Berta Martins

Evidence for CTLA4 as a susceptibility gene for systemic lupus erythematosus.

Several lines of evidence implicate the Cytotoxic T Lymphocyte Antigen 4 (CTLA4) gene in susceptibility to autoimmune disease. We have examined the association of systemic lupus erythematosus (SLE) with polymorhisms within the CTLA4 gene that were previously proposed to regulate CTLA-4 function: a single nucleotide polymorphism (SNP) in position +49 of exon 1 and a dinucleotide repeat in the 3′ untranslated region (3’UTR). The 3′UTR repeat showed a significant association with SLE, with one allele conferring susceptibility and another conferring protection to the disease. The associated alleles do not support previous suggestions of an allele size-dependent effect of the 3’ UTR polymorphism in autoimmunity development and instead suggest that it is in linkage disequilibrium with a true causative locus. No association of the exon 1 SNP with SLE was found in our population. Given the conflicting results obtained in different studies on the association of SLE with this polymorphism, we performed a meta-analysis including seven previously published studies and the present one. Significantly increased and decreased risks for SLE were found for carriers of the G allele and the A allele, respectively. The functional characterization of disease-associated CTLA4 gene variants is now required to elucidate their role in the pathogenesis of SLE and other autoimmune diseases.

Compensatory T-Cell Regulation in Unaffected Relatives of SLE Patients, and Opposite IL-2/CD25-Mediated Effects Suggested by Coreferentiality Modeling

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4+CD25bright T-cells that were regularly 70–90% Foxp3+. We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

HLA class II alleles as markers of tuberculosis susceptibility and resistance

BACKGROUND Not every individual exposed to Mycobacterium tuberculosis becomes infected. One host genetic factor, involved in modulating the immune response that has been studied in many ethnic groups is the association of human leukocyte antigens (HLA) with susceptibility to tuberculosis (TB). OBJECTIVE To investigate the association between TB, HLA-DRB1 and HLA-DQB1 alleles in a Portuguese population. METHODS HLA-DRB1 and HLA-DQB1 gene polymorphisms were analyzed by PCR-SSP in 92 TB patients, and 82 healthcare professionals without TB but exposed on a daily basis to infectious patients for more than two years (healthy exposed - HE). Tuberculin skin test reaction (TST), was positive in 69 individuals (all over 15 mm) in the HE group (HE+) and negative in thirteen (HE-). RESULTS HLA-DRB1*14 frequency is higher in the TB patients group (7 % vs. 0; p = 0.038) than in HE+. CONCLUSIONS No genetic marker clearly indicative of disease susceptibility or resistance was identified in this study. However, HLA-DRB1*14 was more frequent in TB patients suggesting that it may be involved in the evolution infection towards active TB in our population.

Cognitive Functioning in Behcet's Disease

The impact of Behçets disease on higher cognitive functions is still poorly understood. We proposed (1) to characterize the neuropsychological profile of Behçets disease patients with (Neuro‐BD) and without (BD) neurological manifestations; (2) to identify which clinical, psychopathological, and genetic variables are related to neuropsychological performance; and (3) to explore the association between cognitive functioning and neuroimaging findings in BD patients. Fifteen Neuro‐BD and 35 BD patients in the nonactive phase of their illness underwent a neurological examination, performed a comprehensive battery of neuropsychological tests, and answered the hospital anxiety and depression scale. Human leukocyte antigen (HLA)‐B*51 genotyping was also performed. Patients’ neuropsychological performances were compared to those of healthy demographically matched subjects. Within one month from the testing date, a subset of 20‐BD patients underwent a magnetic resonance imaging (MRI) scan. Fifty‐three percent of Neuro‐BD and 40% of BD patients were impaired at least on one neuropsychological measure (i.e., digit span–forward). Poorer cognitive functioning in Neuro‐BD was associated with parenchymal involvement, whereas in BD it was related to presence of white matter changes in the frontal lobes, history of headache complaints, or higher levels of anxiety and depression. Current prednisone intake had a positive impact on neuropsychological performance. Disease duration, time since onset of neurological manifestations, or presence of HLA‐B*51 allele had no significant influence. Our results indicate that Behçets disease may affect cognitive abilities in the absence of overt neurological symptoms. These findings point to an insidious course of neurological involvement.

Protective role of the HLA-A*02 allele in Portuguese patients with multiple sclerosis.

Background Multiple sclerosis (MS) is associated with human leukocyte antigen (HLA) HLA–DRB1*15. Recent evidence that CD8 T cells are implicated in MS suggests that HLA class I may also contribute. An association of HLA–A*02 and A*03 alleles has been described. Objectives We examined the influence of HLA–A*02 and HLA–A*03 in Portuguese patients with MS, independently of HLA–DRB1*15 using a logistic regression model. Conclusions DRB1*15 increased the risk of developing MS and HLA–A*02 decreased the risk. A*03 had no effect. To analyze if HLA–A*02 association was independent from DRB1*15, an interaction between these two alleles was introduced in the model; no significant interaction was found.

HLA in Portuguese systemic lupus erythematosus patients and their relation to clinical features.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease translating the different genetic and environmental factors involved. Polymorphisms at several loci, including the major histocompatibility complex (MHC), have been associated worldwide with SLE, although inconsistencies exist among these studies mainly due to genetic heterogeneity between populations and sample characteristics. The aim of the present study was to investigate in Portuguese SLE the association of HLA‐DRB1 alleles with clinical patterns of the disease and severity. Two hundred eighteen Portuguese patients with SLE—42% of whom had kidney involvement—were studied for HLA‐DRB1. Clinical and laboratory manifestations were correlated with HLA allele frequencies. HLA‐DRB1 * 03 allele frequency was significantly higher in SLE patients—as a whole and as either with or without renal involvement—compared to controls, while HLA‐DRB1 * 09 and DRB1 * 13 allele frequencies were decreased. Regarding the relationship with the presence or absence of specific clinical manifestations, it was only found that HLA‐DRB1 * 08 allele frequency was increased in patients with neurological involvement. No association with the presence or absence of anti‐dsDNA, anti‐sm or antiphospholipid antibodies, or antiphospholipid syndrome, was observed. These results were reproducible when analysis was repeated only with patients with more than 5 years of evolution. As in other populations HLA‐DRB1 * 03 is a susceptibility allele in Portuguese SLE patients, while HLA‐DRB1 * 09 and DRB1 * 13 alleles may be protective alleles, not only for the disease, but for the development of nephritis. No correlations with the different clinical manifestations were found, except with the neurological system.

Association of HLA DQ4‐DR8 haplotype with papillary thyroid carcinomas

Objective  The association of the human leucocyte antigen (HLA) system with thyroid carcinomas is not clear. We sought to relate HLA alleles to susceptibility to papillary thyroid carcinoma (PTC) and also to clinical and pathological characteristics of PTC patients.

Two separate effects contribute to regulatory T-cell defect in SLE patients and their unaffected relatives

Forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)‐2 receptor alpha chain]. Low‐dose IL‐2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE Treg phenotype, we studied its role through developmentally defined regulatory T cell (Treg) subsets in 45 SLE patients, 103 SLE‐unaffected first‐degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25‐encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to Treg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4+FoxP3+CD45RO–CD31+ recent thymic emigrant Tregs. This first component effect influenced the proportions of circulating CD4+FoxP3highCD45RO+ activated Tregs. (2) In contrast, patients and unaffected relatives differed sharply in their activated Treg CD25 state: while relatives as control subjects up‐regulated CD25 strongly in these cells during differentiation from naive Tregs, SLE patients specifically failed to do so. This CD25 up‐regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated Tregs, but not to their circulating numbers. Both effects were found related to T cell IL‐2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early Tregs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up‐regulation upon peripheral Treg activation that is selectively deficient in patients. We expect that Treg‐directed therapies can be monitored more effectively when taking this distinction into account.

Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives: Two effects contribute to SLE Treg defect

Forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)‐2 receptor alpha chain]. Low‐dose IL‐2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE Treg phenotype, we studied its role through developmentally defined regulatory T cell (Treg) subsets in 45 SLE patients, 103 SLE‐unaffected first‐degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25‐encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to Treg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4+FoxP3+CD45RO–CD31+ recent thymic emigrant Tregs. This first component effect influenced the proportions of circulating CD4+FoxP3highCD45RO+ activated Tregs. (2) In contrast, patients and unaffected relatives differed sharply in their activated Treg CD25 state: while relatives as control subjects up‐regulated CD25 strongly in these cells during differentiation from naive Tregs, SLE patients specifically failed to do so. This CD25 up‐regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated Tregs, but not to their circulating numbers. Both effects were found related to T cell IL‐2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early Tregs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up‐regulation upon peripheral Treg activation that is selectively deficient in patients. We expect that Treg‐directed therapies can be monitored more effectively when taking this distinction into account.

Authors' response: Association of HLA DQ4‐DR8 haplotype with papillary thyroid carcinomas

Dear Editor, Drs Machens and Dralle 1 question the significant association of the human leucocyte antigen (HLA)-DQ4 allele with papillary thyroid carcinomas (PTC) reported in our study. Their argument is that differences in age and gender between patients and controls could have biased the results. We acknowledge that our healthy controls were mainly blood donors who are predominantly male and younger than PTC patients. However, we do not think that these two variables, age and gender, could have produced a spurious association between the HLA-DQ4 and PTCs, as suggested by Drs Machens and Dralle, due to four main reasons: (1) the HLA protein complex is encoded by genes on chromosome 6, and therefore there is no difference in the distribution of HLA alleles between the sexes; (2) there were no significant differences of HLA allele distribution in relation to age and gender in our PTC patients; (3) HLA-DQ4 frequency was not increased in our patients with follicular carcinomas who, like patients with PTC, are also predominantly female and older than our healthy controls; and (4) a preliminary study performed in a Turkish population confirmed the HLA-DQ4 phenotype as a risk factor for PTC development. 2