Functional Study Of StAR Gene In A Family With Congenital Lipoid Adrenal Hyperplasia

【Objective】Lipid congenital adrenal hyperplasia(CLAH)is a rare autosomal recessive disorder and the most severe form of congenital adrenal hyperplasia.Mutations in encoding genes of the steroidogenic acute regulatory protein(StAR)have been responsible for LCAH.StAR dysfunction can lead to the first step in steroid hormone synthesis blocked,that is,cholesterol can not be converted to pregnenolone ketone,making cholesterol and cholesterol esters accumulated in bilateral adrenal cortex.The patients were manifested by deficiency of all classes of steroid: glucocorticoid,mineralocorticoids,and sex steroids.but pituitary adrenocorticotropic hormone(ATCH),luteinizing hormone(LH)and plasma reninactivity(PRA)were significantly increased.Most CLAH patients present with adrenal crisis,such as hyponatremia,hyperkalemia,acidosis or even shock in the early infants.Early diagnosis and treatment are effective ways to reduce mortality.In this study,we analyzed the pathogenicity and conservation of a compound heterozygous mutation in a patient with CLAH.Functional experiments of StAR c.229C>T,p.(Gln77X)及 StAR c.659A>G,p.(His220Arg)were performed to determine the effect of two mutations on the activity of StAR.The aim of this study was to provide theoretical basis for the pathogenesis of heterozygous mutation in StAR gene.【Method】A clinical diagnosis of CLAH child as the research object,collecting their clinical data and gene sequencing results.There were compound heterozygous mutations c.229C>T,p.(Gln77X)and c.659A>G,p.(His220Arg)of StAR gene in this patient.Wherein the c.659A>G,p.(His220Arg)is a novel point mutation of StAR gene..The conservativeness and pathogenicity of StAR gene c.659A> G,p.(His220Arg)were predicted by UCSC Genome Brower and Poly Phen-2.Protein Data Bank analyzes the functional domains of StAR and analyzes the pathogenicity of both mutations at the protein level.In this study,nonsteroidogenic COS-7 cells were used as the study object,and the LV5 plasmid was used as vector.StAR wild type,StAR c.229C> T and StAR c.659A> G gene were inserted into the plasmid vector respectively to construct the fusion plasmid.The fusion plasmids were transfected into COS-7 cells by lentivirus,and stable cells containing StAR wild type(positive control),StAR c.229C> T,StAR c.659A> G and no-load plasmid(negative control)were constructed respectively.The STAR stable cells were transiently transfected with P450 scc,adrenodoxin reductase,adrenodoxin.Stable cells were treated with cholesterol(2 mg/L)for 24 h.The content of pregnenolone in the supernatant was detected by ELISA,and the residual activity of StAR was analyzed.The pathogenic mechanism of the compound heterozygous mutation can be further clarified.【Results】UCSC Brower software was used to compare the homology of 220 th amino acids of StAR in human,Ganges RIver monkey,mouse,dog,elephant,opossum,chicken and zebra,which showed that it was highly conserved in evolution.The results of Poly Phen-2 software showed that the StAR gene mutation was benign and the pathogenicity was 0.035.Protein function prediction showed that StAR 220 th amino acids were located in the cholesterol binding domain,and the software did not contain the mutation of StAR p.(His220Arg),proving that the mutation is a novel point mutation of StAR gene.The pathogenicity of StAR p.(Gln77X)was analyzed by online software Protein Data Bank.The results showed that the mutation could produce significantly shortened StAR,resulting in the loss of the functional domain of the protein.Gene function showed that the protein function of StAR c.229C> T mutation was completely lost.The protein activity of StAR c.659A> G mutation was 43.79% of wild type.【[Conclusion] 】In this case,children with StAR gene compound heterozygous mutation,and Wherein the StAR c.659A> G p.(His220Arg)is a novel point mutation.The mutant protein still retains a certain activity(43.79%).Another StAR mutation of c.229C>T,p.(Gln77X)can produce significantly shortened StAR,resulting in almost complete loss of functional domain of the protein,which leads to the clinical manifestations of the classic CLAH.