusted p < 0.01). Fifty-three significant oncogenic or tumor-suppressive DEGs were identified in a single histotype. PPAR signaling and lipolysis regulation in adipocytes were significantly enriched in ILC tumors. CDH1 protein had the highest differential abundance (AUC: 0.85). Moreover, BTG2, GSTA2, GPR37L1, and PGBD5 amplification was associated with poorer OS in ILC compared with no alteration. RIMS2, NACA4P, MYC, ZFPM2, and POU5F1B amplification showed a lower overall survival in patients with IDC. miR-195 showed an IDC-specific downregulation, causing overexpression of CCNE1. Integrative multi-omics supervised analysis identified 296 differentially expressed genes that successfully distinguished IDC and ILC histotypes. CONCLUSIONS: Our findings identify novel molecular candidates that potentially drive and modify the disease differentially among these histotypes.

Pharmacogenomic landscape of Indian population using whole genomes.

Ethnic differences in pharmacogenomic (PGx) variants have been well documented in literature and could significantly impact variability in response and adverse events to therapeutics. India is a large country with diverse ethnic populations of distinct genetic architecture. India's national genome sequencing initiative (IndiGen) provides a unique opportunity to explore the landscape of PGx variants using population-scale whole genome sequences. We have analyzed the IndiGen variation dataset (N = 1029 genomes) along with global population scale databases to map the most prevalent clinically actionable and potentially deleterious PGx variants among Indians. Differential frequencies for the known and novel variants were studied and interaction of the disrupted PGx genes affecting drug responses were analyzed by performing a pathway analysis. We have highlighted significant differences in the allele frequencies of clinically actionable PGx variants in Indians when compared to the global populations. We identified 134 mostly common (allele frequency [AF] > 0.1) potentially deleterious PGx variants that could alter or inhibit the function of 102 pharmacogenes in Indians. We also estimate that on, an average, each Indian individual carried eight PGx variants (single nucleotide variants) that have a direct impact on the choice of treatment or drug dosing. We have also highlighted clinically actionable PGx variants and genes for which preemptive genotyping is most recommended for the Indian population. The study has put forward the most comprehensive PGx landscape of the Indian population from whole genomes that could enable optimized drug selection and genotype-guided prescriptions for improved therapeutic outcomes and minimizing adverse events.

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Pharmacogenomic landscape of COVID-19 therapies from Indian population genomes.

Aim: Numerous drugs are being widely prescribed for COVID-19 treatment without any direct evidence for the drug safety/efficacy in patients across diverse ethnic populations. Materials & methods: We analyzed whole genomes of 1029 Indian individuals (IndiGen) to understand the extent of drug-gene (pharmacogenetic), drug-drug and drug-drug-gene interactions associated with COVID-19 therapy in the Indian population. Results: We identified 30 clinically significant pharmacogenetic variants and 73 predicted deleterious pharmacogenetic variants. COVID-19-associated pharmacogenes were substantially overlapped with those of metabolic disorder therapeutics. CYP3A4, ABCB1 and ALB are the most shared pharmacogenes. Fifteen COVID-19 therapeutics were predicted as likely drug-drug interaction candidates when used with four CYP inhibitor drugs. Conclusion: Our findings provide actionable insights for future validation studies and improved clinical decisions for COVID-19 therapy in Indians.

Protocol for a prospective, observational, deep phenotyping study on adipose epigenetic and lipidomic determinants of metabolic homoeostasis in South Asian Indians: the Indian Diabetes and Metabolic Health (InDiMeT) study.

INTRODUCTION: We describe the rationale and broad study design of the Indian Diabetes and Metabolic Health (InDiMeT) study, a new prospective, observational study incorporating extensive epigenetic (DNA methylation) and lipidomic signatures to examine their association with the dysregulation of adipose de novo lipogenesis (DNL) in South Asian Indians. The InDiMeT study aims to use a case-control design to identify genetic and modifiable-environmental-lifestyle associated determinants of (1) epigenomic (DNA methylome) dysregulation of adipose DNL in type 2 diabetes mellitus (T2DM) adipose tissue, (2) identify correlates of epigenomic (DNA methylome) dysregulation of adipose DNL in peripheral blood mononuclear cells (PBMCs) from T2DM subjects and (3) elucidate plasma lipidomic correlates of adipose DNL in T2DM that can be used as biomarkers of adipose tissue dysfunction. METHODS AND ANALYSIS: The InDiMeT study will involve recruitment of 176 normoglycaemic and T2DM individuals who will be undergoing laparoscopic surgery for clinical conditions. Extensive phenotyping of the subjects will be conducted and DNA methylome and lipidomic measurements will be made. The adipose DNL pathway genes are likely to be hypermethylated in patients with T2DM with corresponding reduction of gene expression. Correlates of epigenomic (DNA methylome) dysregulation of adipose DNL pathway in PBMCs and their adipose and plasma lipidomic signatures in T2DM subjects could act as early markers of development of T2DM. ETHICS AND DISSEMINATION: For the InDiMeT study, ethical approval for addressing the specific aims has been obtained from the Institutional Ethics Committee, St John's Medical College and Hospital, St John's National Academy of Health Sciences, Bangalore. Findings from this study will be disseminated through scientific publications in peer-reviewed journals, research conferences and via presentations to stakeholders, patients, clinicians, public and policymakers through appropriate channels.

Gut microbiota profiles of young South Indian children: Child sex-specific relations with growth.

Gut microbiota has been implicated as a modifier of childhood growth. Here, 16S rRNA sequencing-based fecal microbiota profiles of 18-24 month old Indian children were evaluated (n = 41), in relation to their anthropometric parameters, intestinal permeability, body composition and total energy expenditure. Pathway analyses were conducted to assess microbial functions related to stunting, underweight and wasting. The fecal microbiota was enriched in Prevotella 9, Bifidobacterium and Escherichia-Shigella. Weight, weight-for-age Z-scores (WAZ) and weight-for-length Z-scores (WLZ), along with age, acted as covariates of microbiota variation specifically in boys (n = 23). Bifidobacterium longum subsp longum abundance was positively associated with WAZ while Bifidobacterium bifidum and Bifidobacterium breve abundances were negatively associated with age. The lipopolysaccharide biosynthesis pathway was upregulated in stunted (n = 16) and wasted (n = 8) children. Findings from this study indicate that child sex may be a critical modifier of the role of gut microbiota on childhood growth.

Measuring vitamin B-12 bioavailability with [13C]-cyanocobalamin in humans.

BACKGROUND: Vitamin B-12 deficiency is widespread in many parts of the world, affecting all age groups and increasing with age. It is primarily due to a low intake of animal source foods or malabsorption. The measurement of bioavailability of vitamin B-12 is etiologically important in deficiency but is limited due to the use of radioactive isotopes like [57Co]- or [14C]-cyanocobalamin. OBJECTIVES: The aim of this study was to measure the bioavailability of [13C]-cyanocobalamin in humans and to assess the effect of parenteral replenishment of vitamin B-12 on the bioavailability. METHODS: We synthesized a stable isotope-labeled vitamin B-12, [13C]-cyanocobalamin, using Salmonella enterica by providing [13C2]-ethanolamine as a sole carbon source. After purification and mass spectrometry-based characterization, its oral bioavailability was measured in the fasted state with high and low oral doses, before and after parenteral replenishment of vitamin B-12 stores, from the kinetics of its plasma appearance in a 2-compartment model. RESULTS: [13C]-cyanocobalamin was completely decyanated to [13C]-methylcobalamin describing metabolic utilization, and its plasma appearance showed early and late absorption phases. At a low dose of 2.3 µg, the mean bioavailability was 46.2 ± 12.8 (%, mean ± SD, n = 11). At a higher dose of 18.3 µg, the mean bioavailability was 7.6 ± 1.7 (%, mean ± SD, n = 4). Parenteral replenishment of the vitamin B-12 store in deficient individuals prior to the measurement resulted in a 1.9-fold increase in bioavailability. CONCLUSIONS: Vitamin B-12 bioavailability is dose dependent and at a low dose that approximates the normal daily requirement (46%). The stable isotope method described here could be used to define the etiology of deficiency and to inform the dietary requirement in different physiologic states as well as the dose required for supplementation and food fortification. This trial was registered at the Clinical Trials Registry of India as CTRI/2018/04/012957.

A Temporal Map of Gene Expression Pattern During Zebrafish Liver Regeneration.

Zebrafish is increasingly being used to study liver injury and regeneration. However, very little is known about molecular players that respond to injury and those important for liver regeneration. We use a metronidazole nitroreductase (MTZ-nfsb)-based system to selectively ablate hepatocytes in adult zebrafish to create a model for liver injury and regeneration. In this study, we generate a comprehensive temporal map of gene expression changes during regeneration through RNA sequencing of liver samples at various stages of injury and regeneration. Analyzing these data, we find that soon after injury the immediate early transcription factor MYC induces a battery of genes that respond to the MTZ-induced ROS by activating oxido-reductase pathways and apoptosis machinery. Immediately after injury, liver cells downregulate many functional genes, including complement protein synthesis, bile acid, and lipid biosynthesis, in a concerted manner. At 6 days postinjury, we find a dramatic induction of cholesterol biosynthesis and protein folding machinery, with expression levels returning to predamage levels by 8 days, suggesting an important role for these pathways in liver regeneration. This chronological transcriptomic map of liver regeneration in zebrafish would serve as a framework for further studies in understanding, and for screening for compounds that augment liver regeneration.

Population-scale genomics-Enabling precision public health.

The current excitement for affordable genomics technologies and national precision medicine initiatives marks a turning point in worldwide healthcare practices. The last decade of global population sequencing efforts has defined the enormous extent of genetic variation in the human population resulting in insights into differential disease burden and response to therapy within and between populations. Population-scale pharmacogenomics helps to provide insights into the choice of optimal therapies and an opportunity to estimate, predict and minimize adverse events. Such an approach can potentially empower countries to formulate national selection and dosing policies for therapeutic agents thereby promoting public health with precision. We review the breadth and depth of worldwide population-scale sequencing efforts and its implications for the implementation of clinical pharmacogenetics toward making precision medicine a reality.

Methods to Study Long Noncoding RNA Expression and Dynamics in Zebrafish Using RNA Sequencing.

Long noncoding RNAs (lncRNAs) belong to a class of RNA transcripts that do not have the potential to code for proteins. LncRNAs were largely discovered in the transcriptomes of human and several model organisms, using next-generation sequencing (NGS) approaches, which have enabled a comprehensive genome scale annotation of transcripts. LncRNAs are known to have dynamic expression status and have the potential to orchestrate gene regulation at the epigenetic, transcriptional, and posttranscriptional levels. Here we describe the experimental methods involved in the discovery of lncRNAs from the transcriptome of a popular model organism zebrafish (Danio rerio). A structured and well-designed computational analysis pipeline subsequent to the RNA sequencing can be instrumental in revealing the diversity of the lncRNA transcripts. We describe one such computational pipeline used for the discovery of novel lncRNA transcripts in zebrafish. We also detail the validation of the putative novel lncRNA transcripts using qualitative and quantitative assays in zebrafish.

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.

Pharmacogenomic survey of Qatari populations using whole-genome and exome sequences.

The Arabs represent one of the most genetically heterogeneous populations characterized by a high prevalence of Mendelian disorders due to consanguinity. Population-scale genomic datasets provide a unique opportunity to understand the epidemiology of variants associated with differential therapeutic response. We analyzed publicly available genomic data for 1005 Qatari individuals encompassing five subpopulations. The frequencies of known and novel pharmacogenetic variants were compared with global populations. Impact of genetic substructure on the pharmacogenetic landscape of the population was studied. We report an average of three clinically actionable pharmacogenetic variants with FDA-recommended genetic testing per Qatari individual regardless of their genetic ancestry. We observed extensive differences in the frequencies of clinically actionable pharmacogenetic variants among the Qatari subpopulations. Our analysis revealed 3579 deleterious pharmacogenetic variants potentially altering the function of 1163 genes associated with 1565 drugs. This study has thus compiled the first comprehensive landscape of pharmacogenetic variants for any Arab population.