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Previous issue date: 2007 === Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) === Os objetivos de nosso trabalho foram: 1) investigar a relação entre
polimorfismos dos genes CD28, ICOS, CTLA4, PDCD1, FAS, TGFB1, IFNG, IL6,
IL10, TNFA e a ocorrência de carcinoma do colo uterino; 2) investigar a relação
entre nível de expressão dos genes CD28, CTLA4, ICOS, ICOSL, CD80, CD86,
GZMB (granzima B) em biópsias de carcinoma de colo uterino, no momento do
diagnóstico, e a evolução do tumor após o tratamento. Materiais e Métodos: 1) A
determinação dos polimorfismos foi realizada através dos métodos PCR-RFLP,
para os SNPs: CD28 int3 (+17 T>C), CTLA4 promoter (-319 C>T), CTLA4 exon 1
(+49 A>G), ICOS 3’UTR (+1564 T>C), PDCD1 exon 5 (+7785 C>T) e FAS
promoter (-670 G>A) e PCR-SSP para TNFA promoter (-308 G>A), IL6 promoter
(-174 G>C), IFNG int1 (+874 A>T), TGFB1 codon 10 (+869 T>C), codon 25 (+915
G>C) and IL10 promoter (-1082 A>G), (-819 C>T), (-592 C>A) SNPs. Um
algoritmo foi desenvolvido para identificar genótipos de SNPs isolados e/ou em
combinações de 2 e 3 genótipos associados com a doença 2) Expressão gênica
foi determinada, por PCR em tempo real, em biópsias de carcinoma cervical de
pacientes posteriormente classificadas em dois grupos: 10 pacientes sem
envidência do tumor (evolução clínica boa) após 6 meses do final do tratamento e
8 pacientes nas quais o tumor foi detectado (evolução clínica ruim) neste período.
Resultados: 1) Nenhuma associação significante foi observada com os SNPs
analisados isoladamente. A análise do multi-locus revelou, em pacientes, maiores
frequências de três combinações de três genótipos (CD28 +17 int3(TT)/IFNG+874
int1(AA)/TNFA-308(GG), CD28+17 int3 (TT)/IFNG+874 Iint1(AA)/PDCD1 +7785
exon5 (CT), CD28 int3(TT)/IFNG int1(AA)/ICOS 1564 3’UTR (TT)) (p<0,01).
Entretanto, a contribuição do terceiro polimorfismo (TNFA, ICOS, or PDCD1) não
foi confirmada (p=0,1). Confirmamos maior frequência, em pacientes, da
combinação CD28(TT)/IFNG(AA) nos três grupos juntos, e observamos uma
contribuição principal desta combinação na associação com a doença (OR = 2,07,
p=0,0011). 2) A expressão intra-tumoral de GZMB estava aumentada em biópsias
de pacientes com estadio IIIB que apresentaram uma evolução clínica ruim
(p=0,034). A expressão gênica das moléculas CD28, ICOS, ICOSL, CD80, CTLA4
e CD86 não mostrou diferenças significantes entre as biópsias de pacientes de
grupos de evolução clínica boa e ruim. Conclusões: 1) Nossos resultados
sugerem efeito epistático entre os genes CD28 e IFNG na susceptibilidade do
câncer de colo uterino. Outros trabalhos em estudos independentes aplicando
abordagem multi-locus são importantes para a elucidação da contribuição de
polimorfismos genéticos para a susceptibilidade a esta doença. Além disto, o
algoritmo desenvolvido pode ser aplicado a estudos sobre fatores genéticos de
susceptibilidade a outras doenças complexas. 2) Altos níveis de mRNA de GZMB
em tumores no estádio IIIB correlacionaram-se com menor sobrevida livre do
tumor nos 6 meses após o tratamento. A expressão deste gene, se os resultados
obtidos forem confirmados por um estudo independente, pode vir a se tornar um
marcador de prognóstico para resposta a tratamento de carcinoma de colo
uterino. === Background
Cervical cancer is a complex disease with multiple environmental and genetic determinants.
The most important environmental factor is infection by high-risk types of human papillomavirus
(HPV). Considering that only a small proportion of HPV-infected women develop cervical cancer,
polymorphisms in immune response genes that might be involved in virus elimination, or in the
immune response against the tumor, are considered important candidates to influence cervical cancer
susceptibility.
Results
In the present study we aimed to search association between polymorphisms in immune
response genes and cervical cancer, using both single-locus and multi-locus analysis approaches. A
total of 14 SNPs (Single Nucleotide Polymorphisms) distributed into CD28, CTLA4, ICOS, PDCD1,
FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals
from three independent case/control sets: the first two sets comprised White individuals (one group
with 82 cases and 85 controls, and the other with 83 cases and 85 controls) and the third was
constituted by Non-White individuals (64 cases and 75 controls). No significant association was
observed with any individual SNP. The multi-locus analysis revealed higher frequencies, in cancer
patients, of three three-genotype combinations (CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG),
CD28+17(TT)/IFNG(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)
(p<0.01, Monte Carlo simulation). Noticing that all these three genotype combinations contained two
genotypes in common, i.e. CD28(TT) and IFNG(AA), we hypothesized that this two-genotype
combination could have a major contribution to the observed association. To address this question,
we analyzed frequencies of CD28(TT),IFNG(AA) genotype combination in the three groups combined
and observed its increase in patients (p=0.0011 by Fisher’s exact test). In a second Monte Carlo
simulation of three-genotype combinations where CD28 (TT) and IFNG (AA) were always present, we
verified that the contribution of a third polymorphism did not reach statistical significance (p=0.1).
Conclusions
Further analysis suggested that gene-gene interaction between CD28 and IFNG might be
important for susceptibility to cervical cancer.
Background
There is overwhelming evidence that prolonged infection with oncogenic human
papillomavirus is the major factor associated with development of cervical cancer [1]. However,
considering that only a relatively small proportion of infected women develop cervical cancer, it is
conceivable that other environmental and/or genetic factors play a role in the susceptibility [2]. Since
the immune response has an important role in the defense against the virus, as well as against the
tumor, polymorphisms in genes that potentially affect the immune response are candidates to
influence the susceptibility to cervical cancer.
A series of publications on polymorphisms of HLA class II genes and cervical cancer and/or
its precursor lesions, cervical intraepithelial neoplasia (CIN), show that some HLA alleles are
associated with protection, while others are associated with susceptibility [3–10]. These associations
are probably explained by the role of HLA II molecules in presenting viral- or tumor- derived epitopes
to T CD4+ cells. More recently, interesting findings were reported concerning resistance/susceptibility
to cervical cancer mediated by combinations of killer immunoglobulin-like receptor (KIR) and their
HLA class I ligands [11].
As polymorphisms in genes coding for cytokines, cytokine receptors, co-stimulatory
molecules may affect the immune response [12–14], they are natural candidates to influence the
susceptibility to various diseases, including cancer.
The majority of the studies of association between immune response genes and cervical
cancer have analyzed single nucleotide polymorphisms (SNPs) in genes coding for tumor necrosis
factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 6 (IL-6), interleukin 10 (IL-10), and
transforming growth factor beta (TGF-β) [15–18]. Although some significant associations have been
reported in some studies, they were not consistently confirmed in other studies [19–23].
Bearing in mind that molecules involved in the immune response do not act in isolation, but
form a complex network of interacting proteins, the net immunologic response is most probably the
product of variation in many polymorphic genes. Therefore, it is of great importance to test the
combination of polymorphisms in different genes as a risk factor for diseases [24–27]. Any multi-locus
approach, however, directly or indirectly faces the problem that has been referred as “curse of
dimensionality” because, although the number of polymorphisms under study may not be very large,
the number of possible combinations between them turns to be extremely high. Some statistical
approaches for this problem were recently reviewed [28].
The purpose of this study was to investigate association between invasive cervical cancer and
polymorphisms in genes coding the following immune response molecules: CD28, CTLA-4 (cytotoxic
T-lymphocyte-associated protein 4), ICOS (inducible T cell co-stimulator), PDCD1 (programmed cell
death receptor-1, also called PD-1) FAS, TNFα, IL-6, IFNγ, TGFβ1 and IL-10. In addition to the
analysis of association with each polymorphism, we searched for association with two- and threepolymorphism
combinations, utilizing a new statistical approach. === BV UNIFESP: Teses e dissertações
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