Mutation Identification And Functional Analysis Of Disease Gene In Four Chinese Families With Mendelian Susceptibility To Mycobacterial Disease

Disseminated Bovis Bacille Calmette-Guerin(BCG)infection Diseases,also knowns as the Mendelian Susceptibility Mycobacterial Disease(MSMD),it is a rare congenital immunodeficiency disease.BCG is a bovine attenuated live vaccine,which is injected within 24 hours after birth so that the body can establish specific immunity against tuberculosis bacillus to prevent tuberculosis in children.The clinical phenotype of the disease usually involves the spread of BCG vaccination sites,lymph nodes,skin and soft tissue,lung tissue,liver and bone marrow,etc.In this study,medical genetic analysis was carried out on four families of disseminated BCG infection from Hubei province,China.Pathogenic gene mutations have been found in those family.The effects of these mutations on protein functions were further studied through western blot and protein localization analysis.In addition,the molecular mechanism of pathogenesis was also clarified.We found that four unrelated patients with MSMD carried different new mutations in four different disease genes.The patient of family 1 carries a heterozygous mutation c.2071 A >G(p.Met691Val)in STAT1 gene,which is autosomal dominant inheritance.The patient of family 2 carries a deletion mutation(818-821delTTAA)in IFNGR1 gene,which results in open reading frames shifted and producing truncated protein.The patient of the family3 carries a heterozygous mutation c.235C>A(p.Pro79Thr)in IFNGR2 gene,sequencing results of 100 normal control showed that the control individuals do not carry these mutations.The patient of family 4 carries a homozygous missense mutation c.751G>C(p.Gln251His)in NEMO gene,which is a x-linked recessive disease gene.We do the mutation screening in 100 control show that the control individuals do not carry these mutations.The oligonucleotide polymorphism database shows that four pathogenic mutations are not single nucleotide polymorphisms(SNPs)and the amino acid on the mutation site are highly conserved during evolution.Western Blot and GFP tagged proteins localization experiments were performed.Western Blot results show that the mutation of Met691 Val in STAT1 gene results in decreased expression level of STAT1 protein.GFP tagged proteins localization experiment results show that mutation affect the localization of STAT1 protein,which may affect the SH2 domain structure of STAT1 protein.Therefore cannot form gamma activation factor(GAF),also cannot enter the nuclei combining immune response to gene promoter region gamma activation sequence(GAS),and can't start IFN-gamma resistance mycobacterium immune pathways related gene expression.Thus,this study on screening the disease genes mutations of MSMD,not only enriches the pathogenic gene mutation spectrum of STAT1,IFNGR1,IFNGR2,and NEMO but also help to decipher the mechanism of primary infection and host resistance.Also,provide a new target for prenatal diagnosis and genetic diagnosis and offers a new goal for prevention or intervention after infection.