Clinical Presentation And Mutational Spectrum In A Series Of 166 Patients With Classical 21-hydroxylase Deficiency From South China

Background:Classical 21-hydroxylase deficiency（21-OHD）,due to mutations in the cytochrome P450 family 21 subfamily A member 2（CYP21A2）gene,is the most common type of congenital adrenal hyperplasia（CAH）.The frequency of common micro-conversions of CYP21A2 gene varies greatly among different ethnic groups,and the disparity between genotype and phenotype is also reported in different races.At the same time,the location of the highly homologous inactivated pseudogene（CYPZIA1P）is quite close to the CYP21A2,conversions between these two genes happened frequently,which adds new complexities in detecting mutations in the CYP21A2 gene.There is almost no description of the mutation spectrum of the CYP21A2 gene in a large cohort in South China.Objective:In this retrospective study,the hormone level,molecular and bone age data obtained from 166 classical CAH patients due to 21OHD were analyzed,which will provide physicians with clinical experiences.We are aimed to discuss the effect of Sanger sequencing and multiplex ligation-dependent probe amplification（MLPA）method on detecting the mutations of CYP21A2 gene.We also want to explore the mutation spectrum of the CYP21A2 gene in a large cohort in South China,and to determine the correlation between genetype and phenotype,which will attribute to future prenatal disgnosis and genetic counselling.Methods:A total of 166 Chinese patients diagnosed as classical CAH due to 21-OHD according to clinical data,hormonal level and gene analysis,were recruited in Guangzhou Women and Children’s Medical Center from August 2014 to May 2017.We analyzed clinical and molecular data of these 166 patients with classical CAH.CYP21A2 gene mutations in these 99 salt wasting（SW）patients and 67 simple virilizing（SV）patients were detected.Patients with disease causing mutations were classified into four groups in order to analyze the genotype-phenotype concordance.Prenatal genetic diagnoses were conducted in six families with a history of CAH.Results:1.In our study,166 patients were diagnosed with classical CAH,among whom 99（59.6%）presented SW form and 67（40.4%）presented SV form.All patients,both SW and SV forms,had elevated levels of 170HP（>25 ng/mL）.In patients with SW form,elevated levels of AND（>35nmol/L）,low serum sodium levels（<135 mmol/L）and abnormal levels of T,COR,ACTH were also detected.Elevated serum potassium levels were discovered in most patients,except for 10 patients whose potassium levels appeared to be in normal range（3.5-5.5 mmol/L）.Patients with SV form had almost normal electrolyte levels,despite some had abnormal hormone levels.2.In the 332 alleles,the most frequent mutation was I2G（42.5%）,followed by large gene deletions or conversions（23.8%）,p.I173N（12.7%）,p.R357W（6.0%）,P.Q319X（5.1%）,p.GlllVfs*21（3.0%）,exon 6 I237N,V238E,M240 K cluster（E6）（0.9%）and c.923dupT（0.3%）.With respect to two forms in classic CAH,the rankings were a little bit different.Specifically,referring to the patients with SW form,more common mutations were I2G（42.9%）,large gene deletions or conversions（28.3%）,p.R357W（8.1%）.But for the SV form,more frequent mutations were I2G（41.8%）,p.I173N（29.1%）,large gene deletions or cornversions（17.2%）.Other rare mutations,accounting for 8.4%,among them,variants p.S126X,c.1209₁210insT,p.C429X,p.V70 L and c.840delG were not described in the other cohort,and could not be found ingnomAD,1000Genomes data and ExAC data.CYP21A2 gene duplications linked to the mutation Q319X were found in one patient.3.Most of the patients（74.7%）presented compound heterozygous genotype,while about a quarter（25.3%）were homozygous genotype.There were almost 24 CYP21A2 genotypes（only common mutations）in 166 CAH patients.4.Four novel variants p.S126X,p.C429X,c.1209₁210insT and c.840delG are expected to result in premature stop codons（PTCs）and hence they are likely pathogenic variants.5.Genotype agreed well with the phenotype,especially in group null（92.3%）which was expected to be SW form,and group B（91.7%）which was expected to be SV form.Yet in group A,only 60 out of 84 demonstrated the expected SW form,and the rest showed SV form,suggesting genotype-phenotype correlation disparities.6.In six families with a history of CAH,prenatal genetic diagnosis was carried out.Most fetuses were found to be heterozygous mutations carriers,except one normal individual.All babies were born healthy,and none of them were recalled by newborn screening.Conclusion:1.Micro-conversion mutation IVS2-13A/C>G（I2G）was the most frequent mutation in both SW form（42.9%）and SV form（41.8%）in our large cohort,and large gene deletion or large gene conversion also commonly resulted in classical CAH.2.Rare mutations only account for 8.4%of all alleles,among them four novel variants p.S126X,p.C429X,c.1209₁210insT and c.840delG were responsible for the clinical presentations.3.CYP21A2 gene duplications linked to the mutation Q319X were found in our cohort,though these cases were rather rare.4.Genotype and phenotype correlated well with each other in both group null and B,but the phenotypic divergence mainly existed in group A.5.Sanger sequencing combined with MLPA method could detect most mutation types in the CYP21A2 gene effectively.