Analysis Of Gene Mutation Hotspots In 21-hydroxylase Deficiency Congenital Adrenal Hyperplasia In Yunnan

Objective(s): 21-hydroxylase deficiency(21-OHD)is a rare autosomal recessive monogenic disorder with significant ethnic and regional differences in frequency.Genetic diagnosis is essential for disease typing and precise treatment of 21-OHD patients,and there are differences in the hotspots of CYP21A2 mutations carried by different regional populations.In this study,72 patients with 21-OHD and some family members in Yunnan were collected for genetic testing,with the aim of:1.Mapping of hot-spot genetic mutation in the 21-OHD population in Yunnan.2.To screen the population for new mutations that have not yet been reported.3.To analyze whether there are blind areas in the current genetic diagnosis strategy for 21-OHD patients in Yunnan,and to optimize the design of new assays to screen for gene mutations in such blind areas.Methods: Peripheral blood samples from patients with 21-OHD and their immediate family members who attended the First People’s Hospital of Yunnan Province from 2017 to 2022 and agreed to undergo genetic testing were collected and screened for gene mutations using agarose gel electrophoresis,Sanger sequencing,MLPA detection,construction of allelic monoclonal vectors,and long-read sequencing after DNA extraction,PCR-specific amplification,and statistical analysis.Results:1.23 patients with 21-OHD were included in the study,and after genetic testing,20 patients carried pathogenic mutations,with a confirmed positive rate of 86.96%.2 patients had no pathogenic mutations detected on CYP21A2,and 1 patient had a mutation of undetermined clinical significance;46 patients were carriers of pathogenic genes in the non-patient population.The number of alleles carrying pathogenic or suspected pathogenic mutations detected in 72 patients and their immediate family members was 128,the mutation detection rate was 88.89%(128/144),34 mutations were detected,the frequency of genes with microconversion was41.67%(52.17% in patients and 45.00% in carriers),the frequency of genes with The frequency of genes with large fragment deletions/conversions was 25.68%(36.96% in patients and 21.25%in carriers),and the frequency of genes with other point mutations was 18.75%(26.09% in patients and 18.75% in carriers).The most frequent mutations in the examined population were c.293-13A/C>G(10.24%)(15.22% in patients and 10.00% in carriers),c.518T>A(8.33%)(10.87% in patients and 8.75% in carriers),and large fragment deletions and pathogenic fusion genes.3.The population examined in this study carried six pathogenic CYP21A1P/CYP21A2 fusion genes,the most common being the classic chimeras CH-5(4.86%)(gene frequency of 8.70% in patients and 3.75% in carriers),CH-6(3.47%)(gene frequency of 2.17% in patients and 5.00%),CH-1(3.47%)(6.52% gene frequency in patients and 2.50% in carriers).There was one case of21-OHD in a patient(gene frequency of 2.17%)and one case in a carrier(gene frequency of1.25%)carrying attenuated chimeric CH-4(1.39%).4.The assay revealed multiple genetic recombinations or conversions of CYP21A1 P to CYP21A2 carried on a single allele in patients and carriers,with 18.75% of patients and 52.21%of carriers having 2 or more genetic recombinations or conversions of the allele.5.Two de novo mutations were detected.c.592G>A(0.69%),predicted as benign mutations by Poly Phen-2,FATHMM,Mutation Taster;ACMG rating was benign(BS1,BS2,BP4,BP5).c.472C>T(0.69%),predicted as probable by Poly Phen-2 pathogenic mutation,FATHMM,Mutation Taster predicted as benign mutation;ACMG rating was clinical significance unclear(VUS).6.The detection efficiency of gene mutations by different detection methods was different.In this study,the detection efficiency of agarose gel electrophoresis was 17.65%,that of Sanger sequencing was 38.24%,that of MLPA assay was 35.29%,and that of constructing allelic monoclonal vectors and long-read sequencing were 100%.Conclusion:1.The most common pathogenic 21-OHD gene mutation in the Yunnan population in this study were c>293-13A/C>G,c>518T>A,and large fragment deletions and pathogenic gene fusions,in that order.2.The most common pathogenic CYP21A1P/CYP21A2 fusion gene type in the examined population was CH-5,whose clinical phenotype was associated with 21-OHD of SW type.3.There are multiple genetic recombination/conversion events including benign and pathogenic transitions on a single allele in the examined population,with potential pathogenic risk.4.The novel mutation c.592G>A in this study has a higher probability of being a benign mutation;the c.472C>T mutation is of clinical significance is not clear yet.5.The currently used genetic diagnosis strategies of specific primer amplification electrophoresis and Sanger sequencing combined with MLPA detection cannot detect structural mutations in cis or trans caused by gene recombination,nor can they detect mutations masked by gene homology.In contrast,the construction of allelic monoclonal vectors and long-read sequencing can simultaneously obtain information on single nucleotide variants,gene structure variants,gene copy number variants and gene haplotypes of the tested genes,which have high detection rates and accurate results for gene mutations,and therefore these two detection methods have great potential for scientific and clinical applications.6.In view of the high frequency of deletion/conversion of large segments of genes in the tested population,the genetic screening method for patients and carriers of 21-OHD in Yunnan should be selected by constructing allelic monoclonal vectors or long-read sequencing.