Clinical Analysis And Genotype-phenotype Study Of 21-hydroxylase Deficiency

ObjectiveCongenital adrenal hyperplasia due to 21-hydroxylase deficiency(21-OHD)is one of the most common autosomal recessive disorders,and accounts for 90%-95%of all congenital adrenal hyperplasia cases.Clinically,phenotypical expression of21-OHD is highly variable,ranging from severe or classical,to mild late onset or non-classical.Classical 21-OHD includes the salt-wasting(SW)and simple virilizing(SV)forms.Currently,the clinical diagnosis of 21-OHD,prenatal testing and neonatal screening are mainly dependent on biochemical tests.However,due to a number of factors,these biochemical markers have limitations in the diagnosis of21-OHD,often leading to misdiagnosis.The pathogenic gene of 21-OHD is the CYP21A2 gene,which encoding 21-hydroxylase.Genetic testing can better confirm the clinical diagnosis.More than 200 disease-causing mutations of CYP21A2 have been known to date.A good correlation is most often observed between mutations and the clinical and biological phenotype of the patients.However,some discrepancies have been reported.In this study,11 children with 21-OHD from Tianjin and surrounding areas were enrolled in the clinical and genetic studies,and 10 parents of 5 children were also tested for genetic testing.The aim of this study was:1.To improve the insight and diagnosis of 21-OHD of clinicians by analyze the clinical characteristics,related biochemical markers,and auxiliary examination results of children with 21-OHD.2.To investigate the mutational spectrum of CYP21A2 gene mutation in children with 21-OHD in Tianjin and surrounding areas.3.To investigate the correlation between genotype and phenotype in eleven 21-OHD patients,providing a reliable basis for clinical diagnosis,genetic counseling and prenatal diagnosis.Methods1.Clinical and biochemical data of 11 unrelated 21-OHD patients from May 2013 to May 2018 in Tianjin Children’s Hospital were retrospective analysis.2.Peripheral blood of 11 patients with 21-OHD and 10 parents were collected.Genomic DNA was extracted from the blood samples.Locus-specific PCR,direct sequencing of PCR amplification products,and multiplex ligation-dependent probe amplification were applied to detected pathogenic CYP21A2 gene.3.According to the CYP21A2 genotype,11 patients with 21-OHD were divided into different groups for clinical phenotypic prediction.The relationship between genotype and phenotype was analyzed by comparing the actual phenotypes.Results1.Of eleven 21-OHD proband,6 were classified as SW phenotype and 5 were SV phenotype.Diarrhea,vomiting,and no weight gain were the most common symptoms in patients with SW 21-OHD,and the median age of presentation was33 days.Abnormal genital dysplasia was the most common symptoms in patients with SV 21-OHD,and the median age of diagnostic was 5 years old.Of the 11 patients,8 had pigmentation.Five female patients had genital ambiguities,the sex of one was misclassified and identified as female by chromosome karyotype test.During the follow-up period,4 patients with SW 21-OHD were admitted to hospital for adrenal crisis due to infection.2.Related examination results of 21-OHD patients: 11 patients were tested for serum 17-Hydroxyprogesterone(17-OHP),adrenocorticotropic hormone(ACTH)and cortisol.All patients had elevated levels of 17-OHP and ACTH,and 5 patients had reduced cortisol levels.Six children with SW 21-OHD had hyponatremia and hyperkalemia.Ten patients underwent adrenal ultrasonography or computer tomography examination,and 7 of them had unilateral or bilateral adrenal hyperplasia.Four patients with SV 21-OHD underwent X-ray examination of bone age.The results showed that the bone ages were earlier than the actual ages.3.Twenty-two mutant alleles were detected in a total of 22 alleles in 11 patients,with the mutation detection rate 100%.Six types of mutations were detected in 11 children,including three point mutations(c.293-13C/A>G,c.518T>A,c.1069C>T)and three large fragment deletions(Exon 1,3,4,6,7 deletion,Exon4,6,7 deletion,Exon 1,3,4 deletion),the most common mutation is c.293-13C/A>G(59.1%),followed by c.518T>A(22.7%).The most common mutation in patients with SW was c.293-13C/A>G(83.3%),and the most common mutation in patients with SV was c.518T>A(50%).4.The positive predictive value(PPV)of genotype and clinical phenotype in SW and SV were 85.7% and 100%,respectively.The total PPV of 11 patients was90.9%.Conclusions1.The classic 21-OHD was more common than the non-classical ones in childhood.The patients with SW 21-OHD had earlier onset than that with SV 21-OHD.Diarrhea,vomiting,and no weight gain were the most common symptoms in patients with SW 21-OHD.Abnormal genital dysplasia was the most common symptoms in patients with SV 21-OHD.Most children have pigmentation.A diagnosis can be made in combination with clinical manifestations and biochemical markers such as elevated 17-OHP and ACTH.Patients with SW21-OHD should prevent infection.2.A total of 6 types of mutations were detected,including c.293-13C/A>G,c.518T>A,c.1069C>T and 3 different large fragment deletions.The most common mutation in Tianjin and its surrounding areas was c.293-13C/A>G,followed by c.518T>A.3.There was a significant correlation between genotype and phenotype in this study.The PPV of genotype and clinical phenotype in SW and SV were 85.7% and100%,respectively.The total PPV of 11 patients was 90.9%.