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Master's Dissertation
DOI
10.11606/D.5.2013.tde-05112013-163219
Document
Author
Full name
Natália Umetsu Sobrinho
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Liphaus, Bernadete de Lourdes (President)
Andrade, Luis Eduardo Coelho
Vasconcelos, Dewton de Moraes
Title in Portuguese
Caracterização molecular dos componentes C1q, C4 e C2 do sistema complemento em pacientes pediátricos com lúpus eritematoso sistêmico
Keywords in Portuguese
Adolescente
Complemento C1q/deficiência
Complemento C2/deficiência
Complemento C4/deficiência
Criança
Genes
Lúpus eritematoso sistêmico
Mutação
Abstract in Portuguese
Objetivo: Realizar a caracterização molecular dos genes C1q, C4 e C2 em pacientes com lúpus eritematoso sistêmico juvenil (LESJ). Métodos: Quatro pacientes com LESJ e deficiências de C1q,C4 e/ou C2 foram selecionados. O paciente P1 apresentava níveis séricos indetectáveis de C1q e níveis normais de C3 e C4; paciente P2 níveis baixos de C2 e C4 no soro; P3 apresentava níveis baixos de C2 e normais de C3 e C4 e P4 constantes níveis baixos de C4 e níveis normais de C1q, C2 e C3 no soro. Foram sequenciados os genes C1q e C2. Células mononucleares dos pacientes P1, P3 e P4 e de três indivíduos saudáveis foram cultivadas, estimuladas com interferon gama e incubadas por 36 horas e PCR quantitativo (qRTPCR) foi realizado para verificar a expressão de mRNA. Resultados: A caracterização molecular do gene C1q (P1) mostrou trocas heterozigotas na cadeia A (c.276 A>G Gly) e na cadeia C (c.126 C>T Pro). Foram observadas também duas trocas de base em homozigose na região 5'UTR (c. -159 T>G) e na região 3'UTR (c*78 A>G) da cadeia B. O qRT-PCR mostrou que a expressão de mRNA de C1qA no paciente P1 sem estimulo estava 1,3 vezes mais baixo e com estímulo de interferon gama estava 1,6 vezes mais expresso se comparado aos indivíduos saudáveis. A expressão de mRNA de C1qB sem estímulo foi 2,2 vezes mais baixo e com estímulo foi 1,5 vezes mais expresso quando comparados aos controles. Para C1qC os indivíduos controles não expressaram mRNA porém o paciente P1 apresentou pequena expressão com e sem estímulo. O sequenciamento do gene C2 dos pacientes P2 e P3 apresentou 100% de similaridade com a sequência de referência com exceção da deleção de 28pb no exon 6 (deficiência heterozigota de C2 do tipo I). A expressão de mRNA de C2 do paciente P3 foi sem estímulo 23 vezes mais baixo e com interferon 4,2 vezes menos expresso quando comparado aos controles. P4 apresentou 2 copias de C4A e 3 copias de C4B e no qRT-PCR o gene C4B estava 14 vezes menos expresso sem estímulo e com estímulo estava semelhante aos controles. Conclusões: As trocas de base em homozigose nas regiões 5'UTR (região promotora) e 3'UTR (região de estabilização do mRNA) na cadeia B do gene C1q podem ter modificado a região de transcrição do mRNA pois sua expressão sem estimulo foi baixa. Outras investigações são necessárias para relacionar as variações do gene C1q encontradas com os níveis séricos indetectáveis de C1q. A deficiência de C2 do tipo I heterozigota pode levar a redução na expressão de mRNA e pode estar presente em pacientes lúpicos com níveis séricos detectáveis de C2. Por fim, a baixa expressão de mRNA de C4B mostrou que as dosagens séricas e a avaliação do número de copias podem não ser suficientes para estabelecer a deficiência de C4
Title in English
Molecular characterization of complement components C1q, C4, and C2 in pediatric patients with Systemic Lupus Erythematosus
Keywords in English
Adolescent
Child
Complement C1q/deficiency
Complement C2/deficiency
Complement C4/deficiency
Genes
Lupus erythematosus systemic
Mutation
Abstract in English
Objective: To perform the molecular characterization of C1q, C4 and C2 genes in patients with Juvenile Systemic Lupus Erythematosus (JSLE). Methods: Four patients with JSLE and C1q, C4 and/or C2 deficiencies were chosen. Patient P1 had undetectable C1q serum level and normal levels of C3 and C4; Patient P2 had decreased levels of C2 and C4 serum while P3 had decreased C2 with normal C3 and C4 levels. Lastly P4 had repeated decreased C4 and normal C1q, C2 and C3 serum levels. C1q and C2 genes were sequenced. Peripheral mononuclear cells from patients P1, P3 and P4 and from three healthy individuals were both cultivated and stimulated with interferon gamma and a quantitative PCR (qRT-PCR) was also performed to verify mRNA expression. Results: C1q molecular characterization for P1 revealed heterozygous silent mutations in A chain (c.276 A>G Gly) and in C chain (c.126 C>T Pro). Additionally, in B chain two homozygous single-base exchanges were detected in the 5´UTR (c. -159 T>G) and 3'UTR region (c*78 A>G). The qRTPCR revealed that C1qA gene mRNA expression without stimulation was decreased 1.3 times and with interferon gamma was 1.6 times more expressed compared with controls samples. C1qB gene expression without stimulation was 2.2 times decreased and when stimulated was 1.5 times more expressed. Controls did not expressed C1qC gene and patient P1 had low expression both with and without stimulation. P2 had 2 copies of C4A and 1 copy of C4B. C2 gene sequencing (P2 and P3) showed 100% match with referenced sequence, with exception to 28bp deletion at the exon 6 (heterozygous C2 deficiency type I). C2 mRNA expression from P3 without stimulation was 23 times decreased and with interferon was 4.2 times decreased compared with controls. P4 had 2 copies of C4A and 3 copies of C4B. The qRT-PCR were performed only in C4B gene showed without stimulation a 14 times decreased expression and with interferon stimulation the expression were similar to controls. Conclusions: The two homozygous single-base exchanges in 5'UTR and 3'UTR that correspond to the promoter region and stabilization mRNA region in B chain of C1q gene, may have modified mRNA transcription as its expression was decreased without stimulation. Further analysis is necessary to relate C1q gene variations and undetectable serum C1q. In addition, heterozygous C2 deficiency type I may lead to reduced mRNA expression and may be present in JSLE patients with detectable C2 levels. Finally, the decreased C4B gene expression showed that serum dosage and gene copy number may not be sufficient to assess C4 deficiency
 
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Publishing Date
2013-11-06
 
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