Whole exome and targeted gene sequencing to detect pathogenic recessive variants in early onset cerebellar ataxia.

Over 100 genetically distinct causal known loci for hereditary ataxia phenotype poses a challenge for diagnostic work-up for ataxia patients in a clinically relevant time and precision. In the present study using next-generation sequencing, we have investigated pathogenic variants in early-onset cerebellar ataxia cases using whole exome sequencing in singleton/family-designed and targeted gene-panel sequencing. A total of 98 index patients were clinically and genetically (whole exome sequencing (WES) in 16 patients and targeted gene panel of 41 ataxia causing genes in 82 patients) evaluated. Four families underwent WES in family based design. Overall, we have identified 24 variants comprising 20 pathogenic and four likely-pathogenic both rare/novel, variations in 21 early onset cerebellar ataxia patients. Among the identified variations, SACS (n = 7) and SETX (n = 6) were frequent, while ATM (n = 2), TTPA (n = 2) and other rare loci were observed. We have prioritized novel pathogenic variants in RARS2 and FA2H loci through family based design in two out of four families.

Genetics of Ataxias in Indian Population: A Collative Insight from a Common Genetic Screening Tool.

Cerebellar ataxias (CAs) represent a group of autosomal dominant and recessive neurodegenerative disorders affecting cerebellum with or without spinal cord. Overall, CAs have preponderance for tandem nucleotide repeat expansions as an etiological factor (10 TREs explain nearly 30-40% of ataxia cohort globally). The experience of 10 years of common genetic ataxia subtypes for ≈5600 patients' referrals (Pan-India) received at a single center is shared herein. Frequencies (in %, n) of SCA types and FRDA in the sample cohort are observed as follows: SCA12 (8.6%, 490); SCA2 (8.5%, 482); SCA1 (4.8%, 272); SCA3 (2%, 113); SCA7 (0.5%, 28); SCA6 (0.1%, 05); SCA17 (0.1%, 05), and FRDA (2.2%, 127). A significant amount of variability in TRE lengths at each locus is observed, we noted presence of biallelic expansion, co-occurrence of SCA-subtypes, and the presence of premutable normal alleles. The frequency of mutated GAA-FRDA allele in healthy controls is 1/158 (0.63%), thus an expected FRDA prevalence of 1:100 000 persons. The data of this study are relevant not only for clinical decision making but also for guidance in direction of genetic investigations, transancestral comparison of genotypes, and lastly provide insight for policy decision for the consideration of SCAs under rare disease category.

BRAF V600E and TERT promoter mutations in paediatric and young adult papillary thyroid cancer and clinicopathological correlation.

Objectives The primary objective of this study was to determine the prevalence of BRAF V600E and TERTpromoter mutations in paediatric and young adult patients with papillary thyroid carcinoma (PTC) and the secondary objective, to assess their association with clinicopathological features. Methods Patients ≤20 years who underwent surgery for differentiated thyroid cancer (DTC) from 2005 to 2018 were consecutively enrolled for BRAF V600E and TERTpromoter mutations analysis and records analysed for the association of aggressive features. Univariate analysis and multivariate logistic regression were used to identify the independent predictors of BRAF V600E mutations. Results Among 100 patients with DTC, 68 patients were ≤18 years and the remaining 30 patients were >18 years of age with a median age of 17 years (IQR 14-19 years) 98 patients had PTC and 2 had FTC. BRAF V600E mutation was present in 14/98 (14.3%) PTC and TERTpromoter mutation noted in none. Multivariate analysis identified RAI refractoriness (OR:10.57, 95% CI: 2.6 to 41.6, P-0.0008) as an independent factor associated with BRAF V600E mutation. 17 patients with distant metastases were negative for both BRAF V600E or TERTpromoter mutation. No significant association was observed between age, gender, PTC variants, extra-thyroidal extension, lymphovascular invasion, multifocality, RAI administration and event rate with BRAF V600E mutation. Irrespective of BRAF V600E mutation, radioiodine refractory status (p-0.0001) had a reduced EFS probability. Conclusion In paediatric & young adult PTC, TERTpromoter mutation is absent and BRAFV600E mutation is not associated with distant metastasis. The prevalence rate of the BRAF V600E mutation is much lower compared to adult PTC patients.

RET gene mutation analysis and long-term clinical outcomes of medullary thyroid cancer patients.

OBJECTIVE: Medullary thyroid carcinoma is a rare, potentially aggressive tumour, with relatively worse prognosis than well-differentiated thyroid cancer. We evaluated the long-term outcomes and prognosis of medullary thyroid carcinoma patients at a single institution in India and compared outcomes based on results of RET protooncogene mutation analysis. METHODS: Data were retrieved through a prospectively maintained thyroid cancer database from 1998 to June 2019, and medullary thyroid carcinoma patients were recruited. RET gene mutation status (exon 10-16) was assessed. Patient with a minimum follow-up of 12 months was eligible to be part of the long-term outcome analysis. RESULTS: Out of 149 peripheral blood samples, 42 were positive for RET gene mutation (prevalence of 28.1%). The median follow-up duration was 48 months, ranging from 12 to 240 months. Long-term clinical outcomes of 113 patients were assessed. Two deaths were noted in this series. Both 5- and 10-year survival was cent per cent. Overall survival was 98.2% (97.3% in RET positive and 98.7% in RET negative group). Progression-free survival was 55.4% in total (60% in RET positive and 53.3% in RET negative group). No statistically significant difference was found between RET positive and RET negative groups concerning overall survival (P = 0.6011) and progression-free survival (P = 0.5140). Univariate analysis revealed high calcitonin (>10 pg/mL), stage IV disease, and presence of lymph nodal metastasis to be significant predictors of disease recurrence, however, multivariate analysis demonstrated the presence of lymph node metastases as the only significant predictor of recurrence (P = 0.0005). CONCLUSIONS: Medullary thyroid carcinoma patients had relatively favourable long-term outcomes. Long-term survival was similar irrespective of RET mutation status. Presence of lymph node metastases appeared to be the strongest predictor of overall and progression-free survival, followed by Calcitonin level and stage of the disease.

Long-term Clinicopathological Features of a Family with Multiple Endocrine Neoplasia Type 2A Caused by C634R RET Gene Mutation.

Type 2 multiple endocrine neoplasia (MEN2A) is a variant of hereditary medullary thyroid carcinoma (MTC). MEN2A is characterized by the presence of the following: MTC, hyperparathyroidism, and pheochromocytoma (PHEO). The pathogenesis includes RET proto-oncogene mutation; the most frequently observed mutation is in exon 11 codon 634. We report pedigree of a large Indian family involving three generations including 21 members with MEN2A, in whom RET mutation status was determined. We then analyzed their clinical follow-up details, with a median duration of follow-up of 60 months (range: 9-276 months). Calcitonin (Ctn) levels were routinely checked during the follow-up. The index case was found to carry p.C634R mutation involving exon 11 of the RET gene. RET mutation was positive in 12 members in the family (12/21, i.e., 57%), was negative in 7 patients, and was not tested in 2 patients, as they were not available for the genetic test. Thirteen were clinically affected with MTC and 10 members had PHEO. At the last follow-up, the median Ctn level was 14.3 pg/mL (range: 2-12655 pg/mL). Four patients developed lymph nodal recurrence during follow-up, for which they underwent re-operations with median duration to recurrence being 48 months (range: 9-156 months). We highlight in this article that early diagnosis, adequate surgery, and appropriate genetic counseling with genetic screening are essential to improve the outcome of persons with MTC. Every case of MTC should be seen as familial or index case of hereditary MTC unless otherwise RET mutation excludes it.

Single-step blood direct PCR: A robust and rapid method to diagnose triplet repeat disorders.

OBJECTIVE: DNA extraction prior to polymerase chain reaction (PCR) amplification in genetic diagnoses of triplet repeat disorders (TRDs) is tedious and labour-intensive and has the limitations of sample contamination with foreign DNA, including that from preceding samples. Therefore, we aimed to develop a rapid, robust, and cost-effective method for expeditious genetic investigation of TRDs from whole blood as a DNA template. METHODS: Peripheral blood samples were collected from 70 clinically suspected patients of progressive ataxia. The conventional method using genomic DNA and single-step Blood-Direct PCR (BD-PCR) method with just 2µl of whole blood sample were tested to amplify triplet repeat expansion in genes related to spinocerebellar ataxia (SCA) types 1, 2, 3, 12 and Friedreich's ataxia (FRDA). Post-PCR, the allele sizes were mapped and repeat numbers were calculated using GeneMapper and macros run in Microsoft Excel programmes. RESULTS: Successful amplification of target regions was achieved in all samples by both methods. The frequency of the normal and mutated allele was concordant between both methods, diagnosing 37% positive for a mutation in either of the candidate genes. The BD-PCR resulted in higher intensities of product peaks of normal and pathogenic alleles. CONCLUSIONS: The nearly-accurate sizing of the normal and expanded allele was achieved in a shorter time (4-5h), without DNA extraction and any risk of cross contamination, which suggests the BD-PCR to be a reliable, inexpensive, and rapid method to confirm TRDs. This technique can be introduced in routine diagnostic procedures of other tandem repeat disorders.

Transforming growth factor-ß1 (C509T, G800A, and T869C) gene polymorphisms and risk of ischemic stroke in North Indian population: A hospital-based case-control study.

BACKGROUND: Transforming growth factor-beta 1 (TGF-ß1) is a multifunctional pleiotropic cytokine involved in inflammation and pathogenesis of cerebrovascular diseases. There is limited information on the association between variations within the TGF-ß1 gene polymorphisms and risk of ischemic stroke (IS). The aim of this study was to investigate the association of the TGF-ß1 gene (C509T, G800A, and T869C) polymorphisms, and their haplotypes with the risk of IS in North Indian population. METHODS: A total of 250 IS patients and 250 age- and sex-matched controls were studied. IS was classified using the Trial of Org 10172 in Acute Stroke Treatment classification. Conditional logistic regression analysis was used to calculate the strength of association between TGF-ß1 gene polymorphisms and risk of IS. Genotyping was performed using SNaPshot method. RESULTS: Hypertension, diabetes, dyslipidemia, alcohol, smoking, family history of stroke, sedentary lifestyle, and low socioeconomic status were found to be associated with the risk of IS. The distribution of C509T, G800A and T869C genotypes was consistent with Hardy-Weinberg Equilibrium in the IS and control groups. Adjusted conditional logistic regression analysis showed a significant association of TGF-ß1 C509T (odds ratio [OR], 2.1; 95% CI; 1.2-3.8; P = 0.006), G800A (OR, 4.4; 95% CI; 2.1-9.3; P < 0.001) and T869C (OR, 2.6; 95% CI; 1.5-4.5; P = 0.001) with the risk of IS under dominant model. Haplotype analysis showed that C509-A800-T869 and T509-G800-C869 haplotypes were significantly associated with the increased risk of IS. C509T and T869C were in strong linkage disequilibrium (D' =0.51, r2 = 0.23). CONCLUSION: Our results suggest that TGF-ß1 polymorphisms and their haplotypes are significantly associated with the risk of IS in North Indian population.