Comparing rare variants versus common in the pathogenesis of nonalcoholic fatty liver disease: a whole exome sequencing approach.

BACKGROUND/PURPOSE: Genome-wide association studies have reported the association of common variants with nonalcoholic fatty liver disease in genes, namely, PNPLA3/TM6SF2/MBOAT7/HSD17B13, across ethnicities. However, the approach does not identify rarer variants with a higher effect size. We therefore sequenced the complete exonic regions of patients with nonalcoholic steatohepatitis and controls to compare rare and common variants with a role in the pathogenesis. METHODS: This is a prospective study that recruited 54 individuals with/without fatty infiltration. Patients with biopsy-proven nonalcoholic steatohepatitis and persistently elevated liver enzymes were included. Controls were with normal CT/MR fat fraction. DNA was isolated from whole blood, amplified (SureSelectXT Human All Exon V5 + UTR kit) and sequenced (Illumina). Data were filtered for quality, aligned (hg19), and annotated (OpenCRAVAT). Pathogenic (Polyphen-2/SIFT/ClinVar) variants and variants reported to be associated with NAFLD based on published literature were extracted from our data and compared between patients and controls. RESULTS: The mean age of controls (N = 17) and patients (N = 37) was 46.88 ± 6.94 and 37.46 ± 13.34 years, respectively. A total of 251 missense variants out of 89 286 were classified as pathogenic. Of these, 106 (42.23%) were unique to the patients and remaining (n = 145; 57.77%) were found in both patients and controls. Majority (25/37; 67.57%) patients had a minimum of one or more rare pathogenic variant(s) related to liver pathology that was not seen in the controls. CONCLUSION: Elucidating the contribution of rare pathogenic variants would enhance our understanding of the pathogenesis. Including the rarer genes in the polygenic risk scores would enhance prediction power.

Lesser prevalence of polyps/WNT-dysregulation and concomitant upregulation of gamma-catenin/MYC point to alternate pathways in colorectal cancer in India.

Colorectal cancer (CRC) is known to follow adenoma carcinoma sequence (ACS) in majority of the tumors and the driver variants and associated pathways are well delineated. However, most of the published data are from the west and information in other ethnicities is sparse. We therefore comprehensively evaluated the CRC tumors from Indian ethnicity for the prevalence of ACS. In this cohort study, clinical data of 100,497 patients who attended hospital between 2013 and 2018 were accessed. Tumors from patients (n = 130) with CRC who were treated primarily by surgery were included. DNA and RNA were isolated to assess variants (direct sequencing) and WNT-pathway dysregulation in genes related to ACS. Global gene expression was generated and analyzed on microarrays (Affymetrix; N = 10) and next generation sequencing platforms (Illumina; N = 25). Gene expression at mRNA (qRT-PCR) and protein level (IHC) of JUP/CTNNB1/MYC were assessed. Correlation between expression of JUP and MYC was evaluated by Karl Pearson's correlation coefficient. The prevalence of polyps was 16.75%, while 18.26% variants in APC/CTNNB1, 20.00% in KRAS, and 18.33% WNT dysregulation were noted. Interestingly, 29/60 (48.33%) tumors showed only MYC upregulation with normal APC/CTNNB1 expression. Global gene expression and validation in an independent tumor cohort confirmed concomitant upregulation of JUP (gamma-catenin) & MYC (r = 0.71; p = 0.001) at mRNA and protein in sizeable number of tumors (45/96; 46.88%). Our study provides evidence for limited prevalence of ACS in the Indian ethnicity. Preventive colonoscopies for early identification and management of CRC may not be an effective strategy in this ethnicity.

MicroRNAs as a therapeutic target in IgA nephropathy in Indian population.

Immunoglobulin A nephropathy (IgAN) is the most frequent glomerular disease with rapid development to end stage renal disease, requiring renal replacement therapy. Genome-wide studies suggest geographical variations in genetic susceptibility to IgAN and disease progression. Specific 'candidate genes' were indicated to correlate with different functions that are involved in the pathogenesis of renal conditions. MicroRNAs (miRNAs/miRs) have a major role in mRNA degradation or translation repression, thereby regulating the expression of their target proteins. Previously, a small number of miRNAs were reported to have direct associations with IgAN. In the present study, new miRNAs linked to IgAN were identified in the Indian population. The miRNA was isolated from kidney biopsies of patients with IgAN (n=6) and healthy control tissue from patients with renal cell carcinoma (n=6). The sequencing results indicated that the miRNA percentage acquired from controls and patients with IgAN was 5.61 and 4.35%, respectively. From the results, 10 upregulated and 15 downregulated miRNAs were identified. Of the 25 differentially expressed miRNAs (DEMs), miR-181a-5p, miR-28-3p, let-7g-5p, miR-92a-3p and miR-30c-5p were not reported previously. Furthermore, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the target genes of the DEMs were mainly enriched in pathways such as cancer, ErbB signalling, proteoglycans in cancer, Hippo signalling and MAPK pathways. The newly identified miRNAs may impact the behaviour of tissues or IgA deposition by regulating signalling pathways, which forms a basis for future studies aimed at improving the diagnosis and care of patients with IgAN in the Indian community.

Newly Diagnosed Crohn's Disease Patients in India and Israel Display Distinct Presentations and Serological Markers: Insights from Prospective Cohorts.

Background: Crohn's disease (CD) incidence is rising in India. However, features of newly diagnosed patients with CD in this population are largely unknown. The Indo-Israeli IBD GastroEnterology paRtnership (TiiiGER) aimed to investigate differences in presentation among patients with newly diagnosed CD in India and Israel, and to explore phenotype−serotype correlations. Methods: A prospective observational cohort study of consecutive adults (>18 years) conducted in two large referral centers in India and Israel (2014−2018). Clinical data, an antiglycan serological panel, and 20 CD-associated genetic variants were analyzed. Outcomes: complicated phenotype at diagnosis and early complicated course (hospitalizations/surgeries) within 2 years of diagnosis. Results: We included 260 patients (104, Indian (65.4%, male; age, 37.8); 156 Israeli (49.4%, male; 31.8, age)). Median lag time from symptoms onset to diagnosis was 10.5 (IQR 3−38) vs. 3 (IQR 1−8) months in Indian vs. Israeli patients (p < 0.001). Complicated phenotype at diagnosis was observed in 48% of Indian and 30% of Israeli patients (p = 0.003). Complicated phenotype was associated with higher anti-Saccharomyces cerevisiae antibody (ASCA) seropositivity rate among Israeli patients (p < 0.001), but not among Indian patients. Antiglycan serology did not correlate with the tested genetic variants. Early complicated course occurred in 28 (18%) Israeli and 13 (12.5%) Indian patients. The time from diagnosis to complication was comparable (log rank p = 0.152). Antiglycan serology did not correlate with a complicated early course in either cohort. Conclusions: There are significant differences in patients presenting with newly diagnosed CD in India and Israel, including phenotype and distinct biomarkers at diagnosis. These differences suggest different genetic and environmental disease modifiers.

Clinical outcomes in individuals hospitalized with SARS-CoV-2 Delta variant (B.1.617.2) who had been vaccinated with Covishield (ChAdOx1) and Covaxin (BBV-152).

Background: Covishield (ChAdOx) and Covaxin (BBV-152) are the mainstream vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) used in India and a few other countries. Objective: To assess the clinical outcomes of patients hospitalized with coronavirus disease 2019 (COVID-19) who had been vaccinated with either Covishield or Covaxin. Methods: This prospective, single-centre, observational cohort study of 1160 patients hospitalized with COVID-19 was conducted between April and June 2021. Severity of disease at admission and during hospitalization, requirement for intensive care unit (ICU) admission and ventilatory support, inflammatory markers (C-reactive protein, ferritin, lactate dehydrogenase, D-dimer), neutralizing antibody levels and mortality were assessed in vaccinated and unvaccinated patients. Results: More than 90% of patients in this study harboured the Delta variant (Pango lineage B.1.617.2) of SARS-CoV-2. Severity of disease at admission and during hospitalization (3.44% vs 7.51%; P=0.0032) and requirement for ICU admission and ventilatory support (2.83% vs 5.86%; P=0.0154) were significantly lower in vaccinated patients compared with unvaccinated patients. Vaccinated patients also had significantly (P<0.0001) higher antibody levels and lower inflammatory marker levels compared with unvaccinated patients. A subset of vaccinated, deceased patients mounted minimal antibody response ['non-responders': 4.53 (standard deviation 1.40) AU/mL]. Conclusion: These results demonstrate the effectiveness of Covishield and Covaxin against severe disease in patients hospitalized with COVID-19 with breakthrough infections caused by the Delta variant. Strategies targeting non-responders are desirable to minimize morbidity and mortality.

High-resolution HLA genotyping identifies alleles associated with severe COVID-19: A preliminary study from India.

INTRODUCTION: Human leukocyte antigen (HLA) variability has been demonstrated to be associated with susceptibility/severity of COVID-19. High-resolution HLA genotyping to identify alleles associated with severe COVID-19 in an Indian cohort was performed. METHODS: Quantitative reverse-transcription polymerase chain reaction-confirmed SARS-CoV-2-positive patients with mild/moderate/severe disease (n = 54) and asymptomatic (n = 42) were recruited and genotyped for 11-HLA loci on MiSeq using NGSgo®-MX11-3 and analyzed (NGSengine; GenDx). RESULTS: A significant difference in alleles between the groups was identified for HLA-C*04:01:01:01, HLA-DRB5*01:01:01:02, HLA-DQA1*03:01:01:01, HLA-DPB1*04:01:01:41, and HLA-DPA1*01:03:01:02. Alleles namely, HLA-C*04:01:01:01 (OR: 5.71; 95% CI: 1.2-27.14; p = .02), HLA-DRB5*01:01:01:02 (OR: 2.94; 95% CI: 1.1-7.84; p = .03), DQA1*03:01:01:01 (OR: 22.47; 95% CI: 1.28-393.5; p = .03), HLA-DPB1*04:01:01:41 (OR: 9.44; 95% CI: 0.5-175.81; p = .13), and HLA-DPA1*01:03:01:02 (OR: 8.27; 95% CI: 2.26-30.21; p = .001) were associated with severe COVID-19. CONCLUSION: Genotyping for these alleles will enable identification of individuals at risk of severe disease and stratification for preferential vaccination.

Whole-Exome Sequencing Identifies a Variant in Phosphatidylethanolamine N-Methyltransferase Gene to be Associated With Lean-Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases with simple steatosis on one end and hepatocellular carcinoma on the other. Although obesity is a known risk factor for NAFLD, individuals with normal body mass index (BMI) also have hepatic fatty infiltration, now termed "lean-NAFLD". It represents a distinct entity with a strong underlying genetic component. The present study aimed to sequence the complete exonic regions of individuals with lean-NAFLD to identify germline causative variants associated with disrupted hepatic fatty acid metabolism, thereby conferring susceptibility to NAFLD. METHODS: Whole blood was collected from patients with lean-NAFLD (n = 6; BMI < 23.0 kg/m2) and matched lean controls (n = 2; discovery set). Liver fat was assessed using acoustic radiation force impulse (ARFI) imaging. Patients with ultrasound-detected NAFLD (n = 191) and controls (n = 105) were part of validation set. DNA was isolated, and whole-exome sequencing (WES) was performed in the discovery cohort (Ion Proton™; Ion AmpliSeq™ Exome RDY Kit). Data were analyzed (Ion Reporter software; Life Technologies), and variants identified. Validation of variants was carried out (Taqman probes; Real time-PCR). Student's t test and Fisher's exact test were used to analyze the statistical significance. RESULTS: Although WES identified ∼74,000 variants in individual samples, using various pipelines. variants in genes namely phosphatidylethanolamine N-methyltransferase (PEMT) and oxysterol-binding protein-related protein10 (OSBPL10) that have roles in dietary choline intake and regulation of cholesterol homeostasis, respectively, were identified (discovery set). Furthermore, significant differences were noted in BMI (p = 0.006), waist/hip circumference (p > 0.001), waist/hip ratio (p > 0.001), aspartate aminotransferase (p > 0.001), alanine aminotransferase (p > 0.001), and triglycerides (p = 0.002) between patients and controls. Validation of variants (rs7946-PEMT and rs2290532-OSBPL10) revealed that variant in PEMT but not OSBPL10 gene was associated (p = 0.04) with threefold increased risk of NAFLD in lean individuals. CONCLUSION: Our results demonstrate the association of rs7946 with lean-NAFLD. WES may be an effective strategy to identify causative variants underlying lean-NAFLD.

PRSS1 (R122H) mutation in an Indian family with low penetrance is associated with pancreatitis phenotype.

Mutations in PRSS1 gene namely R122H and N29I cause hereditary pancreatitis. They are autosomal dominant with a high penetrance (80%) reported in North American, North-east Asian, and North European ethnicities. However, the mutations are reportedly absent in Indian, African, and South American ethnicities. We report here for the first time a family from India that is positive for R122H mutation in the PRSS1 gene. The proband is symptomatic with chronic pancreatitis, however, the father although heterozygous for R122H is asymptomatic.