Bases moleculares del lupus eritematoso sistémicoIdentificación de marcadores genéticos

Resumo

Systemic lupus erythematosus (SLE) is the prototype of systemic autoimmune disease, with a complex pathogenesis involving multiple genetic and environmental factors. It is characterized by a diverse array of clinical symptoms, indicative of widespread immune-mediated damage. It is also a heterogeneous disease, presenting differently from patient to patient and with no single clinical or immunological feature required to make a formal diagnosis. The pathogenesis behind the disease remains unclear. The main immunological feature is uncontrolled formation of autoantibodies, leading to excess formation of immune complexes which deposit in different tissues, causing inflammation and tissue damage. The disease primarily affects women in their reproductive years and the estimated prevalence varies between 12 and 64 cases per 100.000 inhabitants in European-derived populations, with a higher prevalence, in general, in non-European-derived populations. For at least 30 years we have known that there is a strong genetic component to SLE: a disease concordance of 2-5% in dizygotic compared with 30-50% in monozygotic twins and a sibling risk ratio (¿s) of 20-29. Progress in identifying these genetic factors was initially slow. Most of the genetic factors proposed to date to be involved in SLE have been analyzed by association studies in unrelated patients and controls. Until recently, the investigations of genetic effects in SLE have been mostly association studies of HLA genes. The alleles HLA-DRB1*0301, HLA-DRB1*1501 y HLA-B8 in the MHC have been consistently associated in SLE. The publication of the human genome sequence in 2001 have stimulated renewed interest in genetic. In the last years, genome-wide linkage studies have shown several loci out of the MHC region with association to SLE, and this has accelerated over the last 12 months with the publication of several high-density genome wide association studies (GWAS) and the identification of several novel candidate genes through fine mapping studies. The main aim in this doctoral thesis has been to try to identify new genetic markers of susceptibility to SLE using case-control association studies of candidate genes, thus helping to elucidate the genetic basis of this complex disease. The strategies used to select the candidates genes to study have been mainly two: positional, based on genes that have previously been found in regions of susceptibility to the disease through linkage studies or genes that are found in areas associated with susceptibility in animal models of SLE; and functional, which is to select genes that may have an implication in the pathophysiological mechanisms of the disease as well as genes involved in inflammatory or autoimmune related diseases. To perform these studies we have used several cohorts of SLE patients and healthy controls from different Spanish regions (Granada, Málaga, Sevilla, Lugo and more recently Sabadell, Valencia and Oviedo) and on time worked in collaboration with various international groups from Germany, Italy, Sweden, Argentina and Mexico. Some of the genes studied in this thesis were selected by the strategy of candidate genes. Among these we studied genes involved in the inflammatory response, because this is a fundamental mechanism in the initiation and perpetuation of the disease. In this group were selected TLR2, TLR4, TLR5 and TLR7 genes, which are surface receptors that play an essential role in the activation and regulation of the innate and adaptative immune response leading to activation of a large number of inflammatory mediators through the activation of the transcription factor NF¿B. Taking into account the important role of the transcription factor NF¿B in the activation of the inflammatory response, we decided also study the NFKB1 gene, that encodes the p50 subunit of the molecule NF¿B, with susceptibility to SLE. Fc¿Rs genes are essential mediators of inflammatory effects, cytotoxic antibodies and immune complexes and relate the innate immune system and acquired one another. Within this group we studied the FCRL3 gene, which belongs to this family of genes (FcgR) and they are believed to have a vital role in increasing the presence of self-reactive B cells. In addition, within these genes involved in inflammatory response we selected a number of cytokines that promote a Th1 response and thus lead to a situation of inflammation, such as genes of the family of the interleukin 12 (IL-12) : IL12B, IL12RB1, IL23A and IL23R genes and two genes that encodes two pro-inflammatory cytokines: IL18 and MIF genes. Regard the MHC region, where the classical HLA genes are, there are also a large numbers of genes very close together characterized of being extended haplotypes that may influence in the susceptibility to disease by increasing the presentation of the immunogenic peptide epitopes in the periphery, resulting in increased activation of T cells, or the presentation of ineffective autoantigen in the thymus, giving rise to more aggressive T cells or less number of regulatory T cells (Treg). The genes studied in this region were the BTNL2 and MICA genes. Other group of selected genes was genes implicated in the regulation of T and B cells. We have selected in this group the PTPN22 gene, which encodes a lymphoid-specific phosphatasa (Lyp). PTPN22 is a key molecule regulating TCR signalling in T lymphocytes and in recent times has been considered as an important gene in autoimmunity. Other selected gene is CD24, which belong to a family of proteins involved in signal transduction by members of the protein tyrosine kinases (PTKs) and is involved in the activation and differentiation of T and B cells. Finally, we selected a series of genes that have been identified through the new GWAS as genes involved in SLE susceptibility and are also implicated in the pathogenesis of the disease. Under this heading, the studied genes were: BANK1, a gene involved in signalling through the BCR; STAT4, a gene that encodes for a transcription factor that mediates the expression of genes in important disease pathways; TRAF1, which is suggested to be a negative regulator of TNF-Receptor signalling and C5, a central component of the complement pathway. Our results show that genes which encodes the pro-inflammatory cytokines MIF and IL-18 are associated with susceptibility to SLE. We have found that the -173C allele and the -173C-CAAT7 haplotype of the MIF gene are associated with high risk to development SLE. In addition, these variations lead to an increased production of MIF which leads to a series of inflammatory processes that trigger the onset of the autoimmune disease. On the other hand, the IL18 -1297C allele was also associated with susceptibility to SLE. Besides, additional functional studies conducted revealed the importance of this allele in the expression of IL-18. Regard to genes involved in T and B cells regulation, we note that the PTPN22 R620W gene polymorphism, which alters the regulation of T cells and has been proposed as a common susceptibility marker to several autoimmune diseases, is strongly associated with susceptibility to SLE in our population. In addition, we found that the CD24 A57V polymorphism is also associated with risk to SLE, and this association has been replicated in an independent cohort from Germany. Within the genes selected by GWAS, we found that the BANK1 and STAT4 genes (works done in collaboration with Dr. Marta Alarcón-Riquelme group, Uppsala University, Sweden), which are implicated in T and B cells regulation, are associated with SLE. Our analysis suggests that three genetic variants of the BANK1 gene, either individually or as haplotypes, confer susceptibility for SLE and functional studies have demonstrated the functional relevance of two of them. With regard to STAT4, we found five polymorphisms associated with SLE in the Spanish population and four of them have been replicated in five independent populations. In addition, we do find a correlation with expression levels of STAT4 in peripheral blood cells and the three most associated SNPs. Finally, in the TRAF1/C5 locus we have found a polymorphism (rs10818488) associated with susceptibility to SLE and this association was replicated in an independent cohort from Crete. This latest work was conducted in collaboration with Dr. Rene Toes group, Leiden University, The Netherlands. Regarding to the rest of genetic variants tested, we did not find evidences of association with the candidate genes TLR2, TLR4, TLR5, TLR7, NF¿B, FCRL3, IL12B, IL12RB1, IL23A, IL23R, MICA and BTNL2 and SLE. Therefore, we suggest that these genes do not play a critical role in genetic predisposition to SLE in our population.