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

Abstract

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.

Keywords:

• T-ARMS-PCR
• CBS
• Homocystinuria
• IEM
• Pakistan

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the institute “National Institute for Biotechnology and Genetic Engineering”.

Consent to participate

Informed written consent was obtained for all the studied participants from their parents/guardian/head of special education centers.

Consent to publish

Consent has also been taken from the participants to publish this data for the welfare of humankind.

Author contributions

“All authors contributed to the study conception and design. Fazli Rabbi Awan conceptualized and designed the study. Muhammad Wasim and Haq Nawaz Khan collected samples of affected families. Adila Khalil conducted laboratory experiments and wrote initial draft. Muhammad Wasim and Haq Nawaz Khan edited the manuscript. Hina Ayesha provided all the clinical information of the patients. Mutational analysis was performed by Adila Khalil, Haq Nawaz Khan, and Muhammad Wasim. All authors have read the manuscript and Fazli Rabbi Awan finalized the manuscript for submission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by a research project, “Diagnosis of treatable inborn metabolic disorders of intellectual disability” [Project No. CRP/PAK14-02; Contract No. CRP/14/012] funded by the International Centre for Genetic Engineering and Biotechnology (ICGEB), Italy).