Development of a Genetic Screening Method for Polyglutamine Expansion Spinocerebellar Ataxias in Universiti Kebangsaan Malaysia Medical Centre
Keywords:
Spinocerebellar Ataxia, polyglutamine, PCR, trinucleotide repeat, Sanger sequencingAbstract
Background and Aims: Spinocerebellar ataxia (SCA) is a progressive neurodegenerative disorder that encompasses a range of conditions characterised by the gradual deterioration of walking associated with poor coordination of hand movements, eye movements and speech. Currently, 40 types of SCA have been identified with different gene mutations. This study aims to develop locally a simple, easy and cost-effective genetic screening method to diagnose SCA types 1, 2, 3, 6 and 7 that are caused by a polyglutamine (polyQ) expansion. Materials and methods: Twenty-four subjects comprises 13 patients, 6 family members and 5 healthy controls from Universiti Kebangsaan Malaysia Medical Centre (UKMMC) gave informed consent to be included in this study. DNA samples were extracted from the blood samples of these subjects and the SCA genes, namely ATXN1 (SCA 1), ATXN2 (SCA 2), ATXN3 (SCA 3), CACNA1A (SCA 6) and ATXN7 (SCA 7), were amplified at the polyQ regions using previously published primers. The polymerase chain reaction (PCR) products for each SCA type were visualised by agarose gel electrophoresis and Sanger sequenced to validate the number of polyQ estimated. Results: The results obtained from PCR followed by gel electrophoresis confirmed that all five SCA genes were successfully amplified using the protocol developed. Using the protocol, pathological polyQ expansions were detected in ATXN1 of one patient, in ATXN2 of another patient, and in ATXN3 in 3 patients. In addition, the protocol identified 2 children of patients genetically diagnosed elsewhere to have expanded polyQ in ATXN3 and CACNA1A, and also have expanded polyQ mutations similar to their parents. Sanger sequencing results verified that the subjects had pathological polyQ expansion as visualized by gel electrophoresis. No subjects were found to have an expanded polyQ in ATXN7 and no healthy controls were found to have a pathological expanded polyQ. Conclusion: In conclusion, the PCR method used in this study successfully amplified the polyQ regions in 5 SCA genes and Sanger sequencing confirmed clinically diagnosed SCA patients to have pathological polyQ expansion. Thus, the present study validates the PCR protocol developed to be used as a tool for screening SCA subtypes.
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