Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is autosomal recessive disorder of cortisol biosynthesis. Genetic defects in CYP21A2 cause 21OHD. The aim of this study was to determine spectrum of mutations in CYP21A2 in a large cohort and analyze the genotype-phenotype correlation to assess predictive characteristics of genotype. We investigated a total of 113 patients with 21OHD. Next-generation sequencing and Multiplex ligation-dependent probe amplification of the CYP21A2 gene were performed in patients and their parents. The genotypes were categorized into Groups 0, A, B, and C according to the residual 21-hydroxylase activities. In this study, the group A was divided into two subgroups as A1 and A2. Three novel variants were found. The genotype–phenotype correlation of the mutation classification was 91.5%. Positive predictivity of subgroups A1 was higher than groups A and subgroups A2. Our study reports genotype–phenotype correlations in the largest 21OHD cohort in Turkey. This correlation sustained when we analyzed our data in combination with metadata from other published studies. This study confirms that CYP21A2 genotyping with next-generation sequencing and MLPA can accurately and reliably confirm the diagnosis of 21OHD. We propose a new classification by dividing group A into two new subgroups to better predict the phenotype. In light of this very high genotype-phenotype correlation, with their ever-increasing availability, declining cost, and turnaround time, we propose that molecular genetic studies can be more economical and practical alternative to the current initial diagnostic laboratory studies based on assays of intermediary steroid metabolites.

21-羟化酶缺乏症（21OHD）引起的先天性肾上腺增生（CAH）是皮质醇生物合成的常染色体隐性遗传疾病。CYP21A2的遗传缺陷导致21OHD。这项研究的目的是确定一个大型队列中CYP21A2的突变谱，并分析基因型与表型的相关性，以评估基因型的预测特征。我们共调查了113例21OHD患者。CYP21A2的下一代测序和多重连接依赖性探针扩增基因在患者及其父母中进行。根据残留的21-羟化酶活性，将基因型分为0，A，B和C组。在这项研究中，A组被分为两个子组，即A1和A2。发现了三个新颖的变体。突变分类的基因型-表型相关性为91.5％。A1组的阳性预测率高于A组和A2组。我们的研究报告了土耳其最大的21OHD队列中基因型与表型的相关性。当我们结合其他已发表研究的元数据分析数据时，这种相关性得以维持。这项研究证实了CYP21A2下一代测序和MLPA进行基因分型可以准确可靠地确认21OHD的诊断。我们通过将A组分成两个新的亚组来提出新的分类，以更好地预测表型。鉴于这种非常高的基因型与表型相关性，以及它们的可用性不断提高，成本下降和周转时间长，我们建议分子遗传学研究可以替代当前的基于中间物测定的初始诊断实验室研究，更加经济实用类固醇代谢物。