STR AMPLIFICATION USING MULTIPLEX - PCR TECHNIQUE IN LINKAGE ANALYSIS FOR POMPE DISEASE DIAGNOSIS

Abstract

Objective: To establish and refine a Multiplex-PCR protocol for the simultaneous amplification of short tandem repeat (STR) markers closely linked to the GAA gene, enabling linkage-based genetic diagnosis of Pompe disease. Subjects and methods: The study was conducted on a nuclear family comprising a father, mother, and three children, one of whom had Pompe disease and carried the homozygous GAA gene variant c.1933G>C (p.Asp645His). A Multiplex-PCR assay was optimised to simultaneously amplify multiple STR markers flanking the GAA locus. STR genotyping was performed by capillary electrophoresis and compared with Sanger sequencing results. Results: The optimised Multiplex-PCR assay successfully amplified six STR markers: D17S802, D17S1806, D17S784, D17S928, D17S78.0, and D17S78.57. Capillary electrophoresis revealed specific amplification of the STR markers with clear signal profiles. The STR genotyping results were fully concordant with the Sanger sequencing results. Conclusion: A Multiplex-PCR method for the amplification of STR markers linked to the GAA gene was successfully established for linkage analysis. This technique has potential applications in preimplantation genetic diagnosis of Pompe disease.