A total of 22 unrelated Chinese CAH patients in Tianjin Children’s Hospital were recruited in this study from 2011 to 2019. The diagnoses of 22 patients were confirmed by molecular genetic testing. The classification of patients was based on a retrospective review of patients’clinical manifestations together withelectrolyte and hormonal levels. All patients (including 18 males and 4 females) came from 22 unrelated families, whose parents were unconsanguineous. The age of onset ranges from 4 hours to 53 days. Total 22 patients presented with classical forms and were all classified as SW form.Informed consent was obtained from all patients(or their parents) and in accordance with the Ethics Committee of Tianjin Children’s Hospital.
Locus-specific PCR and direct sequencing
Genomic DNA of patients and their parents were extracted from peripheral blood using Blood Genomic DNA Mini Kit (Cowin Bio, Beijing, China) according to the instruction. The volume of DNA is 100ul, concentration up to 10ng/uL above, stored at -20℃. Four primers were synthesized to amplify CYP21 genes listed in Table 1. The CYP21A2 gene forward and reverse primers were Pl and P2 respectively, and the pseudogeneCYP21A1Pspecific forward and reverseprimers were P3andP4. Amplicon of primers P3 and P2 was CYP21A1P/ CYP21A2 chimeric gene, and the amplicon of P1 and P4 was CYP21A2/ CYP21A1Prearrangement product. The procedure setting of PCR was described in . The amplicons were detected by 1.5% agarose gel electrophoresis to confirm the PCR quality and distinguish the gene deletion/conversion mutations. The DNA product was purified from agarose gel using Gel Extraction Kit (Cowin, Beijing, China) and sent to GENEWIZ company(Beijing, China) for Sanger sequencing.
Restriction endonuclease analysis
To ensure the locus-specificity of each reaction, the four amplification products above were performed by EcoRI enzyme based on the different cleavage sites of CYP21A2 and CYP21A1P genes. Restriction was carried out in the final volume of 10uL, containing 10×Buffer 1uL, PCR amplicon 6uL and EcoRI enzyme 0.5uL. Each product was digested for 2 hours at 37℃. Digestion products were separated by 1% agarose gel electrophoresis.
MLPA was performed using the SALSA MLPA probemix P050-C1 CAH kit (MRC-Holland, Amsterdam, The Netherlands) to detect large gene deletions and conversions. This P050 kit contains 37 probes, including 8 probes for CYP21A2 gene (exons 1, 3, 4, 6 and 7), 4 probes for CYP21A1P gene (exons 1, 3, 4 and 7), 6 probes for TNXB gene, 1 probe for ATF6B gene and 8 reference probes. The procedure was carried out according to the manufacturer’s instructions. Original volume of DNA was 5ul (100ng). The PCR products were detected using an ABI 3130 Genetic Analyzer (Applied Biosystems, USA) for capillary electrophoresis detection after multiplex PCR amplification reaction. The raw data was analyzed by using Coffalyser software (MRC Holland).
Classification of patients based on genotypes
The phenotypic categorization of 21-OHD was mainly based on the degree of decrease of 21-hydroxylase caused by different gene mutations. The enzyme of SW formwas completely inactive and that of SV form and NC form was partially and only slightly inactive respectively. Genotypes were grouped according to the strategy described by Wang et al. and Speiser et al. to predict phenotypes [15-16]. The patients were divided into five groups, including group Null, group A, group B, group C and group D. The group Null included patients carrying homozygous deletion, homozygous mutation or compound heterozygous mutation that could lead to completely inactive 21-hydroxylase. The group A was composed of the patients with homozygous IVS2-13A/C> G (I2G) mutation or a compound heterozygous mutation consisting of I2G and a Null group mutation. Patients with homozygous p.I173N mutation or heterozygous p.I173N combined with a mutation from group Null or group A composed the group B. Group C was consisted of patients harboring homozygous p.P31L and p.V282L mutations (resulting in remaining 20-60% enzymatic activity) or heterozygous state combined with a mutation from group Null, A or B. Lastly, patients carrying the uncertain significance mutations were categorized into group D. Genotypes of group Null and A were predicted to be associated with SW form. Genotypes in group B may be correlated with SV form, and that of group C may be related with NC form. However, phenotypes in group D could not be predicted.