title: Prevalence, distribution and functional significance of the -237C to T polymorphism in the IL-12Rβ2 promoter in Indian tuberculosis patients.
abstract: Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the -237 polymorphic site in the 5' promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis.
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Vikas Kumar
authors.initials: VK
authors.lastname: Verma
authors.forename: Vibha
authors.initials: V
authors.lastname: Taneja
authors.forename: Anand
authors.initials: A
authors.lastname: Jaiswal
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Sharma
authors.forename: Digamber
authors.initials: D
authors.lastname: Behera
authors.forename: Vishnubhatla
authors.initials: V
authors.lastname: Sreenivas
authors.forename: Shyam Singh
authors.initials: SS
authors.lastname: Chauhan
authors.forename: Hanumanthappa Krishna
authors.initials: HK
authors.lastname: Prasad
mesh_terms: D044466:Asian People / Q000235:genetics*; D001483:Base Sequence; D050990:GATA3 Transcription Factor / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007194:India; D016898:Interferon-alpha / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053707:Receptors, Interleukin-12 / Q000235:genetics*; D014397:Tuberculosis, Pulmonary / Q000235:genetics*
chemical_list: D050990:GATA3 Transcription Factor; C508439:IL12RB2 protein, human; D016898:Interferon-alpha; D012333:RNA, Messenger; D053707:Receptors, Interleukin-12; D015847:Interleukin-4