Genetic Analysis And Prenatal Diagnosis Of Congenital Adrenal Hyperplasia Due To 21-hydroxylase Deficiency

Congenital adrenal hyperplasia(CAH)is an autosomal recessive disorder.About 90-95% of all CAH cases are due to 21-hydroxylase deficiency(21OHD).The 21-hydroxylase enzyme is encoded by the CYP21A2 gene which is adjacent to a pseudogene,CYP21A1 P.The CYP21A2 gene shares with its pseudogene CYP21A1 P 98% identical coding sequences and 96% identical intron sequences.To date,a great number of different mutations in the CYP21A2 gene have been found.First of all,we established a reliable and rational method aiming to detect mutations in the CYP21A2 gene,including multiplex ligation-dependent probe amplification(MLPA)for large deletions,locus-specific polymerase chain reaction(PCR)/restriction endonuclease analysis for conversion mutations and Sanger sequencing of entire CYP21A2 gene for point mutations.Using this method,we investigated 105 patients with clinically diagnosed or suspected 21 OHD.95 patients with CYP21A2 gene mutations were confirmed the diagnosis of 21 OHD,and 10 without the detection of CYP21A2 gene mutations were excluded.We characterized 190 CYP21A2 alleles from 95 patients with gene mutations that 51 alleles(26.84%)carried large gene deletions and conversions and 139 alleles(73.16%)carried point mutations.All of the mutations were inherited from parents after genotyping 86 parents,excepted for 9 parents unavailable.Our results show that the stepwise genetic testing can identify mutations in the CYP21A2 gene and can be used to help confirm the diagnosis of 21 OHD.Secondly,we performed CYP21A2 gene mutation analysis and prenatal diagnosis in families affected with CAH due to 21 OHD for genetic counseling and postnatal interventions.Using the same method as mentioned above,we analyzed CYP21A2 gene mutations in 25 families with CAH,in which 15 with probands and 10 without.After we successfully characterized 50 pathogenic alleles of CYP21A2 in 25 pedigrees,we performed prenatal diagnosis for 28 fetuses at risk of 21 OHD in these families.As a result,20 fetuses were unaffected with 21-OHD and 8 fetuses were affected.These results suggest that it was feasible to perform the method on our patients and carriers for CYP21A2 gene mutation analysis and implement prenatally,which can provide appropriate genetic counseling for these families and postnatal interventions in time.Finally,we developed an approach using capture sequencing in maternal plasma to noninvasive prenatal diagnose(NIPD)CAH due to 21-OHD.14 plasma samples were collected from 12 families,including four plasma sample collected during the first trimester.Targeted capture sequencing was performed using genomic DNA from the parents and child trios to determine the pathogenic and wild-type alleles associated with the haplotypes.Maternal plasma DNA was also sequenced to determine the fetal inherited alleles using hidden Markov model-based haplotype linkage analysis.The effect of fetal DNA fraction and sequencing depth on the accuracy of NIPD was investigated.The lower limit of fetal DNA fraction was 2% and the threshold mean sequence depth was 38,suggesting potential advantage if used in early gestation.The CYP21A2 genotype of the fetuses were accurately determined in all the 14 plasma samples as early as day 1 and 8 weeks of gestation.The results suggest the accuracy and feasibility of NIPD of 21-OHD using a small target capture region with a low threshold for fetal DNA fraction and sequence depth.Our NIPD method is cost-effective and could be applicable in clinical practice.