Diagnostic exome identifies a novel PRKG2 mutation in a proband with skeletal dysplasia.

This graphic abstract combines pedigree, dysmorphology features, radiographs, and the PRKG2 protein domain, specifically the CNB-A regulatory domain, which harbors a mutation resulting in premature protein termination.

Metabolic and Genetic Evaluation in Children with Nephrolithiasis.

OBJECTIVE: To evaluate metabolic and genetic abnormalities in children with nephrolithiasis attending a referral center in North India. METHODS: The patients aged 1-18 y old with nephrolithiasis underwent biochemical evaluation and whole-exome sequencing. The authors evaluated for monogenic variants in 56 genes and compared allele frequency of 39 reported polymorphisms between patients and 1739 controls from the GenomeAsia 100 K database. RESULTS: Fifty-four patients, aged 9.1 ± 3.7 y were included. Stones were bilateral in 42.6%, familial in 33.3%, and recurrent in 25.9%. The most common metabolic abnormalities were hypercalciuria (35.2%), hyperoxaluria (24.1%), or both (11.1%), while xanthinuria (n = 3), cystinuria (n = 1), and hyperuricosuria (n = 1) were rare. Exome sequencing identified an etiology in 6 (11.1%) patients with pathogenic/likely pathogenic causative variants. Three variants in MOCOS and one in ATP7B were pathogenic; likely pathogenic variants included MOCOS (n = 2), AGXT, and SLC7A9 (n = 1, each). Causality was not attributed to two SLC34A1 likely pathogenic variants, due to lack of matching phenotype and dominant family history. Compared to controls, allele frequency of the polymorphism TRPV5 rs4252402 was significantly higher in familial stone disease (allele frequency 0.47 versus 0.53; OR 3.2, p = 0.0001). CONCLUSION: The chief metabolic abnormalities were hypercalciuria and hyperoxaluria. A monogenic etiology was identified in 11% with pathogenic or likely pathogenic variants using a gene panel for nephrolithiasis. Heterozygous missense variants in the sodium-phosphate cotransporter SLC34A1 were common and required evaluation for attributing pathogenicity. Rare polymorphisms in TRPV5 might increase the risk of familial stones. These findings suggest that a combination of metabolic and genetic evaluation is useful for determining the etiology of nephrolithiasis.

Retinoblastoma genetics screening and clinical management.

BACKGROUND: India accounts for 20% of the global retinoblastoma (RB) burden. However, the existing data on RB1 gene germline mutations and its influence on clinical decisions is minimally explored. METHODS: Fifty children with RB underwent complete clinical examination and appropriate multidisciplinary management. Screening of germline RB1 gene mutations was performed through next-generation sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. The mutation and non-mutation groups were compared for clinical parameters especially severity, progression and recurrence. RESULTS: Twenty-nine patients had bilateral RB (BLRB) and 21 had unilateral RB (ULRB). The genetic analysis revealed 20 RB1 variations in 29 probands, inclusive of 3 novel mutations, known 16 mutations and heterozygous whole gene deletions. The mutation detection rate (MDR) was 86.2% in BLRB and 19% in ULRB. Associations of disease recurrence (p = 0.021), progression (p = 0.000) and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors were observed in the mutation group. Clinical management was influenced by the presence of germline mutations, particularly while deciding on enucleation, frequency of periodic follow up and radiotherapy. CONCLUSIONS: We identified novel RB1 mutations, and our mutation detection rate was on par with the previous global studies. In our study, genetic results influenced clinical management and we suggest that it should be an essential and integral component of RB-care in India and elsewhere.

Seven novel genetic variants in a North Indian cohort with classical homocystinuria.

Classical homocystinuria is the most common cause of isolated homocystinuria. The variants of the CBS gene remain unidentified in Indian children with this disorder. Based on the hallmark clinical features, family history, and/or biochemical clues for classical homocystinuria, 16 children below the age of 18 years were evaluated by Sanger sequencing of the coding exons of CBS gene with flanking intronic regions. The common C677T variant of the MTHFR gene was also screened by restriction fragment length polymorphism. Fifteen children were clinically suspected of having classical homocystinuria and one asymptomatic child with positive family history. Only seven children had biochemical features of classical homocystinuria. Sanger sequencing of the CBS gene confirmed 15 different pathogenic or likely pathogenic variants in 14 cases. Of these, seven variants were novel (three frameshift deletions, two nonsense, one missense, one splice site variant) and were predicted to be deleterious by Mutation Taster software. Seven cases were homozygous, another six were compound heterozygous, and one case was single heterozygous in the study. None of the three most frequent mutations reported worldwide viz., I278T, G307S, and IVS 11-2A>C were found in our cohort. No variants were detected in the exons 2, 8, 12, and 14 as compared to reported literature. Eleven out of 15 variants were associated with the conserved catalytic domain of the CBS polypeptide. The MTHFR polymorphism C677T was observed in heterozygous state in six cases. Our study reports the detailed genotype and seven novel variants in the CBS gene, causing classical homocystinuria in Indian children. The genetic analysis will help to offer accurate genetic counseling, prenatal diagnosis, and development of mutation-based novel therapeutic strategies.

Mutational Landscape of Esophageal Squamous Cell Carcinoma in an Indian Cohort.

Esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal cancer in India. Cigarette smoking and chewing tobacco are known risk factors associated with ESCC. However, genomic alterations associated with ESCC in India are not well-characterized. In this study, we carried out exome sequencing to characterize the mutational landscape of ESCC tumors from subjects with a varied history of tobacco usage. Whole exome sequence analysis of ESCC from an Indian cohort revealed several genes that were mutated or had copy number changes. ESCC from tobacco chewers had a higher frequency of C:G > A:T transversions and 2-fold enrichment for mutation signature 4 compared to smokers and non-users of tobacco. Genes, such as TP53, CSMD3, SYNE1, PIK3CA, and NOTCH1 were found to be frequently mutated in Indian cohort. Mutually exclusive mutation patterns were observed in PIK3CA-NOTCH1, DNAH5-ZFHX4, MUC16-FAT1, and ZFHX4-NOTCH1 gene pairs. Recurrent amplifications were observed in 3q22-3q29, 11q13.3-q13.4, 7q22.1-q31.1, and 8q24 regions. Approximately 53% of tumors had genomic alterations in PIK3CA making this pathway a promising candidate for targeted therapy. In conclusion, we observe enrichment of mutation signature 4 in ESCC tumors from patients with a history of tobacco chewing. This is likely due to direct exposure of esophagus to tobacco carcinogens when it is chewed and swallowed. Genomic alterations were frequently observed in PIK3CA-AKT pathway members independent of the history of tobacco usage. PIK3CA pathway can be potentially targeted in ESCC which currently has no effective targeted therapeutic options.

Immunodeficiency, Motor Delay, and Hypouricemia Caused by a Novel Mutation of Purine Nucleoside Phosphorylase Gene in an Indian Infant.

We describe an 11-month-old boy who presented with recurrent respiratory infections from 6 months of age. His elder sister died at 10 months with severe septicemia and meningitis. The boy had a mild motor delay. Investigations revealed T cell deficiency and very low serum uric acid suggestive of purine nucleoside phosphorylase (PNP) deficiency - a rare variant of severe combined immunodeficiency disease. A novel homozygous missense mutation of c.597C>G(p. S199R) of exon 5 on PNP gene confirmed the diagnosis. We suggest that uric acid should be a part of investigation profile for unidentified motor delay, as recurrent infections can be late presentation.

A neoepitope derived from a novel human germline APC gene mutation in familial adenomatous polyposis shows selective immunogenicity.

Familial adenomatous polyposis (FAP) is an inherited condition arising from genetic defects in the Adenomatous polyposis coli (APC) gene. Carriers with mutations in the APC gene develop polyps in the colon and rectum which if not managed, transition into colon cancer. In this study, we identified a novel germline mutation in the APC gene in members of an FAP-affected (Familial adenomatous polyposis) family. This unique heterozygous variant (c.735_736insT; p.Ser246PhefsTer6) was identified in ten out of twenty six family members, ranging in age from 6 to 60 years. Polyps were detected in six of the ten individuals (35-60 years) carrying this mutation. The remaining four members (6-23 years) remain polyp free. A significant fraction of FAP affected individuals eventually develop colon cancer and therapeutic interventions to prevent cancer progression remain elusive. To address this issue, we sought to determine if peptides derived from the novel APC mutation could induce a cytotoxic T cell response, thereby qualifying them as vaccine candidates. Peptides harboring the variant amino acids were first interrogated in silico for their immunogenicity using a proprietary neoepitope prioritization pipeline, OncoPeptVAC. A single 9-mer peptide was predicted to be immunogenic. Remarkably, CD8+ T cells isolated from either an FAP+/ APCmut individual, or from a FAP-/ APCmut individual, failed to respond to the peptide, whereas those from either an unaffected family member (FAP-/ APCwt) or from healthy unrelated donors, showed a robust response, suggesting that CD8+ T cells from individuals carrying this germline APC mutation have been tolerized to the mutation. Furthermore, experimental testing of six additional reported APC gene mutation-derived peptides revealed one of the six to be immunogenic. While not all APC mutant peptides are inmmunogenic, a few qualify as vaccine candidates offering novel treatment opportunities to patients with somatic APC gene mutations to delay/treat colorectal cancer.

Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes.

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Comprehensive genomic analysis identifies pathogenic variants in maturity-onset diabetes of the young (MODY) patients in South India.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.

Retinoblastoma in India : microsatellite analysis and its application in genetic counseling.

OBJECTIVES: This study was conducted with two objectives. The first was to estimate the frequency of loss of heterozygosity (LOH) of the RB1 gene as a mechanism in disease causation in tumors of patients from India. The second objective was to employ RB1 molecular deletion and microsatellite-based linkage analysis as laboratory tools, while counseling families with a history of retinoblastoma (RB). METHODS: DNA was extracted from peripheral blood and tumors of 54 RB patients and their relatives. Eight fluorescent microsatellite markers, both intragenic and flanking the RB1 gene, were used. After PCR amplification, samples were run on an ABI PRISM 310 genetic analyzer for LOH, deletion detection, and haplotype generation. RESULTS: LOH was found in conjunction with tumor formation in 72.9% of RB patients (39/54 patients; p=0.001; 95% CI 0.6028, 0.8417); however, we could not associate various other clinical parameters of RB patients with the presence or absence of RB1 LOH. Seven germline deletions (13% of RB patients) were identified, and the maternal allele was more frequently lost (p=0.01). A disease co-segregating haplotype was detected in two hereditary autosomal dominant cases. CONCLUSION: LOH of the RB1 gene could play an important role in tumor formation. Large deletions involving RB1 were observed, and a disease co-segregating haplotype was used for indirect genetic testing. This is the first report from India where molecular testing has been applied for RB families in conjunction with genetic counseling. In tertiary ophthalmic practice in India, there is an emerging trend towards the application of genetical knowledge in clinical practice.