Estudo do gene btk (bruton's tyrosine quinase) em pacientes com agamaglobulinemia congênita

Abstract

Oliveira, RR. Study of the Btk (Bruton's Tyrosine Kinase) gene in patients with congenital agamaglobulinemia. [Thesis]. Sao Paulo: Interunidades em Biotecnologia USP/Instituto Butantan/IPT; 2008. X-linked Agammaglobulinemia (XLA) is a primary immunodeficiency considered a prototype defect of early B cell development. Approximately 85% of the patients with defects in early B-cell development have XLA, characterized by absent or decreased numbers of mature B cells in peripheral blood and by markedly reduced levels of serum immunoglobulins (IgG < 200 mg/dL, IgM and IgA < 2 standard deviations for the age). Affected boys usually present an increased susceptibility to severe bacterial and enteroviral infections. XLA is caused by mutations in the gene for Bruton's agammaglobulinemia tyrosine kinase (BTK), with codes for a protein BTK expressed in hematopoietic cells. The btk gene is localized in the long arm of the X chromosome at q21.3-22 region. The BTK protein is a member of the Tec family of cytoplasmic tyrosine kinases and plays a vital, but diverse, modulation role in many cellular processes. About 10-15% of the Agammaglobulinemia cases are not X-linked, are autossomal recessive, presenting characteristic phenotypes similar to the XLA, but absence of mutations in the gene btk. Thirty-three male patients were included in this study and analyzed for the presence of BTK mutations. The inclusion of the patients was based on three criteria, according to PAGID and ESID recommendations: absence or less than 2% of circulating B cells; very low levels of serum IgG; and a history of recurrent bacterial infections. In all the thirty-three patients the gene btk was studied by the screening mutation method (SSCP/HA). Possible mutations were characterized by direct sequencing and protein profile expression was analyzed by the real-time PCR technique. The recurrent infections founded in our patients were pneumonias, otitis, sinusitis, sepsis and diarrhea. Patients with a positive family history (parents or brothers) and with the same mutation had distinct clinical presentations. The mutations founded were of missense, nonssense, small deletions/insertions and splicing site alterations types affecting the PH, SH3, SH2 and tyrosine kinase domains of btk gene. The expression levels correlated with the mutation types, since were extremely low in the patients with stop codon mutations, and below 20% in the patients with other mutations, and only one patient presented an expression level of 60%. The data obtained reaffirm the clinical and molecular diversity of XLA, where we observe individuals with the same mutation presenting distinct patterns of manifestations, patients with stop codon mutations with stable clinical condition and a patient, who died during the development of this study, due to a simple amino acid exchange. The molecular and clinical diversity presented by patients with agammaglobulinemia raises objections to develop a gene therapy for these individuals.