Suwanlikit Yossawat, Panthan Bhakbhoom, Chitayanan Pawares, Klumsathian Sommon, Charoenyingwattana Angkana, Chantratita Wasun, Trachoo Objoon
Undergraduate Program in Doctor of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samut Prakarn, 10540, Thailand.
BMC Med Genomics. 2025 Jan 24;18(1):18. doi: 10.1186/s12920-025-02089-5.
Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD CAH) is an autosomal recessive disorder resulting from pathogenic variants in the CYP21A2 gene. The disorder exhibits variable clinical severity, with the classical form manifesting as salt-wasting crisis in neonates, while inducing ambiguous genitalia in females and precocious puberty in males through simple virilization. Identifying at-risk couples during the preconception stage holds significance for optimizing reproductive choices.
This study included 204 unrelated preconception individuals undergoing carrier screening. A robust molecular approach was devised for rapid detection of nine prevalent CYP21A2 pathogenic variants, utilizing Amplification-Refractory Mutation System (ARMS) PCR and mass spectrometry (MS) genotyping. Complementary quantitative real-time PCR (qPCR) and PCR-based Restriction Fragment Length Polymorphism (PCR-based RFLP) assays were employed for comprehensive gene deletion analysis. The concordance of pathogenic variant detection between ARMS-PCR and MS, as well as the consistency observed in molecular insights from qPCR and PCR-based RFLP, fortified the accuracy of our methodologies.
Our combined method could detect common pathogenic variants and large gene deletions with high concordance between ARMS-PCR, MS genotyping, qPCR, and PCR-based RFLP assays. Remarkably, two carriers exhibited significant large-scale deletions, while another manifested a carrier state due to minor-scale gene conversion. The estimated carrier frequency in our cohort using these methods was approximately 1 in 65 individuals.
The methods used for 21-OHD CAH carrier screening offer a reliable, swift, and cost-effective approach for detecting common pathogenic variants and large deletions. Despite some limitations, such as the inability to detect all rare mutations, the techniques provide a practical solution for carrier screening, with an estimated carrier frequency of 1 in 65 in our study population. These findings support the potential adoption of these methods in national carrier screening programs, offering a practical balance between efficiency and affordability.
21-羟化酶缺乏所致先天性肾上腺皮质增生症（21-OHD CAH）是一种常染色体隐性疾病，由CYP21A2基因的致病性变异引起。该疾病临床严重程度各异，典型形式表现为新生儿失盐危象，同时通过单纯性男性化使女性出现生殖器模糊，男性出现性早熟。在孕前阶段识别高危夫妇对于优化生育选择具有重要意义。
本研究纳入204名接受携带者筛查的无亲缘关系的孕前个体。设计了一种强大的分子方法，利用扩增阻滞突变系统（ARMS）PCR和质谱（MS）基因分型快速检测9种常见的CYP21A2致病性变异。采用互补的定量实时PCR（qPCR）和基于PCR的限制性片段长度多态性分析（PCR-RFLP）进行全面的基因缺失分析。ARMS-PCR与MS之间致病性变异检测的一致性，以及qPCR和PCR-RFLP在分子见解上的一致性，增强了我们方法的准确性。
我们的联合方法能够检测常见致病性变异和大片段基因缺失，ARMS-PCR、MS基因分型、qPCR和PCR-RFLP分析之间具有高度一致性。值得注意的是，两名携带者表现出显著的大规模缺失，而另一名由于小规模基因转换表现为携带者状态。使用这些方法估计我们队列中的携带者频率约为每65人中1人。
用于21-OHD CAH携带者筛查的方法为检测常见致病性变异和大片段缺失提供了一种可靠、快速且经济高效的途径。尽管存在一些局限性，如无法检测所有罕见突变，但这些技术为携带者筛查提供了切实可行的解决方案，在我们的研究人群中估计携带者频率为每65人中1人。这些发现支持在国家携带者筛查项目中采用这些方法，在效率和可承受性之间实现了实际的平衡。
