Lan Tian, Wang Jin, Chen Kaibi, Zhang Jianru, Chen Xiaohong, Yao Hui
Department of Genetics, Metabolism and Endocrinology, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
GrandOmics Biosciences Co, Ltd., Beijing, China.
Front Genet. 2024 Nov 1;15:1472516. doi: 10.3389/fgene.2024.1472516. eCollection 2024.
21-Hydroxylase deficiency (21-OHD) is caused by mutations in the gene. Due to the complex structure and the high genetic heterogeneity of the gene, genetic testing for 21-OHD is currently facing challenges. Moreover, there are no comparative studies on detecting mutations by both second-generation sequencing and long-read sequencing (LRS, also known as third-generation sequencing).
To detect variations in 21-OHD patients using targeted capture with LRS method based on the PacBio (Pacific Biosciences) Sequel II platform.
A total of 67 patients with 21-OHD were admitted in Wuhan Children's Hospital. The full sequence of CYP21A2 gene was analyzed by targeted capture combined with LRS based on the PacBio Sequel II platform. The results were compared with those of long-polymerase chain reaction (Long-PCR) combined with multiplex ligation probe amplification (MLPA) detection. Based on the study of 21-hydroxylase activity of common mutations, the patient genotypes were divided into groups of Null, A, B, and C, from severe to mild. The correlation between different genotype groups and clinical typing was observed.
The study analyzed a total of 67 patients. Among them, 44 (65.67%) were males and 23 (34.33%) were females, with a male-to-female ratio of approximately 1.9:1. A total of 27 pathogenic variants were identified in the 67 patients, of which micro-conversion accounted for 61.9%, new variants of accounted for 8.2%; deletion accounted for 22.4% ( single deletion and chimeric / accounted for 12.7%, chimeric / accounted for 9.7%); and duplication accounted for 3.0% ( Gene Duplication). I2G was the most common variant (26.9%). Targeted capture LRS and MLPA combined with Long-PCR detection of mutations showed 30 detection results with differences. The overall genotype-phenotype correlation was 82.1%. The positive predictive rate of the Null group for salt wasting (SW) type was 84.6%, the A group for SW type was 88.9%, the group B for simple virilization (SV) type was 82.4%, and the group C for SV type was 62.5%. The correlation coefficient r between the severity of the phenotype and the genotype group was 0.682 ( < 0.05).
Targeted capture combined with LRS is an integrated approach for detecting mutations, allowing precise determination of connected sites for multiple deletions/insertions and cis/trans configurations without analyzing parental genomic samples. The overall genotype-phenotype correlation for 21-OHD is generally strong, with higher associations observed between genotype and phenotype for group Null, A, and B mutations, and larger genotype-phenotype variation in group C mutations. Targeted capture with LRS sequencing offers a new method for genetic diagnosis in 21-OHD patients.
21-羟化酶缺乏症（21-OHD）由该基因的突变引起。由于该基因结构复杂且遗传异质性高，目前21-OHD的基因检测面临挑战。此外，关于通过二代测序和长读长测序（LRS，也称为三代测序）检测该基因突变的比较研究尚无。
基于PacBio（太平洋生物科学公司）Sequel II平台，采用LRS靶向捕获方法检测21-OHD患者的相关变异。
共纳入67例21-OHD患者于武汉儿童医院。基于PacBio Sequel II平台，采用靶向捕获联合LRS分析CYP21A2基因全序列。将结果与长链聚合酶链反应（Long-PCR）联合多重连接探针扩增（MLPA）检测结果进行比较。基于对常见突变的21-羟化酶活性研究，将患者基因型分为无效型、A、B和C组，从严重到轻度。观察不同基因型组与临床分型之间的相关性。
本研究共分析67例患者。其中，男性44例（65.67%），女性23例（34.33%），男女比例约为1.9:1。67例患者中共鉴定出27个致病变异，其中微转换占61.9%，该基因新变异占8.2%；缺失占22.4%（单个缺失和嵌合体/占12.7%，嵌合体/占9.7%）；重复占3.0%（该基因重复）。I2G是最常见的变异（26.9%）。靶向捕获LRS与MLPA联合Long-PCR检测该基因突变显示有30个检测结果存在差异。总体基因型-表型相关性为82.1%。无效型组对失盐型（SW）的阳性预测率为84.6%，A组对SW型为88.9%，B组对单纯男性化型（SV）为82.4%，C组对SV型为62.5%。表型严重程度与基因型组之间的相关系数r为0.682（P<0.05）。
靶向捕获联合LRS是检测该基因突变的一种综合方法，无需分析亲代基因组样本即可精确确定多个缺失/插入的连接位点和顺式/反式构型。21-OHD的总体基因型-表型相关性一般较强，无效型、A和B组突变的基因型与表型之间关联较高，C组突变的基因型-表型变异较大。LRS测序靶向捕获为21-OHD患者的基因诊断提供了一种新方法。
