MC1R diversity and its role in skin pigmentation variation in West Maharashtra, India.

OBJECTIVES: MC1R polymorphisms have been reported to be under a selective constraint in populations inhabiting high UVR regions such as Africans; however, these patterns are not consistent. Here we analyze the MC1R gene in West Maharashtra, India to see if sequence diversity corresponds to their diverse pigmentary profiles and if MC1R is constrained in dark skinned tribal as compared to lighter skinned caste populations. METHODS: A 2648 bp region of this gene was sequenced in 102 individuals and the data was compared for π, Ï´ diversity indices. Tajima's D was assessed for signatures of purifying selection and MC1R variants were associated with MI measures using the additive, dominant, and recessive models. Pairwise FST was tested among study populations and between study populations and 1000 Genomes regional samples. RESULTS: MC1R diversity was not uniquely patterned among castes and tribes. Non-synonymous variants rs2228479A, rs1805007_T, and rs885479_A showed low variability in these populations. Selection tests did not indicate any constraint on MC1R and pairwise FST were also low among the study populations (-0.0163 to 0.06112). The SNP rs3212359 was significantly associated with MI measures when tested using different association models. CONCLUSIONS: We do not find evidence of a selective constraint on MC1R. The presence of a large number of unique haplotypes and low FST values at this locus suggests that MC1R polymorphisms may not be influencing pigmentation variation among castes and tribes in this region. Observed associations between rs3212359 and MI measures need to be validated through studies on larger samples and in-vitro functional studies.

Genetic diversity of 'Very Important Pharmacogenes' in two South-Asian populations.

OBJECTIVES: Reliable identification of population-specific variants is important for building the single nucleotide polymorphism (SNP) profile. In this study, genomic variation using allele frequency differences of pharmacologically important genes for Gujarati Indians in Houston (GIH) and Indian Telugu in the U.K. (ITU) from the 1000 Genomes Project vis-à-vis global population data was studied to understand its role in drug response. METHODS: Joint genotyping approach was used to derive variants of GIH and ITU independently. SNPs of both these populations with significant allele frequency variation (minor allele frequency ≥ 0.05) with super-populations from the 1000 Genomes Project and gnomAD based on Chi-square distribution with p-value of ≤ 0.05 and Bonferroni's multiple adjustment tests were identified. Population stratification and fixation index analysis was carried out to understand genetic differentiation. Functional annotation of variants was carried out using SnpEff, VEP and CADD score. RESULTS: Population stratification of VIP genes revealed four clusters viz., single cluster of GIH and ITU, one cluster each of East Asian, European, African populations and Admixed American was found to be admixed. A total of 13 SNPs belonging to ten pharmacogenes were identified to have significant allele frequency variation in both GIH and ITU populations as compared to one or more super-populations. These SNPs belong to VKORC1 (rs17708472, rs2359612, rs8050894) involved in Vitamin K cycle, cytochrome P450 isoforms CYP2C9 (rs1057910), CYP2B6 (rs3211371), CYP2A2 (rs4646425) and CYP2A4 (rs4646440); ATP-binding cassette (ABC) transporter ABCB1 (rs12720067), DPYD1 (rs12119882, rs56160474) involved in pyrimidine metabolism, methyltransferase COMT (rs9332377) and transcriptional factor NR1I2 (rs6785049). SNPs rs1544410 (VDR), rs2725264 (ABCG2), rs5215 and rs5219 (KCNJ11) share high fixation index (≥ 0.5) with either EAS/AFR populations. Missense variants rs1057910 (CYP2C9), rs1801028 (DRD2) and rs1138272 (GSTP1), rs116855232 (NUDT15); intronic variants rs1131341 (NQO1) and rs115349832 (DPYD) are identified to be 'deleterious'. CONCLUSIONS: Analysis of SNPs pertaining to pharmacogenes in GIH and ITU populations using population structure, fixation index and allele frequency variation provides a premise for understanding the role of genetic diversity in drug response in Asian Indians.

Measuring the impact of a single dose of ChAdOx1 nCoV-19 (recombinant) coronavirus vaccine on hospital stay, ICU requirement, and mortality outcome in a tertiary care centre.

OBJECTIVE: To comparatively evaluate ICU requirement, length of stay, and mortality between single-dose vaccinated and non-vaccinated hospitalized COVID-19 patients. DESIGN: A retrospective observational study was carried out in a tertiary care hospital in western Indian, from April 1 to June 30, 2021. RESULTS: Of the 569 patients who fulfilled the eligibility criteria and were enrolled in the study, 137 (24.08%) patients had received a single dose of ChAdOx1 nCoV-19 vaccine, while 432 (75.92%) patients had not received any form of vaccination. The overall length of stay in hospital was similar for both groups; however, a significant difference was seen in length of stay in the ward and in the ICU. Vaccinated patients were admitted to the ward for 6.21 ± 3.204 days, while non-vaccinated patients were admitted for 5.56 ± 4.55 days (p < 0.001). The mean length of ICU stay for the 21 vaccinated patients requiring intensive care was 4.47 ± 2.3 days, while that for the 145 non-vaccinated patients was 6.29 ± 2.19 days (p < 0.001). Mortality was observed in four patients in the vaccinated group and in 95 patients in the non-vaccinated group. CONCLUSION: A single dose of ChAdOx1 nCoV-19 vaccine was associated with a significantly lower severity of SARS-CoV-2 infection compared with no vaccination.

GAMUT: A genomics big data management tool.

Efficient analysis of Single Nucleotide Polymorphisms (SNPs) across genomic samples enable in deciphering the relationship between genotype and phenotype. The core principle behind SNP comparison is to arrive at a probable list of variants that can differentiate two sets of data (populations). Such SNPs have direct applications in array design, genotype imputation and in cataloging of variants in regions of interest. We have developed GAMUT (Genomics bigdAta Management Tool), a big data-based solution for efficient run-time comparison of SNPs across large datasets based on partition of samples belonging to different populations taking into account user-defined splits. The tool is based on client-server architecture with MongoDB at the back-end and JSF with PrimeFaces as the front-end. It is readily deployable on wild-fly server as well as a docker container. Spark-based parallel data uploader enables optimal loading times. GAMUT enables dynamic querying of the large datasets consisting of multiple samples using text-based, chromosome position-based as well as gene-name based options. Various charting options like bar and pie charts along with tabular formats are available to ease the analysis of the queried data. The resultant data pertaining to comparison of genomewide SNPs can also be downloaded in different formats like text, html, json for further stand-alone analysis. GAMUT is available for download at: https://github.com/bioinformatics-cdac/gamut.

GenoVault: a cloud based genomics repository.

GenoVault is a cloud-based repository for handling Next Generation Sequencing (NGS) data. It is developed using OpenStack-based private cloud with various services like keystone for authentication, cinder for block storage, neutron for networking and nova for managing compute instances for the Cloud. GenoVault uses object-based storage, which enables data to be stored as objects instead of files or blocks for faster retrieval from different distributed object nodes. Along with a web-based interface, a JavaFX-based desktop client has also been developed to meet the requirements of large file uploads that are usually seen in NGS datasets. Users can store files in their respective object-based storage areas and the metadata provided by the user during file uploads is used for querying the database. GenoVault repository is designed taking into account future needs and hence can scale both vertically and horizontally using OpenStack-based cloud features. Users have an option to make the data shareable to the public or restrict the access as private. Data security is ensured as every container is a separate entity in object-based storage architecture which is also supported by Secure File Transfer Protocol (SFTP) for data upload and download. The data is uploaded by the user in individual containers that include raw read files (fastq), processed alignment files (bam, sam, bed) and the output of variation detection (vcf). GenoVault architecture allows verification of the data in terms of integrity and authentication before making it available to collaborators as per the user's permissions. GenoVault is useful for maintaining the organization-wide NGS data generated in various labs which is not yet published and submitted to public repositories like NCBI. GenoVault also provides support to share NGS data among the collaborating institutions. GenoVault can thus manage vast volumes of NGS data on any OpenStack-based private cloud.

Author Correction: Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Petal abscission in fragrant roses is associated with large scale differential regulation of the abscission zone transcriptome.

Flowers of fragrant roses such as Rosa bourboniana are ethylene-sensitive and undergo rapid petal abscission while hybrid roses show reduced ethylene sensitivity and delayed abscission. To understand the molecular mechanism underlying these differences, a comparative transcriptome of petal abscission zones (AZ) of 0 h and 8 h ethylene-treated flowers from R. bourboniana was performed. Differential regulation of 3700 genes (1518 up, 2182 down) representing 8.5% of the AZ transcriptome was observed between 0 and 8 h ethylene-treated R. bourboniana petal AZ. Abscission was associated with large scale up-regulation of the ethylene pathway but prominent suppression of the JA, auxin and light-regulated pathways. Regulatory genes encoding kinases/phosphatases/F-box proteins and transcription factors formed the major group undergoing differential regulation besides genes for transporters, wall modification, defense and phenylpropanoid pathways. Further comparisons with ethylene-treated petals of R. bourboniana and 8 h ethylene-treated AZ (R. hybrida) identified a core set of 255 genes uniquely regulated by ethylene in R. bourboniana AZ. Almost 23% of these encoded regulatory proteins largely conserved with Arabidopsis AZ components. Most of these were up-regulated while an entire set of photosystem genes was prominently down-regulated. The studies provide important information on regulation of petal abscission in roses.

Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis.

Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ΔBCG TK strain. The protective efficacy of the ΔBCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.

Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle.

BACKGROUND: BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG's efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host's immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. RESULTS: To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. CONCLUSION: These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host's innate immune system to allow it to persist, a property that is important for its protective efficacy.

Identifying signatures of positive selection in pigmentation genes in two South Asian populations.

OBJECTIVES: Skin pigmentation is a polygenic trait showing wide phenotypic variations among global populations. While numerous pigmentation genes have been identified to be under positive selection among European and East populations, genes contributing to phenotypic variation in skin pigmentation within and among South Asian populations are still poorly understood. The present study uses data from the Phase 3 of the 1000 genomes project focusing on two South Asian populations-GIH (Gujarati Indian from Houston, Texas) and ITU (Indian Telugu from UK), so as to decode the genetic architecture involved in adaptation to ultraviolet radiation in South Asian populations. METHODS: Statistical tests included were (1) tests to identify deviations of the Site Frequency Spectrum (SFS) from neutral expectations (Tajima's D, Fay and Wu's H and Fu and Li's D* and F*), (2) tests focused on the identification of high-frequency haplotypes with extended linkage disequilibrium (iHS and Rsb), and (3) tests based on genetic differentiation between populations (LSBL). RESULTS: Twenty-two pigmentation genes fall in the top 1% for at least one statistic in the GIH population, 5 of which (LYST, OCA2, SLC24A5, SLC45A2, and TYR) have been previously associated with normal variation in skin, hair, or eye color. In comparison, 17 genes fall in the top 1% for at least one statistic in the ITU population. Twelve loci which are identified as outliers in the ITU scan were also identified in the GIH population. CONCLUSIONS: These results suggest that selection may have affected these loci broadly across the region.