Development of low-cost in-house tetra-ARMS-PCR assay for the screening of five CBS mutations found in Pakistani homocystinuria patients

Background: Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-β-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. Objective: Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. Materials and methods: In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. Results: The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. Conclusion: T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases.