Abstract：
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders predominantly characterized by impaired corticosteroid synthesis. Clinical phenotypes include hypoadrenocorticism, electrolyte disturbances, abnormal gonadal development, and short stature, of which severe hyponadrenocorticism and salt wasting can be life-threatening. Genetic analysis can help in the clinical diagnosis of CAH. However, the 21-OHD-causing gene CYP21A2 is arranged in tandem with the highly homologous CYP21A1P pseudogene, making it difficult to determine the exact genotypes using the traditional method of multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing or next-generation sequencing (NGS). We applied a long-read sequencing-based approach termed comprehensive analysis of CAH (CACAH) to 48 newborns with CAH that were diagnosed by clinical features and the traditional MLPA plus Sanger sequencing method for retrospective analysis, to evaluate its efficacy in the clinical diagnosis of neonatal CAH. Compared with the MLPA plus Sanger sequencing method, CACAH showed 100 % consistency in detecting SNV/indel variants located in exons and exon-intron boundary regions of CAH-related genes. It can directly determine the cis-trans relationship without the need to analyze parental genotypes, which reduces the time to diagnosis. Moreover, CACAH was able to distinguish different CYP21A1P/CYP21A2 and TNXA/TNXB chimeras, and detect additional variants (CYP21A2 variants c.-121C > T, c.*13G > A, c.*52C > T, c.*440C > T, c.*443 T > C, and TNXB variants c.12463 + 2 T > C, c.12204 + 5G > A). We also identified the TNXB variant c.11435_11524 + 30del alone instead of as a part of the TNXA/TNXB-CH-1 chimera in two newborns, which might be introduced by gene conversion. All of these characteristics enabled clinicians to better explain the phenotype of subjects and manage them more effectively. CACAH has a great advantage over the traditional MLPA and Sanger sequencing methods, showing substantial potential in the genetic diagnosis and screening of neonatal CAH.
摘要：
先天性肾上腺增生（CAH）是一组常染色体隐性遗传疾病，主要特征是皮质类固醇合成受损。临床表型包括肾上腺皮质功能减退，电解质干扰，性腺发育异常，身材矮小，其中严重的轻度肾上腺皮质和盐浪费可能危及生命。遗传分析有助于CAH的临床诊断。然而,引起21-OHD的基因CYP21A2与高度同源的CYP21A1P假基因串联排列，这使得使用多重连接依赖性探针扩增（MLPA）加Sanger测序或下一代测序（NGS）的传统方法难以确定确切的基因型。我们将基于长读数测序的方法称为CAH综合分析（CACAH），对48例通过临床特征诊断为CAH的新生儿和传统的MLPA加Sanger测序方法进行回顾性分析。评价其在新生儿CAH临床诊断中的疗效。与MLPA加Sanger测序法相比，CACAH在检测位于CAH相关基因的外显子和外显子-内含子边界区域的SNV/indel变体方面表现出100％的一致性。它可以直接确定顺反关系，而无需分析亲本基因型，缩短了诊断时间.此外，CACAH能够区分不同的CYP21A1P/CYP21A2和TNXA/TNXB嵌合体，并检测其他变体（CYP21A2变体c.-121C>T，c.*13G>A，c.*52C>T，c.*440C>T,c.*443T>C，和TNXB变体c.12463+2T>C，c.12204+5G>A）。我们还在两个新生儿中单独鉴定了TNXB变体c.11435_11524+30del，而不是作为TNXA/TNXB-CH-1嵌合体的一部分，这可能是通过基因转换引入的。所有这些特征使临床医生能够更好地解释受试者的表型并更有效地管理它们。与传统的MLPA和Sanger测序方法相比，CACAH具有很大的优势，在新生儿CAH的基因诊断和筛查中显示出巨大的潜力。
