Hajer Fourati

Clinical and diagnostic value of ribosomal P autoantibodies in systemic lupus erythematosus

OBJECTIVE To analyse prospectively the diagnostic sensitivity and specificity as well as the clinical relevance of ribosomal P (anti-P) autoantibodies in a large cohort of SLE patients. METHODS The anti-P autoantibodies were evaluated in the serum of 200 Tunisian SLE patients at disease onset and 130 various control subjects by a sensitive immunodot assay. A complete laboratory evaluation and clinical examination were performed in each SLE patient. During the follow-up, the patients were regularly monitored for clinical parameters. Global SLE activity was measured by the ECLAM. RESULTS The sensitivity and specificity of anti-P testing for SLE were 23.5 and 98.4%, respectively. The anti-P-positive samples 14/47 (29.8%), 27/47 (57.4%) and 5/47 (10.6%) were negative for anti-dsDNA, anti-Sm or both antibodies, respectively. The anti-P-positive patients showed more active disease activity and a much higher prevalence of arthritis. An association between IgG aCLs and anti-P antibodies was also found. However, anti-P antibodies were not associated with neuropsychiatric manifestations or lupus nephritis. CONCLUSION This study does not seem to confirm the described association of anti-P antibodies with neuropsychiatric manifestations of SLE. However, it supports the anti-P antibody association with arthritis and disease activity as well as the presence of aCL. Based on our study and other related studies, we propose that, akin to anti-Sm and anti-dsDNA, anti-P antibodies detected by one agreed method may be considered for inclusion as a criterion for the classification of SLE.

Contribution of PTPN22, CD28, CTLA-4 and ZAP-70 variants to the risk of type 1 diabetes in Tunisians.

Type 1 diabetes (T1D) is caused by an immune-mediated destruction of the insulin-producing β-cells. Several studies support the involvement of T cell activation molecules. In order to underline the role of the genes involved in this pathway, we investigated, using the Sequenom MassARRAY platform, polymorphisms of sixteen single-nucleotide polymorphisms (SNPs) belonging to PTPN22, CD28, CTLA-4, and ZAP-70 genes in 76 T1D patients and 162 unrelated healthy controls from Southern Tunisia. We confirmed the association with PTPN22 (rs2476601, Corrected P (Pcorr)=0.002, OR=6.20) and CD28 gene (rs1879877, Pcorr=0.003; OR=4.27 and rs3181096, Pcorr=0.02; OR=1.73). We also identified an association with rs17695937 of ZAP-70 gene (Pcorr=0.02, OR=1.87). Our results suggest a significant effect on T1D susceptibility for A-C-A-G-C and T-C-C-T-A-C haplotypes, of ZAP-70 and CD28 genes, respectively. In addition, (A-G-C) combination of ZAP-70/CD28 gene was significantly increased in T1D patients as compared to controls, suggesting the possible interaction between these genes. These results confirm the involvement of PTPN22 and CD28 genes in the genetic susceptibility to T1D. Interestingly, ZAP-70 seems to contribute to the susceptibility to the disease in our population. However, this finding has to be confirmed in further studies.

Polymorphisms in the IL2RA and IL2RB Genes in Inflammatory Bowel Disease Risk

Associations with different autoimmune diseases of polymorphisms in genes encoding the IL2RA and IL2RB subunits (located in 10p15 and 22q13, respectively), were identified through genome-wide studies. Polymorphisms in these two genes were studied in 107 inflammatory bowel disease (IBD) patients (39 Crohns disease [CD] and 68 ulcerative colitis [UC]) and in 162 ethnically healthy controls from Tunisia (Sfax). Two of the 15 IL2RA single-nucleotide polymorphisms (SNPs) genotyped (rs4749924 and rs706778) were significantly associated with UC (pcorr=0.018 and 0.048, respectively), but no evidence of association with CD was observed. The IL2RA GTCT haplotype was also more frequent in UC patients compared to controls (2.6% vs. 0%; p=0.002). One of the 6 IL2RB SNPs genotyped (rs743776) was significantly associated with CD (pcorr= 0.039), but no evidence of association with UC was observed. No significant association between IL2RB haplotypes was observed among investigated groups. Our study identified markers in the IL2RA and IL2RB genes that are significantly associated with UC and CD, respectively. Our results supporting IL2RA and IL2RB as promising candidate genes for IBD and suggesting a potential role of IL2R in the pathogenesis of IBD, likely involves regulatory T cells.

The CREM gene is involved in genetic predisposition to inflammatory bowel disease in the Tunisian population

The identification of susceptibility genes for inflammatory bowel disease (IBD) is key to understanding pathogenic mechanisms. Recently, the results of genetic association studies have highlighted many loci that are shared among several autoimmune diseases. We aimed to study the genetic epidemiology of polymorphisms in specific genes previously associated with other autoimmune diseases, namely the CREM, STAT4, STAT5a, Stat5b, and IRF5 genes. Twelve polymorphisms in the CREM, STAT4, STAT5a, Stat5b, and IRF5 genes were genotyped in a cohort of 107 IBD patients (39 Crohns disease [CD] and 68 ulcerative colitis [UC]) and 162 controls from southern Tunisia. One CREM single nucleotide polymorphism (SNP) displayed evidence for genetic association with IBD (p=8.7×10(-4), odds ratio [OR]=2.84 [1.58; 5.09]). One STAT4 SNP (p=0.026; OR=1.65 [1.06; 2.58]) exhibited a marginal association with UC but not with CD. No significant association was observed with the SNPs in STAT5a, IRF5, and STAT5b. These results suggest that common variants of the CREM gene are involved in the genetic component conferring general susceptibility to IBD, whereas STAT4 appears to be more specifically associated with UC. This work provides motivation for studies aiming to replicate these findings in larger populations.

Non-HLA autoimmunity genetic factors contributing to Autoimmune Polyglandular Syndrome type II in Tunisian patients

Autoimmune Polyglandular Syndrome Type II (APSII) is characterized by the co-occurrence of clinical insufficiency of at least two endocrine glands. Although, HLA determinants of APSII predisposition have been identified, little attention has been paid to non-HLA genes. Here, we used SNP genotyping in a Sequenom platform and genetic association tests to study a cohort of 60 APSII Tunisian patients presenting highly frequent co-occurrence of Autoimmune Thyroid Disease (AITD) and Type 1 Diabetes (T1D) and lower frequency of Addisons disease (AD). We tested the high a priori possibility that well-established non-HLA autoimmunity loci were involved in APSII and confirmed five association signals to APSII, namely: (1) two T1D-associated SNPs, in CTLA4 and IL2RA, suggest their involvement in T1D pathogenesis in this cohort; (2) two SNPs in STAT4 and IL15 not previously associated to endocrinopathies, are possibly involved in co-occurrence of organ autoimmunity in APSII, and; (3) one SNP in TNF alpha showed association to APSII irrespective of AD. While this work was performed in a relatively small cohort, these results support the notion that the non-HLA genetic component of APSII include genetic factors specific of particular autoimmune manifestations as well as genetic factors that promote the co-occurrence of multiple autoimmune endocrinopathies.

Association of ZAP70 and PTPN6, but Not BANK1 or CLEC2D, with inflammatory bowel disease in the Tunisian population.

Inflammatory bowel diseases (IBDs), consisting of ulcerative colitis (UC) and Crohns disease (CD), are complex disorders with multiple genes contributing to disease pathogenesis. We aimed to identify the associations of genetic variations in the ZAP70, PTPN6, BANK1, and CLEC2D genes encoding for intracellular signaling molecules with IBDs. One hundred seven patients (39 CD and 68 UC) with IBD and 162 healthy control subjects from the Southern Tunisia were recruited. We genotyped 4 single-nucleotide polymorphisms (SNPs) in ZAP70 (rs1020396, rs11686881, rs13420683, and rs17695937), 2 SNPs in PTPN6 (rs7310161 and rs759052), 3 SNPs in BANK1 (rs10516487, rs17266594, and rs3733197), and 1 SNP in CLEC2D (rs3764021). ZAP70 displayed a strong genetic association with CD for rs13420683 [allele C, p=0.003, P(corr)=0.006, odds ratio (OR)=2.25 (1.32; 3.85); genotype CC, p=0.016, P(corr)=0.048, OR=2.57 (1.22; 5.41)]. However, in UC, a weak association with PTPN6 was observed [TT (p=0.01; P(corr)=0.03; OR=2.11 (1.18; 3.76)]. No significant association in the BANK1 and CLEC2D genes was observed. These results suggest the involvement of the ZAP70 and PTPN6 genes in the genetic component conferring a general susceptibility to CD and UC, respectively. This work provides motivation for studies aiming to replicate these findings in larger populations.

HLA-DRB1/DQB1 susceptibility for autoimmune polyglandular syndrome type II and III in south of Tunisia

OBJECTIVES The aim of our study was to investigate the association of HLA-DRB1 and HLA-DQB1 alleles with autoimmune polyglandular syndromes (APS) type II and III in a southern Tunisian population. PATIENTS AND METHODS Sixty-two unrelated patients with APSII (n=20) and APSIII (n=42) and 146 healthy controls were genotyped for HLA class II alleles (DRB1*, DQB1*) by PCR-SSP technique. RESULTS An increased frequencies of HLA-DQB1*03:02 (P=0,02; OR=2.98) in APSII patients, HLA-DRB1*03 (P=310(-6); OR=4.28) and HLA-DQB1*02:01 (P=0.04; OR=1.95) in APSIII patients were found compared to healthy controls. Study of the HLA-DRB1*;DQB1* haplotype frequencies showed a higher occurrence of DRB1*04;DQB1*03:02 and DRB1*03;DQB1*02:01 in APSII patients (P=410(-3); OR=3.31 and P=0.03; OR=2.74 respectively) whereas APSIII was only associated with DRB1*03;DQB1*02:01 (P=7.210(-8), OR=4.71). CONCLUSION Our data suggest that the variation in class II HLA alleles and haplotypes could be a genetic factor involved in the susceptibility of APS syndrome.INTRODUCTION The mosaicism 45, X/46, XY is a gonosomal abnormality characterized by a broad phenotypic spectrum, ranging from women with or without Turner syndrome stigmata, to men apparently normal, passing by the ambiguous phenotypes with variable virilisation of external genitalia. From the histological point of view, several situations may arise. PATIENTS AND METHODS We analyzed the clinical, hormonal, sonographic, and genitographics data, as well as peroperative and histological findings for five cases of mosaicism 45, X/46, XY, and we discussed treatment performed. RESULTS The mean age of patients was 6.6 years, two had a female phenotype with clitoral hypertrophy (one of them had Turner syndrome stigmata), one had a normal male phenotype with bilateral cryptorchidism and two had an ambiguity of external genitalia assigned to male. Short stature was noted for four patients. Surgical exploration concluded to the diagnosis of mixed gonadal dysgenesis for four of our patients. No cases of gonadoblastoma have been reported, for girls a prophylactic gonadectomy was performed, for boys the streak gonad was resected and the dysgenetic testis biopsied and preserved, subject for constant monitoring. CONCLUSION This heterogeneity indicate the importance of an accurate clinical and histological evaluation of any patient presenting with 45, X/46,XY mosaicism.

Association of BANK1 and cytokine gene polymorphisms with type 1 diabetes in Tunisia.

Type 1 diabetes (T1D) is an autoimmune disease (AID) with both genetic and environmental components. We aimed to investigate the genetic association of polymorphisms in genes previously linked with other AIDs, namely BANK1, IL15 and IL2/IL21 region. A total of 76 T1D patients and 162 controls from Southern Tunisia were recruited for a case-control association study investigating the relationship between sixteen SNPs of the BANK1, IL15 and IL2/IL21 gene region and T1D. In the BANK1 gene, G allele and GG genotype of rs3733197 were significantly increased in the group of T1D patients compared to controls. In addition, in the IL15 gene, the minor allele A of rs10519613 polymorphism was significantly higher in patients than in controls. No significant association was found for SNPS in IL2/IL21 gene region. The analysis of the haplotype structure revealed the G-C-A-C-T haplotype of the IL15 gene as associated with a reduction in the risk of developing T1D, while A-T-A-C-T haplotype increased the risk of developing the disease. Furthermore, in the IL2/IL21 region, only one haplotype consisting of eight SNPs was markedly associated with T1D susceptibility. Moreover, G-C combination of the BANK1/IL15 was significantly increased in T1D patients, compared to controls. Our results establish BANK1 and IL15 as new T1D genetic susceptibility factors and replicate the association of the 4q27 region with T1D. Our data agree with the effect previously observed for other autoimmune conditions and delineate a shared underlying mechanism.

Genetic Factors Contributing to Systemic Lupus Erythematosus in Tunisian Patients

Systematic lupus erythematosus (SLE) is a multi system autoimmune disease characterized by autoantibodies production, multi-organ damage and complex genetic inheritance. Multiple genetic and environemental factors contribute to the pathogenesis of this disease. Recent genome-wide studies, have added substantially to the number of genes associated with SLE. We performed a case control study using 138 SNPs in 93 Tunisian patients affected with lupus and 162 healthy controls. All SNPs were genotyped in a Sequenom platform. To confirm some associations, associated SNPs were analyzed using logistic regression which allows the test of association with a given SNP by adjusting for the effect of confounding variables. Association was especially reported with rs3733197 (P=0.0026, OR=2.04), rs17266594 (P=0.046, OR=1.56) in BANK1 gene, rs2070197 (P=0.0016, OR=2.31), rs2004640 (P=0.024, OR=1.54), rs10954213 (P=0.035, OR=1.53) in IRF5 gene and rs7574865 (P=0.017, OR=1.77) in STAT4 gene: previously confirmed SLE susceptibility genes. rs1800629 (P=0.00036, OR=2.26), rs4147359 (P=0.026, OR=1.55) and rs11575812 (P=0.037, OR=1.57) of TNF-α, IR2RA and IL2 genes respectively were also associated with SLE. Haplotypic analysis reported 2 susceptibility haplotypes: TGG (P=0.00421, OR=1.87) in BANK1 and TCA (P=0.00177, OR=2.34) in IRF5 genes. Our results show that numerous genes, some with known immune related function predispose to lupus.

Association of TCR/CD3, PTPN22, CD28 and ZAP70 gene polymorphisms with type 1 diabetes risk in Tunisian population: family based association study.

Type 1 diabetes (T1D) is caused by an immune-mediated destruction of the insulin-producing β-cells. Several studies support the involvement of T cell activation molecules in the pathogenesis of T1D. In order to underline the role of the genes involved in this activation pathway, we investigated, using the Sequenom MassARRAY platform, 45 single-nucleotide polymorphisms (SNPs) belonging to TCR/CD3, CD28, ZAP70, and PTPN22 genes in 59 T1D Tunisian families. In the current study, we identified an association with rs706 (Z score=2.782; p=0.005) of TCRβ gene. We also demonstrated that rs10918706 in the intron of the CD3z gene was associated with increased risk of T1D (Z score 2.137; p=0.032). In the same region, rs2949655 (Z score=2.101; p=0.035) and rs1214611 (Z score=4.036; p=0.00005) showed a genotype association with the risk of T1D. When haplotypes were constructed, GAA haplotype displayed significant association with T1D (Z score=2.135; p=0.032), while GGA haplotype (Z score=-1.988; p=0.046) was negatively associated with the disease. We also identified an association with rs3181096 (Z score=2.177; p=0.029), rs17695937 (Z score =2.111; p=0.034) and rs2488457 (Z score=2.219; p=0.026), respectively of CD28, ZAP70 and PTPN22 genes. In addition, our results suggest a significant effect on T1D susceptibility for AC (Z score=2.30; p=0.02) and CTGGC (Z score=2.309, p=0.02) haplotypes of ZAP70 and PTPN22 genes, respectively. While, the GTCT (Z score=-2.114, p=0.034) and CTAGG (Z score=-2.121, p=0.033) haplotypes of CD28 and PTPN22 genes, may confer protection against T1D. These findings confirm the role of PTPN22 and CD28 involved in the T cell activation pathway in the development of T1D in Tunisian families. Interestingly, ZAP70 and TCRβ/CD3z seem to contribute to the susceptibility to the disease in our population. However, this finding has to be confirmed in further studies.