IDENTIFICAÇÃO DE MUTAÇÃO INÉDITA c.21G>A (W7X) NO GENE IL12RB1 EM PACIENTES COM DOENÇA MENDELIANA DE SUSCETIBILIDADE ÀS MICOBACTERIOSES NO NORTE FLUMINENSE

Abstract

The adverse reaction of BCG vaccine affects children with poor cellular immune response. This deficiency is triggered by an absence of lymphocytes and macrophages, inability to produce O2- or compromised via IL-12-IFN-γ. The decreased production of IFN-γ is one of the causes of Mendelian Susceptibility to Mycobacterial Disease (MSMD). The objective of this research was to investigate the cell populations in peripheral blood (PBMC) of children with episodes of adverse reaction to the vaccine BCG, and the expression of proteins involved in IL-12/IFN-γ axis. Methods: The study included nine children of North Fluminense region of Rio de Janeiro State with adverse reaction to the vaccine BCG. The expression levels of cellular markers for T lymphocytes (CD45+ CD3+ CD4+ CD8+ cells), B-lymphocytes (CD19+), NK cells (CD16+ CD56+) and IL-12Rβ1 (CD212+) and IFNGR1 (CD119+) receptors were quantified by flow cytometry. The production of IFN-γ and IL17-A cytokine in the supernatants of growing blood cells stimulated or not with phytohemagglutinin (PHA) or BCG lysate was assessed by quantitative flow cytometry using microspheres. For identification of pathogenic mutations we used Sanger sequencing, single base sequencing and PCR-RFLP of c.21G mutation> A (rs150172855) in venous blood DNA collected from children with adverse reactions to BCG history and their parents. Results: Four children were diagnosed with MSMD. Two brothers and a third boy were homozygous for the c.21G> A mutation in the IL12RB1 gene, and their parents were heterozygous. The fourth boy died without collecting biological sample, but the parents were heterozygous for the same mutation. There was a significant difference between the ex vivo levels of expression of IL-12Rβ1 in cells from three patients with MSMD and from four without MSMD when compared with healthy control. PBMC of patients with MSMD produced less IL-12Rβ1 after stimulation with PHA when compared with healthy control subjects or subjects without MSMD. Patients without MSMD and with MSMD produced less IFN-γ when PBMC upon stimulation with PHA when compared with healthy control subjects. BCG-stimulated PBMC of control subjects produced more IFN-γ than those from MSMD s or without MSMD. In addition, patients without MSMD produced more IFN-γ than in MSMD patients. There was no difference between the levels of CD4 +, CD8 +, B and NK cells between the three groups. Discussion: Patients with history of adverse reaction to the BCG vaccine produce lower amounts of IFN-γ. However, patients with MSMD showed lower expression of IFN-γ and IL X 12Rβ1 versus patients without MSMD or controls. Thus, the phenotype observed IL 12Rβ1 reduction and IFN-γ reaffirms the diagnostic condition. The homozygous allele c.21G> triggers primary immunodeficiency deficiency of IL-12Rβ1, but low levels of the protein are found on the surface of lymphocytes of affected patients. This is the first report of an IL12RB1 stop-codon mutation with paradoxical cell-surface expression of the receptor. According to in silico analysis, the mutation has a potentially harmful effect on RNA splicing. All carriers of the mutation c.21G>A share the extended CACCAGTCCGG haplotype (positive strand), featuring a founder effect. Conclusion: We identified the novel c.21G> A (W7X) signal-peptide stop mutation in IL12RB1 in four patients with MSMD in the Northern State of Rio de Janeiro. In homozygous carriers, the mutation causes complete IL-12Rβ1 deficiency with paradoxical cell surface protein expression in stimulated lymphocytes. Quantification of IFN-γ and IL 12Rβ1 was extremely important in the diagnosis of MSMD in this cohort of children with episodes of adverse reaction to the BCG vaccine. Early diagnosis of MSMD helped determining the preventive actions in order to reduce mortality and morbidity in these patients.