Comprehensive and Cost-Effective Strategy for Genetic Screening of Congenital Adrenal Hyperplasia (CAH) in India

Background: With substantial challenges in molecular analysis of 21 hydroxylase deficiency and lack of studies in extended panel of genes implicated in CAH, genetic diagnosis is largely unavailable and unaffordable in India. Therefore, we aim to develop a cost-effective screening strategy in CAH using Allele Specific PCR and Targeted Next-Generation Sequencing (NGS). Methods: Long range PCR and restriction digestion were utilized to specifically amplify the CYP21A2 gene whereas multiplex PCR was used to amplify CYP11B1, CYP17A1, CYP19A1 and POR genes. In house developed Allele Specific PCR (ASPCR) for 8 hotspot mutations in CYP21A2 gene and targeted NGS for five genes was carried out. The results were validated using Sanger sequencing and MLPA. Results: Of the 50 patients suspected for 21 hydroxylase deficiency, 64% (n=32) were of Salt Wasting phenotype (SW), 30% (n=15) with Simple Virilizing (SV) phenotype and 6% (n=3) of the study population were suspected for non-classical (NC) CAH. The mutation positive rate of ASPCR was 86% (n=43). Seven patients carried more than two biallelic mutations indicating smaller gene conversions. The predominant mutation identified among the study subjects was I2G splice variant in SW phenotype (38%) and I172N in the SV phenotype (41%). Based on the Long range PCR amplification and restriction digestion we identified one patient with large gene conversion and one patient with large 30kb deletion. These results were confirmed with MLPA. Additionally, utilizing the Targeted NGS we identified five patients with CYP21A2 variants (two patients with novel variants c.1274G>T, c.17_18delTG and two other variants in three patients - c.1451G>C, c.143A>G). We also identified a CYP19A1:c.1142A>T gene variant in a patient who was initially suspected for 21 hydroxylase deficiency. Out of six patients with 11 beta hydroxylase deficiency four patients were positive for homozygous CYP11B1 variants (c.1201-1G>A, c.1200 + 1delG, c.412C>T) and two patients with compound heterozygous variants (c.1024C>T and c.1012dupC, c.623G>A and c.412C>T). Discussion: Utilizing the novel allele specific PCR followed by NGS we identified a total of 96% (48/50) of 21 hydroxylase deficiency patients with homozygous or compound heterozygous mutations and 2% (2/50) were positive for single heterozygous variant in CYP21A2 gene. ASPCR followed by multigene targeted NGS assay for genetic screening in CAH has shown to be a sensitive and specific strategy established in a clinical setting. To best of our knowledge this is the most cost-effective and comprehensive multigene screening carried out in India.