Ravichandran Lavanya, Paul Shriti, Rekha A, Varghese Deny, Parthiban R, Asha H S, Mathai Sarah, Simon Anna, Danda Sumita, Thomas Nihal, Chapla Aaron
Department of Endocrinology, Diabetes and Metabolism, Christian Medical College, Vellore, India.
Department of Medical Genetics, Christian Medical College, Vellore, India.
Indian J Pediatr. 2024 Sep 20. doi: 10.1007/s12098-024-05249-0.
Whole exome sequencing (WES) has emerged as the preferred method for diagnosing a range of Mendelian disorders. Nonetheless, the applicability of WES in genetic diagnosis of 21-hydroxylase deficiency (21-OHD) remains uncertain due to the intricacies involved in molecular analysis of the CYP21A2 gene.
In this case series, authors report the outcomes of couples or families who underwent WES followed by focused sequential strategy (FSS) targeting CYP21A2 gene hotspot mutations and targeted sequencing of genes associated with Congenital Adrenal Hyperplasia (CAH).
This analysis revealed that WES, when compared to FSS, resulted in six false-negative findings out of seven subjects and one false-positive result. These results were corroborated through validation using Multiplex Ligation-Dependent Probe Amplification (MLPA) and Sanger sequencing.
One major limitation of exome sequencing lies in target enrichment, which often achieves less than 95% coverage of the regions of interest, potentially leading to false negatives. This challenge is particularly pronounced when deciphering the complex genetics of 21-OHD, characterized by intricate pseudogene-derived rearrangements and gene conversions. Additionally, next-generation sequencing (NGS) analysis of the CYP21A2 gene is not straightforward due to reads aligning to pseudogene regions, necessitating stringent computational pipelines with defined targets. However, simple genotyping assays have shown a high positive yield of pseudogene-derived mutations in over 80% of cases, while targeted NGS can be valuable in subjects with initially negative results. Therefore, WES is not recommended as the primary testing method for 21-OHD and may be better suited for rare forms of CAH once CYP21A2 mutations have been ruled out.
全外显子组测序（WES）已成为诊断一系列孟德尔疾病的首选方法。然而，由于CYP21A2基因分子分析涉及的复杂性，WES在21-羟化酶缺乏症（21-OHD）基因诊断中的适用性仍不确定。
在本病例系列中，作者报告了接受WES检测，随后采用针对CYP21A2基因热点突变的聚焦序贯策略（FSS）以及先天性肾上腺皮质增生症（CAH）相关基因靶向测序的夫妇或家庭的检测结果。
该分析显示，与FSS相比，WES在7名受试者中产生了6例假阴性结果和1例假阳性结果。通过多重连接依赖探针扩增（MLPA）和桑格测序进行验证，证实了这些结果。
外显子组测序的一个主要局限性在于目标富集，其通常无法实现对感兴趣区域95%以上的覆盖，可能导致假阴性结果。在解读21-OHD复杂的遗传学特征（其具有复杂的假基因衍生重排和基因转换）时，这一挑战尤为突出。此外，由于读取序列与假基因区域比对，CYP21A2基因的下一代测序（NGS）分析并不简单，需要严格的具有明确目标的计算流程。然而，简单的基因分型检测在超过80%的病例中显示出假基因衍生突变的高阳性检出率，而靶向NGS对初始结果为阴性的受试者可能有价值。因此，不建议将WES作为21-OHD的主要检测方法，一旦排除CYP21A2突变，WES可能更适合罕见形式的CAH。
