Mutation Identification And Functional Analysis Of Disease Gene In A Family With Mendelian Susceptibility To Mycobacterial Disease

Mendelian susceptibility to mycobacterial disease(MSMD) is a rare disease Characterized by predisposition to clinical disease caused by weakly virulent mycobacteria, such as BCG vaccines and environmental mycobacteria. It is first reported in 1951. It is primary immunodeficiency disease.We do the genetic study in a family with Mendelian susceptibility to mycobacterial disease, and do the preliminary research to clarify the molecular mechanism of disease by molecular biological and biochemical methods.The proband in the family was diagnosed with MSMD, DNA Sequencing of the disease genes of MSMD identified a novel mutation(c.1228A>G) in the exon15 of STAT1 gene, which leads a substitution of amino acid residue lysine to a glutamic residue(p.K410E). RFLP(restriction fragment length polymorphism) analysis showed that all the affected members in the family carry the mutation and all the unaffected individuals and 100 unrelated normal controls do not carry the mutation. Alignment the sequence of STAT1 protein from different species was performed, which revealed that the asparagine at position 410(K410) is high conserved during the evolution. We also did the in silico analysis of the coding region of STAT1 protein and found that the site of mutated amino acid located in the DNA binding region of the STAT1 protein, and we speculate the mutation might influence the function of the STAT1 protein. Wild-type(WT) and mutant STAT1 gene were constructed into pRc/CMV vector and transiently transfected into HEK293 cells. After stimulated by interferon-gama, HEK293 cells were examined by Immunofluorescence assay and in Electrophoretic Mobility Shift Assay(EMSA). The res ults showed that the translocation of mutant STAT1 protein into nucleus was not affected, while the mutation leads the STAT1 protein decreased capacity to bind with gammaactivating sequences(gamma-activating sequence, GAS), and therefore influencing the normal physiological function of STAT1 protein.Our study identified a novel DNA binding region site mutation(c.1228A>G, p.K410E) in STAT1 gene in a Chinese MSMD family, and clarified the mechanism of the mutation affect the function of STAT1 protein by molecular biological and biochemical methods. Our study expands the spectrum of mutations that cause MSMD and provide the effective theoretical basis for clinical prenatal diagnosis, prevention and treatment of this disease.