A New Causative Gene ARMC5 Mutation Indentified By Whole-exome Sequencing In Primary Bilateral Macronodular Adrenal Hyperplasia

Objective:Pimary bilateral macronodular adrenal hyperplasia(PBMAH)is a rare autosomal dominant disease,characterized by bilateral adrenal hyperplasia with or without clinical manifestations of cortisol hypersecretion.Familial case reports indicate that genetic factors are crucial.To explore the molecular genetic mechanism and further to elucidate pathogenisis of the disease,whole exome sequencing and analysis of clinical features were performed on a total of 4 generations of PBMAH pedigrees.Methods: 1.All members of the pedigree received detailed history questions,laboratory tests,and adrenal CT thin-layer scans.Pedigree genetic lines were also drawn for pedigree analysis.2.Peripheral blood samples of probands and pedigrees were extracted and DNA was extracted from the probands,and DNA was extracted from probands.Three patients and one normal member of the pedigree were sequenced by whole exome.The overlapped 3 patients shared variation except for normal controls Mutation,through bioinformatics analysis software and database comparison screening candidate gene mutation.3.SWISS MODEL online prediction software for suspected pathogenic mutation sites was used to analyze the three-dimensional structure difference and function of wild-type and mutant gene-encoded proteins.4.Sanger sequencing of all members of the pedigree identified genotype and phenotype co-segregation.5.Construction of mutant and wild-type causative gene expression vectors.Results:1.All patients in this pedigree were male,with an average age of onset of 56.6 ± 8.62 years.The genetic pattern was autosomal dominant.Adrenal CT in pedigrees suggested double-sided adrenal hyperplasia with bilateral adrenal gland diameters> 10 mm.However,the clinical phenotype More qualitative,2 patients with clinical Cushing's syndrome,one is subclinical Cushing's syndrome.2.By bioinformatics software such as Poly Phen2,SIFT,Free Bayes and Annovar,208 mutations were screened out and 60 alleles with a genome frequency of less than 0.05 were removed.The benign mutations in Clin Var were removed,and the remaining 56 candidates Mutation site.A review of PBMAH-related literature identified that c.1085G> A on the ARMC5 gene may be a causative mutation in this pedigree.3.Mutation Taster online software predicts the effect of the c.1085G>A mutation on protein function,and the results show that this mutation results in amino acid changes in p.R362 Q and is a pathogenic mutation.4.Sanger sequencing showed that all patients in this pedigree carried the c.1085G> A mutation,and did not carry this mutation in all healthy members,which was consistent with genotypic and phenotypic co-segregation.5.The wild-type ARMC5 gene and the mutant ARMC5 gene R362 Q expression vector were constructed.Conclusion:1.This study reports a PBMAH pedigree of 18 members including 3 patients in 4 generations.Clinical phenotypic analysis revealed phenotypic heterogeneity in their pedigrees.2.This study found a new non-synonymous mutation in ARMC5 gene c.1085G> A(p.R362Q)as a suspectable disease-causing mutation in the PBMAH family.