The Mutation Of PE_PGRS33 Gene In Clinical Isolates Mycobacterium Tuberculosis In Children

Objective:1. To study the distribution of the genotyping and find the effective loci combination of variable number tandem repeats (VNTR) in clinical strains Mycobacterium tuberculosis isolated from pediatrics tuberculosis in Chongqing by multiple locus VNTR analysis (MLVA).2. In vitro and in animal studies have suggested an important role for the Mycobacterium tuberculosis PE_PGRS33 protein in the pathogenesis of tuberculosis. A significant level of PE_PGRS33 gene DNA polymorphism has been found among clinical isolates from adult tuberculosis patients. Association between the PE_PGRS33 gene DNA sequence changes and the clinical and epidemiological phenotypes of adult tuberculosis have been reported. However, the implication of the PE_PGRS33 genetic variations among clinical isolates of M. tuberculosis isolates in pediatric tuberculosis is unknown.3. To understand whether there were mixed infection and the existence feature of PE_PGRS33 gene subpopulations individual in pediatric tuberculosis patients. Methods:1. 24 standardized MLVA loci was used to type the 101 clinical isolated M. tuberculosis strains and results were analyzed by BioNumerics 6.1 software, which including dendrogram and Hunter-Gaston index (HGI) of 24 loci. Then the HGI of different loci set (12 loci, 15 loci, and 24 loci) was evaluated.2. To better understand the role of PE_PGRS33 protein in pediatric tuberculosis pathogenesis, we investigated DNA polymorphisms of PE_PGRS33 gene among 101 pediatric tuberculosis patients'isolates and assessed the relationship between PE_PGRS33 sequence variations and several major clinical characteristics of tuberculosis.3. One hundred and sixteen clonal isolates were obtained from the L-J culture of their sputum/gastric aspirate from 5 pediatric tuberculosis patients and cerebrospinal fluid and there were 3~16 isolates for each patient. All of the clonal isolates were subjected to genotyping with the variable-number tandem-repeat (VNTR) system with 24 loci used PCR and DNA sequencing to identify genetic variations in the PE_PGRS33 gene in comparison with H37Rv.Results:1. 24 loci showed different polymorphism. 101 clinical isolates were typed into one group that displaying 83 genotypes, in which 69 strains (68.32%) were typed 69 genotypes respectively, and 32 strains (31.68%) were typed 14 genotypes which showed cluster rate 17.82%. The HGI of 24 loci was from 0.168 to 0.829, the number of VNTR loci with HGI higher than 0.5 was 16. Three loci set displayed different polymorphism. HGI of 12 loci, 15 loci, and 24 loci set was 0.995, 0.996, and 0.996.2. A total of twelve different PE_PGRS33 alleles were observed among the 101 M. tuberculosis clinical isolates investigated and 59.41% of the isolates had PE_PGRS33 alleles that would result in a change in the amino acid sequence of the PE_PGRS33 protein. Patients having TB meningitis and false negative PPD skin test results were found to be more likely to be infected by isolates having a mutant type of the PE_PGRS33 gene, compared to patients who had no TB meningitis and who had positive PPD-skin test results, respectively.3. One hundred and sixteen M. tuberculosis clonal isolates were identified five genotypes and two types sequenced variations of PE_PGRS33 gene. There was no difference in genotype and sequence variations of PE_PGRS33 gene among the clonal isolates from different parts from a same child.Conclusion:1. The clinical strains of M. tuberculosis isolated from pediatrics tuberculosis in Chongqing show definite polymorphism and the standardization 15 loci VNTR set of MLVA can be used in the molecular epidemiological study of M. tuberculosis in Chongqing. 2. Variations in the PGRS domain of PE_PGRS33 of M. tuberculosis clinical isolates were significantly associated with tuberculous meningitis and PPD skin test-negative pediatric tuberculosis, respectively.3. It implied that each of 5 pediatric patients with tuberculosis had the infection with a single M. tuberculosis strain and the existence feature of PE_PGRS33 gene subpopulations was genetic homogeneity.