Department of Endocrinology, Diabetes and Metabolism, Christian Medical College, Ida Scudder Road, Vellore, Tamil Nadu, India.
Department of Paediatric Endocrinology, Christian Medical College, Ida Scudder Road, Vellore, Tamil Nadu, India.
Eur J Med Genet. 2021 Dec;64(12):104369. doi: 10.1016/j.ejmg.2021.104369. Epub 2021 Oct 27.
Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.
先天性肾上腺皮质增生症（CAH）的基因筛查由于 CYP21A2 基因分型的复杂性而具有挑战性，并且尚未成为常规临床实践中的一线诊断工具。此外，随着大规模平行测序技术的出现，需要研究其在 CAH 相关扩展基因panel 筛查中的应用。在这项研究中，我们建立并利用了一种基于等位基因特异性聚合酶链反应（ASPCR）的方法，对来自印度的 72 例临床诊断为 CAH 的患者的 CYP21A2 基因中的 8 个常见突变进行了筛查，然后对 CYP21A2、CYP11B1、CYP17A1、POR 和 CYP19A1 基因进行了靶向下一代测序（NGS）。通过这些研究，21 羟化酶缺乏症患者中有 88.7%的患者通过 ASPCR 对 8 个 CYP21A2 突变呈阳性。靶向 NGS 检测对 ASPCR 鉴定的所有突变均具有敏感性。在 ASPCR 阴性的患者中利用 NGS，有 5 例研究对象为其他 CYP21A2 变体的纯合阳性：1 例为 c.1274G>T 突变，3 例为 c.1451G>C 突变，1 例为 c.143A>G 突变。1 例患者通过 ASPCR 和 NGS 鉴定为 c.955C>T 和 c.1042G>A 变体的复合杂合子。1 例疑似简单性男性化（SV）21 羟化酶缺乏症的患者 CYP19A1:c.1142A>T 变异呈阳性。CYP11B1 变体（c.1201-1G>A、c.1200+1del、c.412C>T、c.1024C>T、c.1012dup、c.623G>A）在所有 6 例疑似 11β-羟化酶缺乏症的患者中均被鉴定出来。总体突变阳性率为 97.2%。我们的研究结果表明，ASPCR 后进行靶向 NGS 是一种经济有效的综合策略，可用于在临床环境中筛查常见的 CYP21A2 突变和 CAH 基因panel。
