DOI: https://doi.org/10.14710/jbtr.v12i1.30107

Rapid PCR-RFLP Screening of CYP21A2 Variants: Bridging Molecular Diagnostics in a Low-Resource Setting

1Doctor of Medicine and Health Study Program, Faculty of Medicine, Universitas Diponegoro, Indonesia

2Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Diponegoro, Indonesia

3Center for Biomedical Research (CEBIOR), Faculty of Medicine, Universitas Diponegoro, Indonesia

4 Department of Pediatrics, Division of Pediatric Endocrinology, Faculty of Medicine Universitas Diponegoro, Indonesia

5 Center for Biomedical Research, Faculty of Medicine Universitas Diponegoro, Indonesia

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Received: 18 Nov 2025; Revised: 16 Jan 2026; Accepted: 13 Feb 2026; Available online: 29 Apr 2026; Published: 30 Apr 2026.
Open Access Copyright (c) 2026 Journal of Biomedicine and Translational Research
Creative Commons License This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

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Abstract

Background: The 21-hydroxylase deficiency (21OHD) accounted for the majority of autosomal recessive inherited congenital adrenal hyperplasia (CAH) cases. A genetic test could offer a more thorough diagnostic approach because of the wide range of disease’s severity, but it was challenging in a resource-limited setting.

Objective: To apply a simple and cost-effective rapid molecular screening for detecting the most common CYP21A2 variants causing salt-wasting (SW) CAH.

Methods: DNA extraction was conducted using a salting out method from a venous blood sample taken from 49 CAH patients without prior genetic testing. A PCR-RFLP approach was utilized using primer sets specifically designed to anneal to the CYP21A2. Specific restriction enzymes were selected to cleave the DNA sequence, differentiating the wild and mutant type, i.e, the p.Arg357Trp, p.Gln318Ter, IVS2-13A/C>G, and exon 6 cluster. Samples positive for those mutation types detected from DNA sequencing, used as positive controls.

Results: The most common variant was IVS2-13A/C>G, detected in 26 out of 27 patients. Only 6.1% of 49 patients were found to have the p.Gln318Ter variant. Both the p.Arg357Trp and exon 6 cluster carried inconclusive results. All patients detected with the IVS2-13A/C>G and p.Gln318Ter had a phenotype of SW CAH.

Conclusion: The PCR-RFLP was an advantageous method for identifying CYP21A2 variants in a low-resource setting. Nevertheless, the use of multiplex ligation-probe dependent amplification (MLPA) and Sanger sequencing offered a comprehensive analysis to discover novel variants that could help with patients' diagnosis and treatment.

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Keywords: Congenital adrenal hyperplasia; CYP21A2; PCR; RFLP; developing country
Funding: Program Magister menuju Doktor untuk Sarjana Unggul (PMDSU) Ministry of Education and Culture Republic Indonesia scholarship program (No: 601-81/UN7.D2/PP/VI/2024)

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