Khaled Ayed

Distribution of HLA-B*27 subtypes in Tunisians and their association with ankylosing spondylitis.

OBJECTIVE The human leukocyte antigen HLA-B27 is a class I antigen of the major histocompatibility complex strongly associated with ankylosing spondylitis (AS) and other related spondyloarthropathies (SpAs). The mechanism of this association remains unknown. HLA-B27 is a serologic specificity that represents a family of at least 25 different HLA-B27 alleles (2701-2725). These alleles are closely related by nucleotide sequence homology, but differ in ethnic distribution. The purpose of the present study is to investigate the distribution of HLA-B27 alleles in healthy controls and in patients with ankylosing spondylitis (AS). METHODS We selected 160 HLA-B27-positive individuals (39 controls and 121 patients with ankylosing spondylitis). Typing of HLA-B27, and Cw alleles were performed by polymerase chain reaction amplification with sequence specific primers (PCR-SSP), and by serological typing (microlymphocytotoxicity). RESULTS Seven B27 subtypes were identified: B*2702, 03, 04, 05, 07, 09 and B*2714. The distribution of these alleles in the population of patients was B*2702 (47.1%) and B*2705 (47.1%). These subtypes were also detected in 16 (41%) and 16 (41%), respectively of the 39 control subjects. HLA-B*2707 was detected in 4 (3.31%) patients and in 3 (7.6%) controls. B*2704, 09 and B*2714 were relatively rare and were detected in one subject each. No significant differences were noticed in the frequencies and distribution of HLA-B27 alleles between patients and controls. CONCLUSIONS Our results show a restricted number of HLA B27 subtypes associated with AS. B*2702 and B*2705 were equally common in patients and controls. The most prominent B27/Cw haplotypes in the patient groups and controls were B*2702/Cw02022 and B*2705/Cw02022.

Polymorphism in ICAM-1, PECAM-1, E-selectin, and L-selectin genes in Tunisian patients with inflammatory bowel disease.

Background Ulcerative colitis (UC) and Crohns disease (CD) are chronic intestinal disorders characterized by immune dysregulation and leukocytes recruitment into gastrointestinal tract. Cell adhesion molecules (CAM) mediate the extravasation of leukocytes and their accumulation in inflamed intestinal mucosa. Recently, CAM genes have been implicated in determining susceptibility to UC and CD. We investigate seven mutations in CAM: G241R and K469E in ICAM-1, V125L in PECAM-1, G98T, S128R, and L554F in E-selectin and F206L in L-selectin in 197 Tunisian patients (73 with UC and 124 with CD) and 194 controls. These polymorphisms were detected by polymerase chain reaction sequence-specific primers and restriction enzyme analysis. Results A significant increase in allele frequencies of 206L of L-selectin and the associated genotype F/L was observed in both patients with UC and CD compared with controls. Subgroup analysis showed that the L206 allele and F/L206 genotype frequencies were significantly increased in UC patients with left-sided type; whereas, the F/L206 genotype was significant in CD patients with ileocolonic location and stricturing behavior compared with controls. No significant differences in allele or genotype frequencies were observed for ICAM-1 K469E, E-selectin, and PECAM-1 polymorphisms between UC patients, CD patients, and controls. Conclusion We found an association of inflammatory bowel disease with allele L206 of L-selectin gene, whereas genotype L/F was associated with a subgroup of UC (left-sided type) and CD patients with more extensive location of disease and stricturing behavior. However, further studies are needed to confirm our findings.

Cytokine Gene Polymorphisms in Kidney Transplantation

BACKGROUND Acute and chronic rejections remain an important cause of graft loss after renal transplantation. It has been suggested that cytokine genotyping may have a predictive role to identify patients at greater risk of rejection regardless of human leukocyte antigen (HLA) compatibility and/or the presence of anti-HLA antibodies before the renal allograft. OBJECTIVES We sought to investigate polymorphisms of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, interleukin (IL)-10, IL-6, and interferon (IFN)-γ as indices of differential cytokine production in kidney transplantation and to examine their predictive roles for acute or chronic rejection. PATIENTS AND METHODS TNF-α (G/A -308), TGF -β1 (haplotype codon 10/codon 25), IL-10 (haplotype-1082, -819, -592), IL-6 (C/G -174), and IFN-γ (T/A +874) single nucleotide polymorphisms (SNPs) were detected using polymerase chain reaction (PCR)-specific sequence primers (SSP) in 231 kidney transplant recipients (KTR) including 106 treated with mycophenolate mofetil (MMF+). RESULTS We observed no significant associations of any of investigated polymorphism taken alone with acute rejection episodes (ARE) or chronic allograft dysfunction (CAD). Nevertheless, TGF-β1 Low (L) production was correlated with greater graft survival at 20 years (81.8%) compared with intermediate (L) or high (H) levels (56.1%), affect that the difference was not significant (P = .2). Cytokine haplotype analysis in KTR (MMF-) without HLA-mismatches or presynthesized anti-HLA antibodies (n = 32) showed ARE to be significantly more prevalent among the TNF-α*H/TGF- β1*H/IL-10*H production haplotype (75%) compared with the other haplotypes (16%; P = .03). CONCLUSION The presence of TGF-β1-H secretion profile may protect the kidney graft. TNF-α*H/TGF-β1*H/IL-10*H haplotype was associated with a higher risk of ARE and with poorer graft survival.

Cytokine and apoptosis gene polymorphisms influence the outcome of hepatitis C virus infection

BACKGROUND Hepatitis C virus (HCV) infection is thought to be chronic and the factors leading to viral clearance or persistence are poorly understood. This study was undertaken to investigate the possibility of a significant relationship between the spontaneous clearance or the persistence of hepatitis C virus (HCV) infection and cytokine and apoptosis gene polymorphisms in Tunisian patients on hemodialysis. METHODS Polymorphisms of the genes IL-1 (-889 IL-1alpha, -511 and +3954 IL-1beta, IL-1Ra), IL-18 (-137 and -607), IL-12 (-1188) and Apo1/Fas (-670) were determined by PCR-RFLP, PCR-SSP and PCR-VNTR in 100 healthy blood donors and 100 patients infected with HCV and undergoing hemodialysis. The patients were classified into two groups: G1 consisted of 76 active chronic hepatitis patients (positive for HCV RNA) and G2 consisted of 24 hemodialysed patients who spontaneously eliminated the virus (negative for HCV RNA). RESULTS The frequency of genotype association [-137GC/-607CA] IL-18 was higher in G2 (41.7%) than in G1 (15.8%) (P=0.008; OR=0.26; 95% CI, 0.10-0.73). We also found a higher frequency of the AA genotype of the Apo1/Fas gene in G2 (41.6%) than in G1 (17.5%) (P=0.026; OR=3.49; 95% CI, 1.13-10.69). Adjustment for known covariate factors (age, gender and genotype) confirmed these univariate findings and revealed that the genotype association GC-CA of the (-137 and -607) IL-18 gene and the AA genotype of the Apo1/Fas gene were associated with the clearance of HCV (P=0.041 and 0.017, respectively). CONCLUSION The two genotypes GC-CA of the (-137 and -607) IL-18 polymorphism and the AA genotype of the Apo1/Fas gene influence the outcome of HCV infection in Tunisian patients on hemodialysis.

THE INVOLVEMENT OF HLA -DRB1, DQA1, DQB1 AND COMPLEMENT C4A LOCI IN DIAGNOSING SYSTEMIC LUPUS ERYTHEMATOSUS AMONG TUNISIANS

Background Genetic susceptibility to systemic lupus erythematosus (SLE) varies among populations. Few data exist on associations of HLA class II and class III alleles of the major histocompatibility complex (MHC) and susceptibility to SLE in Tunisians. Patients and Methods We compared HLA-DRB1*, DQA1, DQB1* and C4 allotypes in 62 Tunisian SLE patients and 100 matched controls. We also assessed the association of specific alleles with distinct autoantibody profiles in SLE patients. Results HLA-DRB1*0301, -DRB1*1501 and C4AQO alleles were increased in the SLE patients, while the frequencies of HLA-DRB1*04 and DQB1*03 were decreased. HLA-DQA1*0102 and DQA1*0501 were significantly increased in the SLE patients. HLA-DQB1*0201 and DQB1*0602 were more frequent in the SLE patients. C4A*QO and C4B*QO were increased in frequency in the SLE patients compared to the controls, but only C4A null was significantly increased. Eleven of 17 SLE patients with the C4 null allele were HLA-DRB1 *0301 positive. Three of 16 SLE patients with HLA-DRB1*1501 were associated with HLA-DQB1*0501 rather than DQB1 *0602, as has been reported in European SLE patients. Conclusions The MHC class II alleles (DRB1, DQA1, DQB1) and C4 null associations noted in other ethnic groups are also found in Tunisians, suggesting shared susceptibility factors across ethnic lines in predisposition to SLE. In contrast to other ethnic groups, MHC class II alleles are not associated with the presence of specific autoantibodies in Tunisian SLE patients.

Interleukin-18 gene polymorphisms in tunisian patients with inflammatory bowel disease.

Aim: Interleukin (IL)-18 can regulate the Th2-mediated immune response and it may be involved in the pathogenesis of Th1 and Th2 chronic inflammatory diseases. This study sought to detect a possible association between two single nucleotide polymorphisms (SNPs) (–137G/C and –607C/A) in the IL-18 gene promoter region and susceptibility to inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) in the Tunisian population. Methods: The (–137G/C and –607C/A) IL-18 polymorphism was analyzed in 105 patients with CD, 59 patients with UC, and 100 controls using the sequence-specific polymerase chain reaction method. Results: The distribution of allele and genotype frequencies illustrate that the –137G/G genotype frequency was significantly higher in UC than in controls (p value corrected (pc) = 0.038). On the other hand, we found a statistically significant association (pc = 0.033) between genotype AA of the IL-18 gene promoter (–607C/A) polymorphism in UC patients and the distal localization of the lesions. In CD, no significant differences were observed at positions –607 and –137. The analysis of IBD patients according to clinical behavior revealed no difference. Conclusion: The two SNPs at position –607 (C/A) and –137 (G/C) in the promoter region of the IL-18 gene was associated with the development of UC but not CD, providing a strong support for an IBD susceptibility gene in the region surrounding IL-18. It remains to be determined precisely how the IL-18 alleles influence the pathogenesis of IBD.

(AT) Repeat in the 3′ Untranslated Region of the CTLA-4 Gene and Susceptibility to Acute Allograft Rejection in Tunisian Renal Transplantation

Allograft rejection is an immune response relying on the proliferation and the differentiation of T cells. CTLA-4 is a co-stimulatory molecule, expressed on activated T lymphocytes, which has been shown to play a crucial role in the down-regulation of T-cell activation. Herein, we have examined the impart of a genetic marker in the CTLA-4 gene on renal transplant outcomes. A cohort of 144 renal recipients and 100 healthy subjects were genotyped by the fragments analysis method using an automated sequencer. Patients were classified into two groups: Group I included 31 HLA-identical haplotype allograft recipients and Group II, 113 showing one or more HLA haplotype mismatches. Forty patients (27.78%) developed at least one acute rejection episode (ARE): 9 in Group I and 31 in Group II. Before transplantation, 20 patients were lymphocytotoxic antibodies (LCT) positive: 4 Group I, 2 of whom developed an ARE, and sixty in Group II, including 8 with an ARE. The occurrence of an ARE was associated with the presence of LCT before transplantation among the entire cohort of patients (P = .032) and among Group II (P = .037). The allelic frequencies of (AT)n polymorphism did not reveal significant differences between patients and controls. The most prevalent alleles were the 88 bp (51% in controls and 44.44% in patients) and the 106 bp (8% and 10.76%, respectively). We noticed an increase of the 120 bp allele frequency among patients who had undergone an ARE compared with those who did not display this complication (8.75% vs 3.85%). Likewise, among LCT-negative Group I, recipients the incidence of the 120 bp allele was higher in ARE than non-ARE patients. Although the differences were not statistically significant, we propose that the 120 bp allele of the CTLA-4 gene (AT)n microsatellite a predisposes to acute rejection episodes in renal transplantation.

Phylogenetic Analysis of Isolated HCV Strains from Tunisian Hemodialysis Patients

The present study describes the strains of hepatitis C virus (HCV) isolated from Tunisian hemodialysis patients. Thirty-three HCV strains isolated from different dialysis centers in Tunis City were amplified by RT-PCR in a region of the NS5b gene, genotyped by sequencing, and compared to international sequences by phylogenetic analysis. The phylogenetic tree showed that 16 HCV isolates have been identified as subtype 4k (48.5%), 7 as unspecified HCV-4 subtype (21.2%), 5 as subtype 4a et 1b (each 15.2%). The analysis of this tree revealed that the HCV-1b strains were closely related to Anglo-Saxon and European isolates, while the HCV-4 isolates are genetically similar to Egyptian and African strains. Phylogenic analysis of 33 Tunisian isolates with international HCV strains on a region of the NS5b gene demonstrated that the subtype 4k submerged the Tunis city and a new subtype of HCV4 seems to be suspect in this area.

Association between CTLA-4 Gene Promoter (49 A/G) in Exon 1 Polymorphisms and Inflammatory Bowel Disease in the Tunisian Population

Background/Aim: To investigate the possible association between the polymorphism of the CTLA-4 exon 1 +49 A/G and susceptibility to Crohns disease (CD) and ulcerative colitis (UC) in the Tunisian population. Methods: The +49 A/G dimorphism was analyzed in 119 patients with CD, 65 patients with UC, and 100 controls by the polymerase chain reaction–restriction fragment length polymorphism method. Results: Significantly higher frequencies of the CTLA-4 +49A allele and A/A homozygous individuals were observed in patients with CD when compared with controls (pc = 0.0023 and pc = 0.0003, respectively). Analysis of CTLA-4 A/G polymorphism with respect to sex in CD showed a significant difference in A/A genotypes between female patients and controls (pc = 0.0001 and pc = 0.038, respectively). There were no differences in the subgroups of patients with CD. Conclusions: Forty-nine A alleles and AA genotype are associated with CD susceptibility in Tunisians. Other genes involved in the T-cell regulation remain strong candidates for IBD susceptibility and require further investigation.

Role of genetic polymorphisms in factor H and MBL genes in Tunisian patients with immunoglobulin A nephropathy.

The molecular mechanisms of IgA nephropathy (IgAN) remain poorly understood. Several different polymorphic genes have been investigated in order to demonstrate their possible association with this disease. It is evident that mainly alternative and lectin pathways complement activation and play an important role in renal injury of IgAN. This study was conducted to determine eventual deficiencies of factor H in the SCR20 gene region and to look for a possible association between the polymorphism (+54) exon 1 of the MBL gene and the predisposition in Tunisian patients with IgAN. We then evaluated the effects of these FH mutations and/or this MBL polymorphism on nephropathy susceptibility and progression. Polymorphism A/B (+54) in the exon1 of the MBL gene and analysis within the C-terminal domain of the protein SCR20 in the exon 22 of the factor H (FH) gene were conducted in 36 sporadic IgAN Tunisian patients and 117 age and gender matched healthy subjects recruited from blood donors, by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing respectively. The analysis of the Gly54Asp (+54) mutation of the MBL gene according to the criteria of gravity of the IgAN reveals that the patients with genotype AB present more frequently with end-stage renal disease (ESRD) compared with those of genotype AA [OR: 8, CI (1.74–54.49), P = 0.019]. Moreover, the variant allele B was statistically more frequent than the allele A in patients with an association with initial arterial high blood pressure, ESRD and class V of the Haas classification compared to those without this association (P = 0.009). The direct sequencing of exon 22 (SCR 20) of FH gene did not reveal any abnormal mutational deficiency for this factor in all patients and controls. The data did not support the hypothesis that FH is a susceptibility factor for the IgAN. However the data did show there was an association between AB (+54) exon1 MBL genotype and severe sporadic forms of this disease in Tunisian patients. Because of the small number of subjects studied, a much larger cohort of IgAN patients with varying severity of the disease and its progression would seem necessary to confirm these findings.