<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis

Introduction

The goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).

Methods

A long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.

Results

Longitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.

Conclusions

Our data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.     

Genetic polymorphisms in TNFA/TNFR2 genes and Chagas disease in a Colombian endemic population

In this study we investigated the association of functional single nucleotide polymorphisms of tumor necrosis factor-alfa (TNFA) and TNF receptor 2 (TNFR2) genes in determining the susceptibility to Chagas' disease. This study included 313 patients from Colombia serologically positive for Trypanosoma cruzi antigens (cardiomyopathic, N=159; asymptomatic, N=154). Genotypes were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism method. We found a significant difference in the distribution of the TNFA -1031C (p=0.0153, OR=1.69, CI=1.10-2.58) and -308A (p=0.0002, OR=2.60, CI=1.53-4.39) alleles between cardiomyopathic and asymptomatic subjects. In addition, we observed that the TNFR2 +676T allele was monomorphic in our population. Our results suggest that TNFA -1031C and -308A gene polymorphisms may influence the susceptibility to develop chagasic cardiomyopathy in the population under study.     

Frequencies of the tumor necrosis factor gene polymorphisms in the Korean population

Tumor necrosis factor (TNF), a potent immuno-modulator and pro-inflammatory cytokine, has been implicated in many pathological processes. The TNFA and the TNFB genes, which encode TNFalpha and TNFbeta, respectively, are both located on the short arm of chromosome 6 between the class I and class II regions of the HLA complex. A striking feature of the entire HLA complex is a high degree of genetic variation. Two biallelic polymorphisms in the TNFA (- 308G/A) and TNFB (+ 252A/G) genes have been reported to be associated with TNF production and with susceptibility to inflammatory diseases. Population information on polymorphisms is essential for the study of genetic diseases. The aim of this study is to obtain accurate information about polymorphisms in the TNF genes in the Korean population. Allele frequencies of TNFA (- 308G/A) and TNFB (+ 252A/G) were measured in 581 unrelated Korean individuals by PCR-RFLP. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. A significant difference was found for the allele frequencies of TNFA and TNFB gene in Koreans compared with Europeans. The - 308/A allele in the TNFA gene was very rare in Asians (0.008-0.096). The frequency of the - 308/A allele in Koreans was considerably lower than in Europeans (0.120-0.189). Contrary to lower frequency of the -308/A allele, that of + 252/G allele in the TNFB gene was higher than in Koreans (0.445) compared with Europeans (0.29-0.39). The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common inflammatory diseases.     

A second susceptibility gene for developing rheumatoid arthritis in the human MHC is localized within a 70-kb interval telomeric of the TNF genes in the HLA class III region

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with a multifactorial genetic basis. However, pathogenic genes for RA other than the human leukocyte antigen (HLA)-DRB1 gene have yet to be identified. Here, we investigated whether there is a second susceptibility locus for RA within the human major histocompatibility complex using 18 microsatellite markers distributed from the centromeric (HSET) to the telomeric end (P5-15) of the 3.6-Mb HLA region. Statistical studies of associated alleles on each microsatellite locus showed that one pathogenic gene for RA in the HLA region is localized in the DRB1 gene, as expected. Further, a second susceptibility gene of RA was suggested to be present in the HLA class III region, narrowed to 70 kb, that is just telomeric of the TNF gene cluster (TNFA and LTA) and that is located between the microsatellites TNFa and C1-2-A. In this critical segment, four expressed genes have been thus far identified, NFKBIL1 (IkappaBL), ATP6G, BAT1, and MICB, all of which are candidate genes for determining susceptibility to RA. These results exclude the possibility of involvement of the TNFA genes (TNF-alpha) in the development of RA, which was suggested previously to be a strong candidate for RA in the class III region.     

PADI4 Haplotypes Contribute to mRNA Expression,the Enzymatic Activity of Peptidyl Arginine Deaminase and Rheumatoid Arthritis Risk in Patients from Western Mexico

Citrullination is catalyzed by the peptidyl arginine deiminase 4 (PAD4) enzyme, encoded by the PADI4 gene. Increased PAD4 activity promotes the onset and progression of rheumatoid arthritis (RA). This study aimed to evaluate the association of PADI4 haplotypes with RA risk, mRNA expression, and the PAD4 activity in patients with RA from Mexico. Methodology: 100 RA patients and 100 control subjects (CS) were included. Genotyping was performed by PCR-RFLP method, PADI4 mRNA expression was quantified by real-time PCR, the contribution of PADI4 alleles (PADI4_89 G>A, PADI4_90 T>C, and PADI4_92 G>C) to mRNA expression by the ASTQ method, and PAD4 activity by HPLC. Also, the anti-CCP and anti-PADI4 antibodies were quantified by ELISA. Results: The three PADI4 polymorphisms were associated with RA susceptibility (OR = 1.72, p = 0.005; OR = 1.62; p = 0.014; OR = 1.69; p = 0.009; respectively). The 89G, 90T, and 92G alleles have a higher relative contribution to PADI4 mRNA expression from RA patients than 89A, 90C, and 92C alleles in RA patients. Moreover, the GTG/GTG haplotype was associated with RA susceptibility (OR = 2.86; p = 0.024). The GTG haplotype was associated with higher PADI4 mRNA expression (p = 0.04) and higher PAD4 enzymatic activity (p = 0.007) in RA patients. Conclusions: The evaluated polymorphisms contribute to PADI4 mRNA expression and the enzymatic activity of PAD4 in leukocytes. Therefore, the GTG haplotype is a genetic risk factor for RA in western Mexico, and is associated with increased PADI4 mRNA expression and higher PAD4 activity in these patients.     

The MHC2TA -168A>G gene polymorphism is not associated with rheumatoid arthritis in Austrian patients

An association between susceptibility to rheumatoid arthritis (RA) and a common -168A>G polymorphism in the MHC2TA gene with differential major histocompatibility complex (MHC) II molecule expression was recently reported in a Swedish population. The objective of the present study was to replicate this finding by examining the -168A>G polymorphism in an Austrian case-control study. Three hundred and sixty-two unrelated RA cases and 351 sex-matched and age-matched controls as well as 1,709 Austrian healthy individuals were genotyped. All participants were from the same ethnic background. Genotyping was performed using 5' allelic discrimination assays. The association between susceptibility to RA and the -168A>G single nucleotide polymorphism was examined by chi-square test. Comparison was made assuming a dominant effect (AG + GG genotypes versus AA genotype). In contrast to the primary report, the frequency of MHC2TA -168G allele carriers was not significantly different between patients and controls in the Austrian cohort. The homozygous MHC2TA -168 GG genotype was more frequent in matched controls than in Austrian RA patients. There was no association between the presence of RA-specific autoantibodies and the MHC2TA -168 GG genotype. In this cohort of Austrian patients, no association between the MHC2TA polymorphism and RA was found.     

Differential allelic expression of the type II collagen gene (COL2A1) in osteoarthritic cartilage.

Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced < 12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in all three patients, and this allele is more frequent in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA.     

Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study

CD40-CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.     

Polymorphism and signatures of selection in the multimammate rat DQB gene

In order to test if DQB is a good candidate marker to investigate the relationship between major histocompatibility complex genes and pathogens in natural populations of Mastomys natalensis, we assessed the polymorphism and evolutionary history of this gene. Twenty-four individuals were genotyped for exon 2 of DQB using capillary electrophoresis single-strand conformation polymorphism, cloning, and sequencing. We found 21 different alleles. Four individuals show three alleles implying a duplication event in the history of this gene. Each distinct sequence translates to give a distinct amino acid sequence and there are strong signals of positive selection on peptide binding sites. Signals of recombination were found in the sequences suggesting that recombination has played a role in generating allelic diversity. Although trans-taxon polymorphism is present at the interspecific level in DQB exon 2 sequences of Mus species, we did not find any evidence of allele sharing among Muridae genera. This indicates a temporal limit of DQB allele sharing in Muridae of less than 8 Mya. In conclusion, although DQB seems to be a good marker to investigate pathogen-driven selection, the polymorphism of gene copy number may restrict its utility in natural populations.     

Allelic variants of the tumor necrosis factor superfamily as markers of the severity of the course of chronic obstructive lung disease and bronchiectatic disease

The distribution of allelic variants of genes of the TNF superfamily (TNFA and LTA) was studied in 172 patients with chronic obstructive pulmonary disease (COPD), bronchiectatic disease (n = 22), and in healthy individuals (n = 169). Analysis of the TNFA gene locus -308G-->A revealed no differences between the examined groups. Analysis of the LTA gene polymorphic locus +252A-->G showed that in patients with COPD, the frequency of the G allele was significantly higher than that in the control group (chi 2 = 3.98, p < 0.05). The presence of this allele in the genotype was correlated with the degree of COPD severity. Thus, in patients with stage II COPD, heterozygous AG genotype predominated (51.3%), whereas in patients with stage III COPD, the frequency of AG genotype was reduced to 32.7% at the expense of increased frequency of GG genotype (14.6%) (chi 2 = 6.78, p < 0.05; OR = 4.6, CI 1.37-15.96). The distribution of combined TNFA and LTA genotypes was also studied. In the group of COPD patients, the proportion of individuals with a combination of normal GG TNFA genotype and heterozygous AG LTA genotype was significantly higher (28.5% versus 18.4% in control; chi 2 = 4.14, p < 0.05; OR = 1.75, CI = 1.01-3.04). Genotype combinations were characterized at various clinical stages of COPD and bronchiectatic disease (BED). Thus, we have shown for the first time ever that LTA gene alleles and their combinations with the polymorphic variants of the TNFA gene are associated with predisposition to COPD and severity of this disease.     

Gene polymorphisms and serum levels of TL1A in patients with rheumatoid arthritis

Recent findings showed elevated expression of tumor necrosis factor (TNF)-like ligand 1A (TL1A) in rheumatoid arthritis (RA) patients and arthritis mice. However, whether TL1A gene polymorphisms may correlate with RA susceptibility needs to be discussed. This case-control study was performed on 350 RA patients and 556 healthy subjects to identify TL1A genetic variants (rs3810936, rs6478109, and rs7848647) and their possible association with TL1A levels, susceptibility to and severity of RA. Odds ratio and 95% confidence interval were calculated to represent the correlation between TL1A polymorphisms and RA. The TL1A serum levels were evaluated. Results showed that frequencies of TC, TT + TC genotypes of rs3810936, rs7848647 in RA patients were significantly lower in RA patients compared with controls. Patients with C allele showed more severe disease course (disease activity index: erythrocyte sedimentation rate, rheumatoid factor) than in carriers of T allele. However, the allele or genotype frequencies of rs6478109 were not associated with RA. In addition, TL1A genetic variants conferred higher TL1A levels in RA patients compared with controls. In conclusion, these findings indicated an association between TL1A rs3810936, rs7848647 variation and the susceptibility of RA in a sample of Chinese individuals, and TL1A may correlate with severity of RA.     

Rapid screening method of abnormal insulin-receptor gene expression: allele-specific oligonucleotide hybridization by using silent polymorphisms

An asymmetrical reduction in the levels of the insulin receptor mRNA transcribed from one allele was reported in some patients with severe insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM). To detect this abnormality, we designed the less laborious method; Allele-specific oligonucleotide hybridization of the amplified mRNA (cDNA) by using silent polymorphisms in the insulin receptor gene (nucleotide positions at 1686 and 1698). The allelic frequencies of C-1686 and T-1686 were 0.63 and 0.37, respectively (0.60 and 0.40 in 10 normal subjects, and 0.67 and 0.33 in 20 NIDDMs; n.s.). Similarly, the allelic frequencies of A-1698 and G-1698 were 0.47 and 0.53, respectively (0.50 and 0.50 in the normal subjects, and 0.45 and 0.55 in the NIDDMs; n.s.). These results suggest that these two polymorphisms are very common in Japanese. Nineteen (64%) out of 30 cases are heterozygous at one or two position(s), suggesting that it is possible to distinguish the mRNA transcribed from each of two alleles of the insulin receptor gene with using allele-specific oligonucleotide hybridization. Although we successfully measured the ratio of mRNA expression from two alleles of the gene in 20 NIDDMs, there was not patient whose mRNA transcribed from one allele of the insulin receptor gene was extremely decreased. We showed that allele-specific oligonucleotide hybridization method is useful for the screening of abnormal insulin-receptor gene expression.     

Polymorphism in vitamin D receptor and osteoprotegerin genes in Egyptian rheumatoid arthritis patients with and without osteoporosis

1α,25-Dihydroxyvitamin D3 upregulates the expression of the receptor activator of nuclear factor kB ligand (RANKL), and downregulates osteoprotegerin (OPG) expression. We tested the effects of polymorphisms in the vitamin D receptor gene (VDR), and OPG gene in rheumatoid arthritis (RA) patients and healthy controls and their relationship to bone mineral density (BMD) and development of osteoporosis. Three hundred and fifty women were evaluated, 200 women having RA and 150 healthy control. The subjects were genotyped for polymorphism at BsmI in VDR and A163G in OPG genes by polymerase chain reaction followed by restriction fragment length polymorphism analysis. BMD was also measured. In A163G, the G allele increased the risk for RA and for the development of osteoporosis. We found a significant association between lower hip (BMD-h) and genotype variants of VDR (BsmI) and OPG A163G in RA patients with osteoporosis. Our results suggested that OPG A163G polymorphism was associated with RA susceptibility and with the development of osteoporosis in these patients. Also, VDR and OPG genes are important candidates for osteoporosis in RA patients.     

Parasite-mediated selection drives an immunogenetic trade-off in plains zebras (Equus quagga)

Pathogen evasion of the host immune system is a key force driving extreme polymorphism in genes of the major histocompatibility complex (MHC). Although this gene family is well characterized in structure and function, there is still much debate surrounding the mechanisms by which MHC diversity is selectively maintained. Many studies have investigated relationships between MHC variation and specific pathogens, and have found mixed support for and against the hypotheses of heterozygote advantage, frequency-dependent or fluctuating selection. Few, however, have focused on the selective effects of multiple parasite types on host immunogenetic patterns. Here, we examined relationships between variation in the equine MHC gene, ELA-DRA, and both gastrointestinal (GI) and ectoparasitism in plains zebras (Equus quagga). Specific alleles present at opposing population frequencies had antagonistic effects, with rare alleles associated with increased GI parasitism and common alleles with increased tick burdens. These results support a frequency-dependent mechanism, but are also consistent with fluctuating selection. Maladaptive GI parasite ‘susceptibility alleles’ were reduced in frequency, suggesting that these parasites may play a greater selective role at this locus. Heterozygote advantage, in terms of allele mutational divergence, also predicted decreased GI parasite burden in genotypes with a common allele. We conclude that an immunogenetic trade-off affects resistance/susceptibility to parasites in this system. Because GI and ectoparasites do not directly interact within hosts, our results uniquely show that antagonistic parasite interactions can be indirectly modulated through the host immune system. This study highlights the importance of investigating the role of multiple parasites in shaping patterns of host immunogenetic variation.     

HLA and beta-myosin heavy chain do not influence susceptibility to Chagas disease cardiomyopathy

An inflammatory dilated cardiomyopathy occurs in 30% of Chagas' disease patients, chronically infected by Trypanosoma cruzi, while the remaining infected individuals are asymptomatic. Studies have indicated a role for genetic factors in the susceptibility to Chagas' disease cardiomyopathy. In an attempt to identify the genetic factors influencing the development and outcome of Chagas' cardiomyopathy, we compared the frequencies of alleles from two candidate gene loci, class II HLA and a microsatellite marker for the human cardiac beta-myosin heavy chain gene in different clinical groups. Patients were grouped as asymptomatic or with severe or mild cardiomyopathy. The results indicate that the HLA and myosin microsatellite allele profiles in all cardiomyopathy and in asymptomatic groups are similar. In conclusion, these results establish that polymorphism of HLA-DR and -DQ molecules, as well as beta-cardiac myosin, do not influence the susceptibility to different clinical forms of Chagas' disease or the progression to severe Chagas' cardiomyopathy. On the other hand, male sex was identified as a risk factor for progression to the more severe forms of cardiomyopathy (relative risk = 8.75).