A Chinese Young Man With 11?-hydroxylase Deficiency Due To Compound Heterozygosity In CYP11B1 Gene

Background:Congenital adrenal hyperplasia(CAH)is an autosomal recessive disorder caused by defects in various enzymes responsible for the biosynthesis of cortisol and aldosterone in the adrenal glands.Patients with CAH have a wide spectrum of clinical presentation depending on the underlying enzymatic deficiency including 21-hydroxylase,11?-hydroxylase,3?-hydroxysteroid dehydrogenase,and 17a-hydroxylase[1].11?-hydroxylase deficiency(11OHD is the second most common cause of CAH accounting for approximately 5-8%of cases with an incidence of 1:100 000-1:200 000 live births in nonconsanguineous populations(1).11?-OHD may similarly present as classic or non-classic phenotype depending on the degree of clinical severity and percentage loss of enzyme activity[2].The deficiency of 11?-hydroxylase leads to reduced cortisol biosynthesis,increased ACTH secretion,and overproduction of steroid precursors.These precursors are shunted toward androgen synthesis,resulting in hyperandrogenism.Phenotypical expression of classic 11?-OHD leads to virilization of external genitalia in newborn females.The overproduction of reactive androgen also causes precocious pseudopuberty,accelerated somatic growth,and premature epiphyseal closure in both sexes.Theaccumulation of 11-deoxycorticosterone and its metabolites have mineralocorticoid activity,which leads to sodium retention and capacity expansion[3].Two homologous enzymes,11?-hydroxylase and aldosterone synthase,encoded by the CYP11B1 and CYP11B2 genes,respectively.The two genes are 40-kb apart,each comprising nine exons and mapped to chromosome 8q21-22(3,4)(Fig.1A).In contrast to CYP21A2 and its CYP21A1P pseudogene,CYP11B1 and CYP11B2 are both active and do not have a pseudogene.The two encoded homologs,however,have distinct functions in cortisol and aldosterone synthesis,respectively(3).Unlike 21-hydroxylase deficiency in which there is a good correlation of genotype with phenotype,it is difficult to predict the influence of mutations in patients with 11?-OHD on the enzyme activity and clinical phenotype,,due to a small patient quantity[4].However,gene analysis of CYP11B1 can assist in confirmation of CYP11B1 and genetic counseling.Purpose:In this study,a patient present with hypertension and hypokalemia was collected.The patients were identified as classic 11?-OHD.CYP11B1 gene analysis was performed of the patients and their families.Real-time Quantitative PCR was used to identify the genetic form of the patient;the in vitro expression of mRNA was determined using minigene technology to understand the effect of the splice mutation of the patient on mRNA.Method:We reached a purpose of clinical diagnosis of 11OHD through laboratory and imaging examination combined with family data collection.DNA of mononuclear cells DNA were extracted from the peripheral blood of patients and their families in EDTA-anticoagulant tubes.According to the sequence of CYP11B1 gene in the gene bank,software primer premier 6.0 software was used to design specific primers to amplify all the nine exons and exon-intron boundaries,respectively.DNA of the patient and his parents were used as template to amplify.The amplication products were separated and purified by agarose gel electrophoresis followed by sequencing on the and ABI 3130x1 Genetic Analyzer.The sequencing results were compared to the UCSC data using Autoassembler2.0 software to detect the mutation site.The results of gene sequencing showed that the patient was homozygous point mutation,the mother was heterozygous point mutation,and the father did not find the mutation site,so it was speculated that the CYP11B1 gene of the patient had a large deletion mutation.The gene sequencing data showed the patients carring homozygous point mutations,his mother or hybrid point mutations,while his father found no mutations.Thus we speculated that patient inherited large genomic deletions from his father.To comfirm the idea,we designed primers surrounding the mutation point mutation of 150 bp long to detect copy number of the segment by Real-time fluorescent quantitative PCR technology.In addition,to comfirm the effect of the splicing mutation on mRNA,we took advantage of minigene technology,by constructing plasmid and importing into the engineering cells to observe the difference of mRNA expression between wild type and mutate type.Results:3.Clinical diagnosis:The patient was confirmed classical 11-OHD.His family members,including his father,mother,brother and sister are with normal phenotype.4.Results of mutation detection results:(1)the Sanger sequencing showed patient carring CYP11B1 c.595 + 1g>A homozygous mutations.Preliminary bioinformatics analysis(Augustus)showed the mutationc was a splicing mutation,which significantly affected the mRNA splicing.Her mother and sister carries c.595 + 1 g>A hybrid mutation,while his father did not see abnormalities within CYP11B1 gene.5.Results of Real-time fluorescent quantitative PCR:There was a heterozygous deletion in the target segment which spans 3'end of exon3 and 5'end of intron 5,containing the mutated site.6.Results of c.595 + 1 g>A mutation on mRNA splicing:Agarose gel electrophoresis showed that the cDNA segment of the mutation group reversely transcriped from mRNA was longer than that of the wild group by about 100bp.After gene sequencing,it was found that cDNA of the mutant mRNA had a intron 3 more than the wild group suggesting that the splice mutation resulted in the retention of intron 3 in the mature mRNA,which affected the protein coding and enzyme structure.