Evaluation of Single Exon Deletions in DMD/BMD: Technical and Analytical Concerns.

Background: Oftentimes, a variation at the multiplex ligation-dependent probe amplification (MLPA) probe binding site leads to improper hybridrization/ligation of the probe showing up as a deletion of an exon leading to false positive results for the detection of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD). Objective: Investigating cases with single exon deletion using an alternate method [polymerase chain reaction (PCR) or sequencing] for confirmation of the deletion. Methods: We evaluated males with single exon deletion (n = 49) in our study population (2015-2019). Forty-six were confirmed by an alternate method (conventional PCR/Sanger's sequencing) to confirm the deletion. Results: We observed 25.12% single exon deletions in our study cohort. Further evaluation determined a false positive rate of 6.12%. Three out of 49 single exon deletions had a point mutation near the probe-binding site, indicating a false positive result. Single exon deletions, thus, need to be evaluated with extreme caution, and point mutations, if any, need to be characterized to determine the nature of their pathogenicity.

A novel CFTR gene variant - p.Tyr517* associated with cystic fibrosis: a case report.

INTRODUCTION: Cystic fibrosis (CF) is a genetic disease usually diagnosed by clinical findings and abnormal sweat chloride testing. CASE REPORT: We report a case of an 18-month-old Indian female with clinical findings suggestive of CF referred for genetic confirmation. The CFTR gene was sequenced for 23 mutations as per American College of Medical Genetics (ACMG) guidelines for CF and showed presence of a known common heterozygous delF508 (c.1521_1523delCTT, p.Phe508 del) variant. In addition to delF508 variant, exon 10 of CFTR gene also showed a novel variant c.1551C > G, p.Tyr517*, which was classified as "likely pathogenic" based on recent ACMG variant classification guidelines. The presence of compound heterozygous pathogenic variants along with classical clinical findings, confirmed the diagnosis of CF in this patient. CONCLUSION: The novel pathogenic variants (missense/nonsense/deletion/duplication) in CFTR gene are often identified and are associated with CF, thus highlighting the need of comprehensive complete CFTR gene analysis.

A Study of Triplet-Primed PCR for Identification of CAG Repeat Expansion in the HTT Gene in a Cohort of 503 Indian Cases with Huntington's Disease Symptoms.

BACKGROUND: Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder with an average age at onset of 40 years. It is a polyglutamine (polyQ) disorder that is caused by an increase in the number of CAG repeats in the huntingtin (HTT) gene. Genetic tests that accurately determine the number of CAG repeats are performed for confirmation of diagnosis, predictive testing of persons at genetic risk for inheriting HD, and prenatal testing. The aim of our study was to evaluate efficacy of triplet-primed polymerase chain reaction (TP-PCR) for routine diagnosis of HD in suspected cases from India. METHODS: We evaluated a combination of CAG flanking PCR and triplet-primed PCR for estimation of CAG repeats in 503 cases with clinical suspicion of HD. RESULTS: There were 250 cases (49.7%) that showed the presence of expanded alleles, with 241 (47.9%) being fully penetrant alleles and nine (1.8%) in the reduced penetrance category. There were seven juvenile cases with an age of onset of < 20 years, with the longest allele comprising 106 CAG repeats found in an 8-year-old male patient. The results demonstrated an inverse (R = - 0.67) relationship between CAG length and age at clinical onset. CONCLUSION: Our study on pan-Indian cases is one of the largest studies reported so far in India and focuses on the most accurate and comprehensive molecular diagnostic evaluation of HD.

HLA-B27 testing: A journey from flow cytometry to molecular subtyping.

BACKGROUND: Determination of HLA-B27 status plays an important role as adjuvant in suspected cases for diagnosis of Ankylosing Spondilytis (AS). Objectives of this study were to evaluate (i) flow cytometry method in comparison with DNA microarray for HLA-B27 typing and (ii) EUROArray HLA-B27 Direct assay for HLA-B27 allele detection along with discrimination of AS/non-AS subtypes in Indian population. METHODS: A total of 7543 patients with a presumptive clinical diagnosis of AS were referred for screening of HLA-B27. All samples were initially tested by flow cytometry, and based on its findings, 1560 samples were analyzed for the presence of HLA-B27 allele by microarray technology. A subset of samples (n = 200) were further tested by DNA sequencing for identification of HLA-B27 subtypes. RESULTS: Screening of HLA-B27 by flow cytometry reported 1551 positive (20.56%) and 5556 negative (73.65%) cases. Remaining 436 (5.78%) samples were identified within equivocal zone. Of cases (n = 1560) analyzed by microarray method, 1333 (85.44%) and 227 (14.55%) were detected microarray positive and negative, respectively. DNA sequencing identified HLA-B*27:07 as the predominant subtype among cases showing ex2 positivity by microarray method. Of 200 cases, 20 cases (14 of HLA-B*07 and 6 of HLA-B*37) of HLA-B27 cross-reactive subtypes were also identified. CONCLUSION: We recommend DNA typing as a complementary tool along with flow cytometry to accomplish successful HLA-B27 phenotype determination. This is the first study among Indian population to evaluate efficacy of EUROArray to detect B27 allele and its potential to indicate the presence of nondisease-associated alleles in Indian population.