Association of hyperhomocysteinemia with genetic variants in key enzymes of homocysteine metabolism and methotrexate toxicity in rheumatoid arthritis patients.

OBJECTIVES: The study investigated the association between plasma homocysteine, folate and vitamin B12 with 5,10 methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TYMS 2R â†’ 3R) and methionine synthase (MTR A2756G) polymorphisms and methotrexate (MTX) treatment and toxicity in Tunisian Rheumatoid arthritis (RA) patients. METHODS: A total of 185 patients with RA were included. Homocysteine (Hcy) was assessed by fluorescence polarization immunoassay, and folate and vitamin B12 were measured by chemiluminescence immunoassays. The genetic polymorphisms were analyzed by PCR or PCR-RFLP. Hyperhomocysteinemia (HHC) was considered for Hcy > 15 µmol/L. RESULTS: MTHFR C677T polymorphism was associated with HHC in RA patients (multi-adjusted OR, 95% CI 2.18, [1.07-4.57]; p = 0.031). No association was detected with the remaining polymorphisms. Plasma Hcy, folate, and vitamin B12 did not differ according to each polymorphism, or with MTX treatment or toxicity. However, HHC was more prevalent in patients with than those without MTX toxicity (32.7 vs. 16.7%; p = 0.035). CONCLUSIONS: The MTHFR 677TT genotype is an independent risk factor for HHC in Tunisians RA patients. HHC could be a useful marker of MTX toxicity in RA patients.

Measurement of absolute copy number variation of Glutathione S-Transferase M1 gene by digital droplet PCR and association analysis in Tunisian Rheumatoid Arthritis population.

BACKGROUND: The investigation of copy number variations (CNVs) analysis of candidate genes is currently an important research area in modulating human diseases. We aimed to quantify CNVs in glutathione S-transferase M1 (GSTM1) gene and determine its genetic contribution in Tunisian rheumatoid arthritis (RA) and its subsets through an innovative technique for quantification. METHODS: A total of 165 RA cases and 102 healthy controls were included in the study. Using a recently powerful approach of digital droplet PCR (ddPCR), we quantified GSTM1 gene to determine the presence of no, one, or multiple copy number (CN) at high levels of sensitivity and specificity. Odds ratio and Fisher exact test were performed to estimate the association risk for GSTM1CNVs in RA. RESULTS: Copy number identified by ddPCR was 0, 1, and 2 copies per diploid genome. A high frequency of '0' copy was revealed with 54% in RA patients. The deletion ('0' copy) of GSTM1 was found to be a significant risk factor for anti-cyclic citrullinated peptide (anti-CCP) positive RA (OR=4.16, CI95% =[1.17-14.7]). In addition, a lack of association was found when comparing between the CNVs of RA patients and those of controls. CONCLUSION: This study highlights the powerful accuracy of ddPCR for the quantification of CNVs and suggests that the variation in the CN of GSTM1 is associated with anti-CCP positivity in RA. However, it does not indicate a specific role in the susceptibility to the disease in our Tunisian sample.

Anticyclic citrullinated peptide antibody and rheumatoid factor in south Tunisian patients with rheumatoid arthritis: association with disease activity and severity.

OBJECTIVE: To explore relationships between immunological status, clinical features, radiographic damage, disease activity, and functional disability in Tunisian patients with rheumatoid arthritis (RA). MATERIALS AND METHODS: The study was carried out in 112 patients with RA. Demographic characteristics, disease duration, disease activity score 28 (DAS28), the Health Assessment Questionnaire (HAQ), and the Sharp/van der Heijde score were collected. Anticyclic citrullinated peptide antibody (anti-CCP) and rheumatoid factor (RF) were performed. RESULTS: We found that anti-CCP positivity was associated with longer disease duration (P = 0.001), presence of RF (P = 4.89 × 10(-8) ), and night pain positivity (P = 0.025). Patients with positive RF had more night pain and higher anti-CCP positivity (for all P ≤ 0.05). Anti-CCP titer was correlated with disease duration (P = 0.034) and Sharp total score (P = 1.2 × 10(-4) ). Moreover, there was a significant correlation between RF and anti-CCP antibodies titers (P = 0.011). Indeed, DAS28 correlated with HAQ (P = 1.8 × 10(-7) ) and morning stiffness duration (P = 0.045). In multivariate regression analysis, the main factors associated with anti-CCP titers were radiographic damage (P = 1.625 × 10(-4) ) and RF (P = 0.013). For DAS28, only HAQ (P = 2.9 × 10(-4) ) was associated. CONCLUSION: These findings suggest that anti-CCP antibodies are associated with RF and more severe joint damage. Moreover, disease activity is associated with functional disability.

A Tunisian case-control association study of a 6q polymorphism in rheumatoid arthritis.

Rheumatoid arthritis (RA), the most common autoimmune inflammatory disease of the joints, is a multifactorial disease, involving both genetic and environmental risk factors. TNFAIP3 (tumor necrosis factor, alpha-induced protein 3) gene located in a region of genetic susceptibility in RA is an attractive candidate to be involved in autoimmunity disorders. Our aim was to test the single nucleotide polymorphism rs6920220 located near TNFAIP3 in a case-control study in Tunisian population. The rare allele rs6920220-A was reported to have a risk effect on RA in several genome-wide association studies. Our results revealed a trend of an association of rs6920220-A allele with RA and genotypes containing this allele were in a higher proportion in RA patients than in matched controls. These findings have to be confirmed by a replication in largest RA and control groups of the same ethnic origin. TNFAIP3 gene may have a key role in autoimmunity through its action as a negative regulator of the NF-κB pathway. Further functional investigations are required to understand the mechanism by which this gene is involved in the RA pathogenesis.

The R620W polymorphism of the protein tyrosine phosphatase 22 gene in autoimmune thyroid diseases and rheumatoid arthritis in the Tunisian population.

BACKGROUND: Protein tyrosine phosphatase (PTPN22) is involved in the negative regulation of T-cell responsiveness. The association of a coding variant of the PTPN22 gene (R620W) with a number of autoimmune diseases has been described. AIM: The present study investigated whether PTPN22 gene polymorphism was also involved in the genetic predisposition to autoimmune thyroid diseases (AITDs) and rheumatoid arthritis (RA) in a Tunisian case control study. SUBJECTS AND METHODS: DNA samples from 150 patients affected with RA, 204 patients affected with AITDs and 236 healthy controls were genotyped for PTPN22 R620W polymorphism (1858C/T). Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: No significant differences in T allele frequency (2.3% in RA patients and 1% in AITDs patients vs 2.6% in controls; p=0.85 and p=0.08, respectively) and in genotype frequencies were detected between RA patients and controls (p=0.15) and between AITDs patients (p=0.11). Stratifying patients affected with AITDs according to their phenotype (Graves' disease and Hashimoto's thyroiditis) and RA patients according to the presence of rheumatoid factor (RF) and antibodies against cyclic citrullinated peptides (ACPA) did not show any significant association with PTPN22 R620W allele (p>0.05). CONCLUSION: Our data suggest that the PTPN22 C1858T single nucleotide polymorphism has no or minor effect on RA and AITDs susceptibility in the Tunisian population.

Association study of apoptosis gene polymorphisms in mitochondrial diabetes: A potential role in the pathogenicity of MD.

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia and is maternally transmitted. Syndromic MD is a subgroup of MD including diabetic microangiopathy and macroangiopathy, in addition to extrapancreatic disorder. MD is caused by genetic mutations and deletions affecting mitochondrial DNA. This mitochondrial damage initiates apoptosis. In this study, we hypothesized that functional polymorphisms in genes involved in apoptotic pathway could be associated with the development of apoptosis in MD disease and increased its risk. Detection of apoptosis was confirmed on muscle biopsies taken from MD patients using the TUNEL method and the Cytochrome c protein expression level. We genotyped then 11 published SNPs from intrinsic and extrinsic apoptotic pathway and assessed the signification of these polymorphisms in 43 MD patients and 100 healthy controls. We found 10 selected polymorphisms (p53 (rs1042522 and rs17878362), BCL2 (rs2279115), BAX (rs1805419), BAK1 (rs210132 and rs2227925), CASP3 (rs1405937), CASP7 (rs2227310), CASP8 (rs1045485) and CASP10 (rs13006529)) with a potential apoptosis effect in MD patients compared to control population. Specifically, SNPs involved in the intrinsic pathway (p53, BCL2, BAK1 and CASP3) presented the highest risk of apoptosis. Our result proved that apoptosis initiated by mtDNA mutations, can be emphasized by a functional apoptotic polymorphisms associated with high expression of cytochrome c protein and more myofibers with apoptosis in syndromic MD subgroup compared with non-syndromic MD subgroup.

Analysis of two susceptibility SNPs in HLA region and evidence of interaction between rs6457617 in HLA-DQB1 and HLA-DRB1*04 locus on Tunisian rheumatoid arthritis.

Previous genomewide association studies (GWAS) and meta-analyses have enumerated several genes/loci in major histocompatibility complex region, which are consistently associated with rheumatoid arthritis (RA) in different ethnic populations. Given the genetic heterogeneity of the disease, it is necessary to replicate these susceptibility loci in other populations. In this case, we investigate the analysis of two SNPs, rs13192471 and rs6457617, from the human leukocyte antigen (HLA) region with the risk of RA in Tunisian population. These SNPs were previously identified to have a strong RA association signal in several GWAS studies. A case-control sample composed of 142 RA patients and 123 healthy controls was analysed. Genotyping of rs13192471 and rs6457617 was carried out using real-time PCR methods by TaqMan allelic discrimination assay. A trend of significant association was found in rs6457617 TT genotype with susceptibility to RA (P = 0.04, pc = 0.08, OR = 1.73). Moreover, using multivariable analysis, the combination of rs6457617*TT-HLA-DRB1*04+ increased risk of RA (OR = 2.38), which suggest a gene-gene interaction event between rs6457617 located within the HLA-DQB1 and HLA-DRB1. Additionally, haplotypic analysis highlighted a significant association of rs6457617*T-HLA-DRB1*04+ haplotype with susceptibility to RA (P = 0.018, pc = 0.036, OR = 1.72). An evidence of association was shown subsequently in antiCCP+ subgroup with rs6457617 both in T allele and TT genotype (P = 0.01, pc = 0.03, OR = 1.66 and P = 0.008, pc = 0.024, OR = 1.28, respectively). However, no association was shown for rs13192471 polymorphism with susceptibility and severity to RA. This study suggests the involvement of rs6457617 locus as risk variant for susceptibility/severity to RA in Tunisian population. Secondly, it highlights the gene-gene interaction between HLA-DQB1 and HLA-DRB1.

Characterization of copy number variants for CCL3L1 gene in rheumatoid arthritis for French trio families and Tunisian cases and controls.

Analyses of copy number variants (CNVs) for candidate genes in complex diseases are currently a promising research field. CNVs of C-C chemokine ligand 3-like 1 (CCL3L1) gene are candidate genomic factors in rheumatoid arthritis (RA). We investigated CCL3L1 CNVs association with a case-control study in Tunisians and a transmission analysis in French trio families. Relative copy number (rCN) of CCL3L1 gene was quantified by droplet digital PCR (ddPCR) in 100 French trio families (RA patients and their two parents) and in 166 RA cases and 102 healthy controls from Tunisia. We calculated odds ratio (OR) to investigate association risk for CCL3L1 CNVs in RA. rCN identified varied from 0 to 4 in the French population and from 0 to 7 in the Tunisian population. A significant difference was observed in the distribution of these rCNs between the two populations (p = 2.34 × 10(-10)), as when rCN from French and Tunisian RA patients were compared (p = 2.83 × 10(-5)). CNVs transmission in French RA trios allowed the characterization of genotypes with the presence of tandem duplication and triplication on the same chromosome. RA association tests highlighted a protective effect of rCN = 5 for CCL3L1 gene in the Tunisian population (OR = 0.056; CI 95 % [0.01-0.46]). Characterization of CCL3L1 CNVs with ddPCR methodology highlighted specific CN genotypes in a French family sample. A copy number polymorphism of a RA candidate gene was quantified, and its significant association with RA was revealed in a Tunisian sample.

Genetic Determinants of Methotrexate Toxicity in Tunisian Patients with Rheumatoid Arthritis: A Study of Polymorphisms Involved in the MTX Metabolic Pathway.

BACKGROUND AND OBJECTIVE: Methotrexate (MTX) is a disease-modifying anti-rheumatic drug used in the treatment of rheumatoid arthritis (RA). It is the first line drug in the treatment of this disease. However, MTX-related adverse drug reactions (ADRs) are seen in 40 % of the patients. The aim of this study was to determine the impact of six genetic polymorphisms located in five genes encoding proteins involved in the MTX metabolic pathway in Tunisian RA patients and evaluate its association with MTX toxicity. METHODS: Genotyping of 5,10 methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), dihydrofolate reductase (DHFR 19-base pair deletion allele), thymidylate synthase (TYMS 2R/3R), methionine synthase (MTR A2756G) and methionine synthase reductase (MTRR A66G) was performed using PCR and PCR-RFLP method in 141 RA patients treated with MTX. Demographic and clinical characteristics were obtained and ADRs were recorded. Association analyses with regard to MTX toxicity were performed using the χ (2) test, the toxicogenetic risk index (TRI) and the Mann-Whitney U-test. RESULTS: The analysis highlighted a significant association of the T/T genotype of MTHFR C677T polymorphism with increased MTX toxicity. However, the MTHFR A1298C, DHFR 19-base pair deletion allele, MTR A2756G and MTRR A66G polymorphisms were not associated with increased MTX toxicity. The TYMS 2R/3R polymorphism had a protective effect against MTX toxicity. CONCLUSION: The results demonstrated that the C677T polymorphism in the MTHFR gene is associated with MTX toxicity in Tunisian RA patients. In contrast, the TYMS 2R/3R polymorphism is associated with a protective effect against overall MTX toxicity.

Evidence of association between FKBP1B and thyroid autoimmune disorders in a large Tunisian family.

FKBP1B belongs to immunophilins superfamily and functions as a cytosolic receptor protein of FK506. The role of FKBP1B in the immunosuppressive pathway of FK506 is well established. Previously, we reported a strong evidence of linkage between D2S171 microsatellite marker (located in vicinity of FKBP1B gene) and susceptibility to autoimmune thyroid diseases (AITDs). In this study, we report linkage disequilibrium between the dimorphism (C/T) in the 3' untranslated region (3' UTR) of FKBP1B gene and susceptibility to AITDs. DNAs were extracted from a large Tunisian family affected with Graves' disease (GD) and Hashimoto's thyroiditis (HT) and analysed by PCR-RFLP using DraIII restriction enzyme. Our results showed an excess of transmission of the allele C from heterozygous parents to affected offspring (transmission disequilibrium test (TDT) = 4.76; p = 0.012). This suggests a linkage disequilibrium of 3' UTR (C/T) SNP with AITDs. Moreover, The FBAT analysis gives a significant association with the C allele under the recessive model (chi2 = 5.50; p = 0.018). These results support the involvement of FKBP1B gene in the genetic susceptibility to the AITDs development in the studied family.

Allelic structure and distribution of 103 STR loci in a Southern Tunisian population.

Genotypes of 103 short tandem repeat (STR) markers distributed at an average of 40 cM intervals throughout the genome were determined for 40 individuals from the village of BirEl Hfai (BEH). This village of approximately 31,000 individuals is localized in the south-west of Tunisia. The allele frequency distributions in BEH were compared with those obtained for individuals in the CEPH (Centre d'Etude du Polymorphisme Humain) data using a Kolmogorov-Smirnov two-sample test. Fourteen out of the 103 markers (13.2%) showed significant differences (P<0.05) in distribution between the two populations. Population heterogeneity in BEH was indicated by an excess of observed homozygosity deviations from Hardy-Weinberg equilibrium at three loci (P<0.0005). No evidence for genotypic disequilibrium was found for any of the marker pairs. This demonstrated that in spite of a high inbreeding level in the population, few markers showed evidence for a different pattern of allelic distribution compared to CEPH.

A 20 year history of clinical and genetic study of thyroid autoimmunity in a Tunisian multigenerational family: Evidence for gene interaction.

Autoimmune thyroid diseases (AITD), which include Hashimoto thyroiditis (HT), Graves' disease (GD) and primary idiopathic myxoedema (PIM), are recognized by their clinical and genetic heterogeneity. In this study, we have carried on a global approach gathering 20 year genetic and clinical data on a Tunisian multigenerational family (Akr). Our purpose was search for a combined genotype involved in AITD susceptibility using 33 gene polymorphisms. The Akr pedigree is composed of more than 400 members distributed on 10 generations. Clinical follow-up was performed by appreciation of the thyroid gland and measurement of both thyroid hormone and auto antibody levels. We used FBAT software to test for association between gene polymorphisms and AITDs. Clinical follow-up has showed that the number of AITD patients has increased from 25 to 78 subjects subdivided on 51 cases of GD, 22 PIM and 5 HT. Concerning genetic analysis, our study has revealed new gene association when compared with our previous analysis (considering single genes). Thus, PTPN22, TG and VDR gene polymorphisms have became associated with p-values ranging from 4.6  10(- 2) to 4  10(- 3) when considered with other genes on the same chromosome; giving evidence for gene interaction. The most significant association was found with the MHC region (p = 7.15 10(- 4)). Moreover, and among gene polymorphisms explored, our analysis has identified some of them as AITD biomarkers. Indeed, PDS gene polymorphisms were associated with either exophthalmia or goiter (p-values from 10(- 2) to 10(- 3)). In conclusion, our study gives evidence for gene interaction in AITD development confirming genetic complexity of these diseases.

Association study of human leukocyte antigen-DRB1 alleles with rheumatoid arthritis in south Tunisian patients.

The aim of this study is to explore relationship between HLA-DRB1 alleles and the susceptibility and clinical features of rheumatoid arthritis (RA) in the south Tunisian population. We studied 142 RA patients and 123 controls matched for age, sex, and ethnicity. HLA-DRB1 genotyping and HLA-DRB1*04 subtypes were performed using polymerase chain reaction/sequence-specific primers. Association was assessed based on the χ (2) test and odds ratio with 95% confidence interval. For multiple comparisons, p value was corrected (p (c)) with Bonferroni test. Two alleles, HLA-DRB1*04 (p=0.045, p(c)=NS) and HLA-DRB1*10 (p=0.021, p(c)=NS), were found to have increased frequencies in RA patients compared to controls. In contrast HLA-DRB1*08 allele was found to have a decreased frequency in patients compared to controls (p=0.044, p(c)=NS). Molecular subtyping of the most prevalent allele (DRB1*04) revealed increased frequencies of HLA-DRB1*04:05 in patients compared to controls (p=0.013, p(c)=NS) whereas HLA-DRB1*04:02 showed a protective effect (p=0.005, p(c)=0.04). Moreover, stratified analyses indicated statistically significant associations between HLA-DRB1*04 allele and anti-cyclic peptides antibodies positivity (ACPA(+)) and rheumatoid factor positivity (RF(+); p(c)=0.03, for both subgroups), HLA-DRBI*10 and ACPA(+) and the presence of another autoimmune disease (p(c)=0.05 and p(c)=0.007, respectively), and HLA-DRB1*04:05 and RF(+) and erosion (p(c)=0.005 and p(c)=0.049; respectively). A significant decrease in the frequency of the DRB1*04:02 allele was observed in patients with ACPA(+) and RF(+) subgroups (p(c)=0.04 and p(c)=0.02, respectively). Our results showed that there was a trend of positive association of HLA-DRB1*04 and HLA-DRB1*10 with RA as such and significant associations with the disease severity in the south Tunisian population.

Analysis of skewed X-chromosome inactivation in females with rheumatoid arthritis and autoimmune thyroid diseases.

INTRODUCTION: The majority of autoimmune diseases such as rheumatoid arthritis (RA) and autoimmune thyroid diseases (AITDs) are characterized by a striking female predominance superimposed on a predisposing genetic background. The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of several autoimmune diseases. METHODS: We examined XCI profiles of females affected with RA (n = 106), AITDs (n = 145) and age-matched healthy women (n = 257). XCI analysis was performed by enzymatic digestion of DNA with a methylation sensitive enzyme (HpaII) followed by PCR of a polymorphic CAG repeat in the androgen receptor (AR) gene. The XCI pattern was classified as skewed when 80% or more of the cells preferentially inactivated the same X-chromosome. RESULTS: Skewed XCI was observed in 26 of the 76 informative RA patients (34.2%), 26 of the 100 informative AITDs patients (26%), and 19 of the 170 informative controls (11.2%) (P < 0.0001; P = 0.0015, respectively). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 17 RA patients (22.4%), 14 AITDs patients (14.0%), and in only seven controls (4.1%, P < 0.0001; P = 0.0034, respectively). Stratifying RA patients according to laboratory profiles (rheumatoid factor and anti-citrullinated protein antibodies), clinical manifestations (erosive disease and nodules) and the presence of others autoimmune diseases did not reveal any statistical significance (P > 0.05). CONCLUSIONS: These results suggest a possible role for XCI mosaicism in the pathogenesis of RA and AITDs and may in part explain the female preponderance of these diseases.

Lack of association of VDR gene polymorphisms with thyroid autoimmune disorders: familial and case/control studies.

We study the association between three Vitamin D receptor gene polymorphisms (rs10735810, rs1544410, rs731236) and susceptibility to thyroid autoimmune diseases. Seventy-six affected subjects, belonging to a large family, as well as one hundred unrelated Tunisian patients and one hundred healthy Tunisian controls were genotyped. A family-based association test and a standard chi-square test were used to assess association in family and case-control data, respectively. Our results showed no significant association of the Vitamin D receptor gene polymorphisms with the susceptibility to thyroid autoimmune diseases in the family. Moreover, allele frequencies for the three polymorphisms in the Tunisian population were similar to those reported in the Tunisian control population and none was associated with the disease. These results suggest a lack of association between the Vitamin D receptor gene polymorphisms and susceptibility to thyroid autoimmune diseases in Tunisian population, in agreement with data from the UK, but in conflict with studies from the Far East.