Helena Alves

Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene

The tyrosine phosphatase PTPN22 allele 1858T has been associated with rheumatoid arthritis (RA) and other autoimmune diseases. RA is the most frequent of those multifactorial diseases. The RA association was usually restricted to serum rheumatoid factor positive disease (RF+). No interaction was shown with HLA-DRB1, the first RA gene. Many case-control studies replicated the RA association, showing an allele frequency increase of ≈5% on average and large variations of population allele frequencies (2.1–15.5%). In multifactorial diseases, the final proof for a new susceptibility allele is provided by departure from Mendels law (50% transmission from heterozygous parents). For PTPN22–1858T allele, convincing linkage proof was available only for type 1 diabetes. We aimed at providing this proof for RA. We analyzed 1,395 West European Caucasian individuals from 465 “trio” families. We replicated evidence for linkage, demonstrating departure from Mendels law in this subset of early RA onset patients. We estimated the overtransmission of the 1858T allele in RF+ families: T = 63%, P < 0.0007. The 1858T allele frequency increased from 11.0% in controls to 17.4% in RF+ RA for the French Caucasian population and the susceptibility genotype (1858T/T or T/C) from 20.2% to 31.6% [odds ratio (OR) = 1.8 (1.2–2.8)]. In conclusion, we provided the linkage proof for the PTPN22–1858T allele and RF+ RA. With diabetes and RA, PTPN22 is therefore a “linkage-proven” autoimmunity gene. PTPN22 accounting for ≈1% of the RA familial aggregation, many new genes could be expected that are as many leads to definitive therapy for autoimmune diseases.

Primary association of tumor necrosis factor–region genetic markers with susceptibility to rheumatoid arthritis

OBJECTIVE To determine whether tumor necrosis factor (TNF) polymorphisms are associated with susceptibility to rheumatoid arthritis (RA) independently of the HLA-DR shared epitope. METHODS Fifty-two Spanish families with one or more affected members were typed for HLA-DRB1, TNF promoter polymorphisms, and TNFa and TNFb microsatellites. We performed an association analysis comparing transmitted versus not transmitted haplotypes, with or without shared epitope, to determine whether there is an independent effect of TNF genetic markers on RA susceptibility. RESULTS TNFa6,b5 was significantly associated with susceptibility to RA. The haplotypes containing these markers were preferentially transmitted to the affected offspring, even if these haplotypes lacked the HLA-DR shared epitope. TNF promoter polymorphisms were not associated with susceptibility to RA. CONCLUSION The data suggest that TNFa/b is an independent marker of RA susceptibility, pointing to a genetic role of the TNF region in the pathogenesis of RA.

Major histocompatibility complex class I associations in iron overload: evidence for a new link between the HFE H63D mutation, HLA‐A29, and non‐classical forms of hemochromatosis.

Abstract The present study is an analysis of the frequencies of HFE mutations in patients with different forms of iron overload compared with the frequencies found in healthy subjects from the same region. The frequencies of HLA-A and -B antigens and HLA haplotypes were also analyzed in the same subjects. The study population included: 71 healthy individuals; 39 genetically and clinically well-characterized patients with genetic hemochromatosis (HH); and 25 patients with non-classical forms of iron overload (NCH), excluding secondary hemochromatosis. All subjects were HLA-typed and HFE-genotyped by the oligonucleotide ligation assay (OLA). The gene frequencies found for the C282Y and H63D mutations of HFE were respectively: 0.03 and 0.23 in healthy individuals, 0.86 and 0.04 in HH patients, and 0.08 and 0.48 in NCH patients. An expected significant association between HH and HLA-A3 was observed, which was found to be in linkage disequilibrium with the C282Y mutation. A new association was seen, however, between HLA-A29 and NCH, in linkage disequilibrium with the H63D mutation. Again as expected, the HLA-B antigen B7 was associated with HH in linkage disequilibrium with HLA-A3. In addition, the HLA-B antigen B44 was found to be associated with NCH but not in linkage disequilibrium with either A29 or the H63D mutation. In conclusion, a new association of the HFE H63D mutation with forms of hemochromatosis other than HH and a new association between the HLA phenotype A29 and the HFE H63D mutation were found in the same patients. These findings reinforce evidence for the involvement of the major histocompatibility class I in iron metabolism, supporting the notion of a physiological role for the immunological system in the regulation of iron load.

Primary association of a MICA allele with protection against rheumatoid arthritis

OBJECTIVE To determine whether major histocompatibility complex class I chain-related gene A (MICA) polymorphisms are associated with susceptibility to rheumatoid arthritis (RA) independently of the HLA-DRB1 shared epitope (SE). METHODS Fifty-four Spanish families with an affected son or daughter and 211 consecutive RA patients were genotyped for HLA-DRB1, tumor necrosis factor a/b microsatellite alleles, and MICA transmembrane polymorphism. We performed a case-control comparison with the consecutive patients and an independent transmission disequilibrium test with the families. RESULTS The frequency of the MICA 6.0 allele was significantly reduced, compared with controls, in the group of SE+ patients (odds ratio 0.39, P = 0.0005). Additionally, the haplotypes containing this allele were preferentially not transmitted to the affected offspring (9 transmitted of 33; P = 0.007), independent of the presence or absence of an SE either in the same haplotype or in the other haplotype in the progenitor. CONCLUSION These data suggest that the MICA 6.0 allele is an independent marker of protection against RA in the SE+ group of RA patients.

The interferon regulatory factor 5 gene confers susceptibility to rheumatoid arthritis and influences its erosive phenotype

Background Increased expression of type I IFN genes, also referred to as an IFN signature, has been detected in various autoimmune diseases including rheumatoid arthritis (RA). Interferon regulatory factors, such as IRF5, coordinate type I IFN expression. Multiple IRF5 variants were suggested as autoimmunity susceptibility factors. Objective As the linkage proof remains important to establish fully any genetic RA susceptibility factor, the authors took advantage of the largest reported European trio family resource dedicated to RA to test for linkage IRF5 and performed a genotype–phenotype analysis. Methods 1140 European Caucasian individuals from 380 RA trio families were genotyped for IRF5 rs3757385, rs2004640 and rs10954213 single nucleotide polymorphisms (SNP). Results Single marker analysis provided linkage evidence for each IRF5 SNP investigated. IRF5 linked to RA with two haplotypes: the CTA risk haplotype ‘R’ (transmission (T)=60.6%, p=23.1×10−5) and the AGG protective haplotype ‘P’ (T=39.6%, p=0.0015). Linkage was significantly stronger in non-erosive disease for both IRF5 R and P haplotypes (T=73.9%, p=4.20×10−5 and T=19.6%, p=3.66×10−5, respectively). Multivariate logistic regression analysis found IRF5 linked to RA independently of the rheumatoid factor status. IRF5 RR and PP haplotypic genotypes were associated with RA, restricted to the non-erosive phenotype: p=1.68×10−4, OR 4.80, 95% CI 2.06 to 11.19; p=0.003, OR 0.17, 95% CI 0.05 to 0.57, respectively. Conclusion This study provides the ‘association and linkage proof’ establishing IRF5 as a RA susceptibility gene and the identification of a genetic factor that seems to contribute to the modulation of the erosive phenotype. Further studies are warranted to clarify the role of IRF5 in RA and its subphenotypes.

A study of 82 extended HLA haplotypes in HFE -C282Y homozygous hemochromatosis subjects: relationship to the genetic control of CD8+ T-lymphocyte numbers and severity of iron overload

BackgroundIt has been recently demonstrated that CD8+ T-lymphocyte numbers are genetically transmitted in association with the MHC class I region. The present study was designed with the objective of narrowing the region associated with the setting of CD8+ T-lymphocyte numbers in a population of C282Y homozygous hemochromatosis subjects, in whom a high prevalence of abnormally low CD8+ T-lymphocyte counts has been described.MethodsThe study includes 43 C282Y homozygous subjects fully characterized both phenotypically and genotypically. Clinical characterization includes measurements of iron parameters at diagnosis (transferrin saturation and serum ferritin), total body iron stores and T-cell immunophenotyping determined by flow cytometry. Genetic characterization includes HLA class I alleles (A, B and C) and four additional microsatellite markers (D6S265, D6S2222, D6S105 and D6S2239) spanning 5 Megabases in the 6p21.3 region.ResultsEighty-two extended C282Y carrying haplotypes were defined. Single-locus analysis revealed that the HLA-A region was associated with CD8+ T-cell numbers. Multivariate analysis showed that the combinations of the most common HLA-A alleles (HLA-A*03, -A*02 and -A*01) were associated with significantly lower numbers of CD8+ T-lymphocytes (0.30 ± 0.14 × 106/ml), in comparison with subjects carrying only one copy of those alleles (0.46 ± 0.19 × 106/ml) and subjects without any copy of those alleles (0.79 ± 0.15 × 106/ml;p = 0.0001). No differences were observed in CD8+ T-cell counts among control subjects carrying the same combinations of HLA-A alleles (0.47 ± 0.14; 0.45 ± 0.21 and 0.41 ± 0.17 × 106/ml, respectively), therefore not supporting a direct effect of HLA specificity but rather an indirect association with a locus close to HLA-A. Multivariate analysis showed that the combination of the most common HLA-A alleles also have an impact on the clinical expression of HH in terms of iron stores, in males(p = 0.0009).ConclusionThe present study provides evidence supporting an inextricable link between extended HLA haplotypes, CD8+ T-lymphocyte numbers and severity of iron overload in hereditary hemochromatosis(HH). It gives additional information to better define a candidate region involved in the regulation of CD8+ T-lymphocyte numbers. A new evolutionary hypothesis concerning the inheritance of the phenotype of low CD8+ T-lymphocyte numbers associated with particular ancestral HLA haplotypes carrying the C282Y mutation and its implication on the clinical heterogeneity of HH is discussed.

The ITGAV rs3738919-C allele is associated with rheumatoid arthritis in the European Caucasian population: a family-based study.

The integrin αvβ3, whose αv subunit is encoded by the ITGAV gene, plays a key role in angiogenesis. Hyperangiogenesis is involved in rheumatoid arthritis (RA) and the ITGAV gene is located in 2q31, one of the suggested RA susceptibility loci. Our aim was to test the ITGAV gene for association and linkage to RA in a family-based study from the European Caucasian population.Two single nucleotide polymorphisms were genotyped by PCR-restriction fragment length polymorphism in 100 French Caucasian RA trio families (one RA patient and both parents), 100 other French families and 265 European families available for replication. The genetic analyses for association and linkage were performed using the comparison of allelic frequencies (affected family-based controls), the transmission disequilibrium test, and the genotype relative risk.We observed a significant RA association for the C allele of rs3738919 in the first sample (affected family-based controls, RA index cases 66.5% versus controls 56.7%; P = 0.04). The second sample showed the same trend, and the third sample again showed a significant RA association. When all sets were combined, the association was confirmed (affected family-based controls, RA index cases 64.6% versus controls 58.1%; P = 0.005). The rs3738919-C allele was also linked to RA (transmission disequilibrium test, 56.5% versus50% of transmission; P = 0.009) and the C-allele-containing genotype was more frequent in RA index cases than in controls (RA index cases 372 versus controls 339; P = 0.002, odds ratio = 1.94, 95% confidence interval = 1.3–2.9).The rs3738919-C allele of the ITGAV gene is associated with RA in the European Caucasian population, suggesting ITGAV as a new minor RA susceptibility gene.

Association of the X-Chromosomal Genes TIMP1 and IL9R with Rheumatoid Arthritis

Objective. Rheumatoid arthritis (RA) is an inflammatory joint disease with features of an autoimmune disease with female predominance. Candidate genes located on the X-chromosome were selected for a family trio-based association study. Methods. A total of 1452 individuals belonging to 3 different sample sets were genotyped for 16 single-nucleotide polymorphisms (SNP) in 7 genes. The first 2 sets consisted of 100 family trios, each of French Caucasian origin, and the third of 284 additional family trios of European Caucasian origin. Subgroups were analyzed according to sex of patient and presence of anti-cyclic citrullinated peptide (anti-CCP) autoantibodies. Results. Four SNP were associated with RA in the first sample set and were genotyped in the second set. In combined analysis of sets 1 and 2, evidence remained for association of 3 SNP in the genes UBA1, TIMP1, and IL9R. These were again genotyped in the third sample set. Two SNP were associated with RA in the joint analysis of all samples: rs6520278 (TIMP1) was associated with RA in general (p = 0.035) and rs3093457 (IL9R) with anti-CCP-positive RA patients (p = 0.037) and male RA patients (p = 0.010). A comparison of the results with data from whole-genome association studies further supports an association of RA with TIMPL The sex-specific association of rs3093457 (IL9R) was supported by the observation that men homozygous for rs3093457-CC are at a significantly higher risk to develop RA than women (risk ratio male/female = 2.98; p = 0.048). Conclusion. We provide evidence for an association of at least 2 X-chromosomal genes with RA: TIMP1 (rs6520278) and IL9R (rs3093457).

Testing for the association of the KIAA1109/Tenr/IL2/IL21 gene region with rheumatoid arthritis in a European family-based study

IntroductionA candidate gene approach, in a large case–control association study in the Dutch population, has shown that a 480 kb block on chromosome 4q27 encompassing KIAA1109/Tenr/IL2/IL21 genes is associated with rheumatoid arthritis. Compared with case–control association studies, family-based studies have the added advantage of controlling potential differences in population structure. Therefore, our aim was to test this association in populations of European origin by using a family-based approach.MethodsA total of 1,302 West European white individuals from 434 trio families were genotyped for the rs4505848, rs11732095, rs6822844, rs4492018 and rs1398553 polymorphisms using the TaqMan Allelic discrimination assay (Applied Biosystems). The genetic association analyses for each SNP and haplotype were performed using the Transmission Disequilibrium Test and the genotype relative risk.ResultsWe observed evidence for association of the heterozygous rs4505848-AG genotype with rheumatoid arthritis (P = 0.04); however, no significance was found after Bonferroni correction. In concordance with previous findings in the Dutch population, we observed a trend of undertransmission for the rs6822844-T allele and rs6822844-GT genotype to rheumatoid arthritis patients. We further investigated the five SNP haplotypes of the KIAA1109/Tenr/IL2/IL21 gene region. We observed, as described in the Dutch population, a nonsignificant undertransmission of the AATGG haplotype to rheumatoid arthritis patients.ConclusionsUsing a family-based study, we have provided a trend for the association of the KIAA1109/Tenr/IL2/IL21 gene region with rheumatoid arthritis in populations of European descent. Nevertheless, we failed to replicate a significant association of this region in our rheumatoid arthritis family sample. Further investigation of this region, including detection and testing of all variants, is required to confirm rheumatoid arthritis association.

Co-selection of the H63D mutation and the HLA-A29 allele: a new paradigm of linkage disequilibrium?

The major histocompatibility complex (MHC) shows a remarkable conservation of particular HLA antigens and haplotypes in linkage disequilibrium in most human populations, suggesting the existence of a convergent evolution. A recent example of such conservation is the association of particular HLA haplotypes with the HFE mutations. With the objective of exploring the significance of that association, the present paper offers an analysis of the linkage disequilibrium between HLA alleles or haplotypes and the HFE mutations in a Portuguese population. Allele and haplotype associations between HLA and HFE mutations were first reviewed in a population of 43 hemochromatosis families. The results confirmed the linkage disequilibrium of the HLA haplotype HLA-A3-B7 and the HLA-A29 allele, respectively, with the HFE mutations C282Y and H63D. In order to extend the study of the linkage disequilibrium between H63D and the HLA-A29-containing haplotypes in a normal, random population, an additional sample of 398 haplotypes was analyzed. The results reveal significant linkage disequilibrium between the H63D mutation and all HLA-A29-containing haplotypes, favoring the hypothesis of a co-selection of H63D and the HLA-A29 allele itself. An insight into the biological significance of this association is given by the finding of significantly higher CD8+ T-lymphocyte counts in subjects simultaneously carrying the H63D mutation and the HLA-A29 allele.