Altered expression of cytokines, chemokines, growth factors, and soluble receptors in patients with colorectal cancer, and correlation with treatment outcome.

Insofar as they play an important role in the pathogenesis of colorectal cancer (CRC), this study analyzes the serum profile of cytokines, chemokines, growth factors, and soluble receptors in patients with CRC and cancer-free controls as possible CRC signatures. Serum levels of 65 analytes were measured in patients with CRC and age- and sex-matched cancer-free controls using the ProcartaPlex Human Immune Monitoring 65-Plex Panel. Of the 65 tested analytes, 8 cytokines (CSF-3, IFN-γ, IL-12p70, IL-18, IL-20, MIF, TNF-α and TSLP), 8 chemokines (fractalkine, MIP-1ß, BLC, Eotaxin-1, Eotaxin-2, IP-10, MIP-1a, MIP-3a), 2 growth factors (FGF-2, MMP-1), and 4 soluble receptors (APRIL, CD30, TNFRII, and TWEAK), were differentially expressed in CRC. ROC analysis confirmed the high association of TNF-α, BLC, Eotaxin-1, APRIL, and Tweak with AUC > 0.70, suggesting theranostic application. The expression of IFN-γ, IL-18, MIF, BLC, Eotaxin-1, Eotaxin-2, IP-10, and MMP1 was lower in metastatic compared to non-metastatic CRC; only AUC of MIF and MIP-1ß were > 0.7. Moreover, MDC, IL-7, MIF, IL-21, and TNF-α are positively associated with tolerance to CRC chemotherapy (CT) (AUC > 0.7), whereas IL-31, Fractalkine, Eotaxin-1, and Eotaxin-2 were positively associated with resistance to CT. TNF-α, BLC, Eotaxin-1, APRIL, and Tweak may be used as first-line early detection of CRC. The variable levels of MIF and MIP-1ß between metastatic and non-metastatic cases assign prognostic nature to these factors in CRC progression. Regarding tolerance to CT, MDC, IL-7, MIF, IL-21, and TNF-α are key when down-regulated or resistant to treatment is observed.

Links between SNPs in TLR-2 and TLR-4 and idiopathic recurrent pregnancy loss.

Background: Recurrent pregnancy loss is a serious complication of pregnancy and failure of the innate immune system, one part of which are toll-like receptors (TLRs). We hypothesised links between variants of TLR-2 and TLR-4 with recurrent pregnancy loss.Subjects and methods: We recruited 335 women with recurrent pregnancy loss, defined as ≥3 consecutive spontaneous miscarriage of unknown aetiology, and 331 age-matched control women. TLR-2 rs1898830 and rs4696483 and TLR-4 rs2770150, rs1554973 and rs7856729 genotyping were performed by allelic exclusion method (real-time PCR).Result: Of the five tested TLR-2 and TLR-4 tag-SNPs, minor allele frequency of TLR-2 rs1898830 was significantly more frequent in recurrent pregnancy loss patients than in controls. Significantly higher frequencies of homozygous (2/2) TLR-2 rs1898830 (14.1% vs. 8.9%) genotype carriers were seen between recurrent pregnancy loss cases and control women. Haploview analysis identified 1-locus TLR-2 haplotype (GC) that was positively associated with recurrent pregnancy loss. No TLR-4 haplotypes associated with altered recurrent pregnancy loss risk were identified.Conclusion: These findings confirm positive associations of TLR-2 rs1898830 with recurrent pregnancy loss, further supporting a role for TLR signalling in defining pregnancy outcome.

TLR3 and TLR4 SNP variants in the liver disease resulting from hepatitis B virus and hepatitis C virus infection.

BACKGROUND: Chronic infection with hepatitis B (HBV) and C virus (HCV) is linked with a pro-inflammatory state, predisposing to cirrhosis and liver cancer, particularly hepatocellular carcinoma (HCC). A role for Toll-like receptor (TLR) signalling in hepatocarcinogenesis was recently documented. We hypothesised a link TLR3 and TLR4 polymorphisms and HCC, as surrogates for the significance of TLR signalling in the promotion and initiation of HCC. MATERIALS AND METHODS: We recruited 174 HCV-infected patients, 100 HBV-infected patients and 360 healthy control subjects. TLR3 (rs3775290) and TLR4 (rs4986790) genotyping was done by PCR-restriction fragment length polymorphisms (PCR-RFLP), LFTs and AFP by standard routine techniques. Liver fibrosis was assessed clinically by the Fibrotest and Actitest. RESULT: The TLR3 rs3775290 minor T genotype was linked with increased risk of chronic HBV (P = 0.05) and HCV (P = 0.031) infection. The TLR4 rs4986790 minor G genotype was linked with significantly increased risk for HBV/HCV chronic infection (P < 0.001). Subgroups analyses indicated decreased risk of HBV-related HCC in relation to TLR3 rs3775290 CC/CT genotype (P = 0.022), with increased risk ascribed to the minor (T) allele (P = 0.04). Likewise, TLR4 rs4985790 minor (GG) genotype was positively associated with HBV-linked HCC (P < 0.001). Furthermore, a link between TLR3 TT (P < 0.001) andTLR4 GG (P = 0.04) minor genotypes was noted in relation to increased risk of HCV-related disease. CONCLUSION: TLR3 and TLR4 polymorphisms are promising biomarkers of liver cirrhosis and cancer associated with HBV and HCV infection.

FAS A-670G and Fas ligand IVS2nt A 124G polymorphisms are significantly increased in women with pre-eclampsia and may contribute to HELLP syndrome: a case-controlled study.

OBJECTIVE: We evaluated the association between the Fas-670A/G and the Fas ligand FasL IVS2nt 124 A/G polymorphisms and the risk of pre-eclampsia and its complications. DESIGN: A case-controlled study. SETTING: University Hospitals in most areas of Tunisia. POPULATION: We recruited 300 pregnant women who developed pre-eclampsia and 300 age-matched healthy pregnant women from the same hospital. METHODS: Genotyping of Fas-670A/G and the FasL IVS2nt 124A/G gene polymorphisms were conducted using polymerase chain reaction-restriction fragment length polymorphism among our cohort. MAIN OUTCOME MEASURES: Fisher's exact test was used to compare the statistical differences between groups for categorical variables and Student t tests were used for continuous variables. RESULTS: The frequency of the Fas-670G gene variant was significantly increased in women with pre-eclampsia (42%) compared with control women (30%; P < 0.001). Also, a statistically significant difference was obtained in the distribution of the FasL IVS2nt 124G gene variant when comparing women with pre-eclampsia (43%) with controls (30%; P < 0.001). Interestingly, we found that the carriage of Fas-670G was associated with increased liver enzymes, suggesting an increased prevalence of the haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome, a pre-eclampsia complication. CONCLUSION: The Fas-670G and FasL IVS2nt 124G polymorphisms are associated with a higher risk of pre-eclampsia and its complications. TWEETABLE ABSTRACT: Polymorphisms in the Fas and FasL genes are associated with increased risk of pre-eclampsia and HELLP syndrome.

Interleukin-10 rs1800871 (-819C/T) and ATA haplotype are associated with preeclampsia in a Tunisian population.

OBJECTIVES: Interleukin-10 (IL-10) is implicated in several aspects of pregnancy. As genetic predisposition can be involved in the development of preeclampsia, the association between IL-10's promoter region polymorphisms and this pathology has been investigated, although with conflicting results. To date, only a small cohort study (total n = 40) has evaluated this association in the African continent, and none have been conducted in Tunisia. Hence, we evaluated the association between these polymorphisms and the risk of preeclampsia in a large Tunisian cohort. STUDY DESIGN: 345 preeclampsia patients and 300 controls were genotyped for the IL-10 promoter region variants -1082A/G, -819C/T and -592A/C using real-time PCR. MAIN OUTCOME MEASURES: Differences in means were determined by Student's t-test, while intergroup significance was assessed by Pearson χ2 or 2-way ANOVA. Genotypes were tested for Hardy-Weinberg equilibrium (HWE) in the control and cases. Logistic regression analysis was performed in order to determine the odds ratios and 95% confidence intervals associated with the linkage disequilibrium risk. RESULTS: An increased frequency of the -819 T (minor) allele and the -819 T/T genotype was seen in preeclampsia cases. Also, three-locus haplotype (-1082AG/-819CT/-592AC) analysis identified the ATA haplotype as having a higher incidence in women with preeclampsia (OR = 1.48, 95% CI: 1.03-2.11) and this was confirmed by multivariate regression analysis (OR = 1.65, 95% CI: 1.13-2.43) after controlling for covariates. CONCLUSIONS: We suggest that the IL-10 -819 T/T variant and the ATA haplotype, which are associated with low production of IL 10, represent genetic risk factors for preeclampsia in Tunisian women.

Gender differences in the association of ELMO1 genetic variants with type 2 diabetes in Tunisian Arabs.

PURPOSE: Polymorphisms of the engulfment and cell motility 1 (ELMO1) gene were recently associated with type 2 diabetes (T2DM) and its complications. We investigated the association of rs10255208, rs7782979, and rs2041801 ELMO1 gene variants with T2DM in Tunisian Arabs. METHODS: Subjects comprised 900 T2DM patients and 600 normoglycemic controls. ELMO1 genotyping was done by PCR-RFLP; the contribution of ELMO1 variants to T2DM was analyzed by Haploview and regression analysis. RESULTS: Minor allele frequencies of rs7782979 and rs10255208 ELMO1 variants were significantly higher among unselected T2DM cases than controls, and significant differences in the distribution of rs7782979 genotypes were seen between T2DM cases and control subjects, which was seen in male but not female subjects. Three-locus ELMO1 haplotype analysis identified haplotype GAA to be positively associated, and haplotypes GCA, AAA, and GCG to be negatively associated with T2DM. The distribution of these haplotypes was gender-dependent for some (GCA, GCG, AAG), and gender-independent for others (GAA, AAA). This translated into altered risk of T2DM in male or female subjects, which persisted after adjusting for BMI, systolic and diastolic blood pressure, and serum lipid profile. CONCLUSION: These results confirm role for ELMO1 as T2DM susceptibility locus, which appears to be gender-dependent.

Genetic variation in the progesterone receptor gene and susceptibility to recurrent pregnancy loss: a case-control study.

OBJECTIVE: To investigate the association of progesterone receptor (PGR) gene variants with susceptibility to recurrent pregnancy loss (RPL). DESIGN: Retrospective case-control study. SETTING: Outpatient obstetrics and gynaecology clinics. POPULATION: Women with RPL (396), defined as three or more consecutive miscarriages of unknown aetiology, and 361 women used as controls. METHODS: PGR genotyping was performed by the allelic exclusion method (real-time polymerase chain reaction). MAIN OUTCOME MEASURES: PGR single nucleotide polymorphisms (SNPs) and the distribution of their alleles, genotypes and haplotypes. RESULTS: Higher minor allele frequencies (MAFs) for rs590688, rs10895068, and rs1942836 were seen in RPL cases than in controls, which remained significant after controlling for multiple comparisons. Significantly higher frequencies of heterozygous (1/2) rs608995, along with heterozygous (1/2) and homozygous (2/2) rs590688, rs10895068, and rs1942836 genotype carriers, were seen between RPL cases versus controls, respectively, which persisted after controlling for age, body mass index (BMI), and menarche. The increased risk of RPL associated with rs590688 and rs1942836 was dependent on the number of minor alleles, thus suggesting a 'dose-dependent' effect associated with both variants. Varied linkage disequilibrium (LD) was noted between rs590688, rs10895068, rs608995, and rs1942836 PGR variants associated with RPL. Haplotypes with an increased frequency of CGTC and reduced frequency of GGAT were noted in women with RPL, compared with controls, thereby indicating these haplotypes as RPL-susceptible and RPL-protective, respectively. This association persisted after controlling for multiple comparisons, and after adjusting for covariates. CONCLUSIONS: We have confirmed a positive association of specific PGR variants (rs590688, rs10895068, and rs1942836) and PGR haplotypes (ATGCCGTC and ATTCGGTC) with an increased risk of RPL, thereby supporting a role for PGR as an RPL candidate locus. TWEETABLE ABSTRACT: Genetic variants in progesterone receptor gene are associated with increased risk of recurrent pregnancy loss.

HLA DRB1/DQB1 alleles and DRB1-DQB1 haplotypes and the risk of rheumatoid arthritis in Tunisians: a population-based case-control study.

Rheumatoid arthritis (RA) is an inflammatory disease, which affects synovial joints, and is influenced by environmental and genetic factors, in particular the human leucocyte antigen (HLA) system. In our study, we investigated the association of HLA class II DRB1 and DQB1 alleles and DRB1-DQB1 haplotypes with RA susceptibility in Tunisian subjects. Therefore, HLA class II low-resolution genotyping was done in 110 RA patients and 116 controls, with a HLA-DRB1*04 high-resolution typing. Our results showed a strong association between HLA-DRB1*04/DRB1*04:05 alleles and RA presence, which persisted after correcting for multiple comparisons (Pc < 10-3, Pc = 0.020, respectively), in contrast to the protective effect of HLA-DRB1*04:03 allele (Pc = 15.2 × 10-4). However, increased frequency of DQB1*05 (Pc = 0.020) and decreased frequency of DRB1*04:03 subtype (Pc = 0.032) were seen in RF+ patients than controls. Moreover, when RA patients were compared to controls, DRB1*04-DQB1*03 haplotype was associated with RA susceptibility in Tunisians (Pc = 16.8 × 10-5), independently of RF status. Conversely, DRB1*01 allele and DRB1*01-DQB1*05 haplotype was highly present in RF+ vs RF- groups (Pc < 10-3, Pc = 5.6 × 10-3, respectively) and seems to be linked to seropositivity. Investigation of HLA class II alleles and haplotypes association with RA susceptibility with secondary Sjögren's syndrome (sSS) showed a predisposing effect of DRB1*04 (Pc < 10-3) and DRB1*04-DQB1*03 haplotype when RA with sSS/without sSS groups were compared to healthy controls. Our results confirms the association of HLA-DRB1*04, specifically HLA-DRB1*04:05 subtype, and DRB1*04-DQB1*03 haplotype with RA susceptibility in Tunisians, independently of seropositivity or the sSS presence.

Interleukin-1ß, Interleukin1-Ra, Interleukin-10, and tumor necrosis factor-α polymorphisms in Tunisian patients with rheumatoid arthritis.

OBJECTIVES: The aim of this study was to investigate the role of IL-1ß (-511C>T), TNFα (-308 G>A), IL-10 (-1082 G>A) and IL-1RN VNTR polymorphisms in the susceptibility to rheumatoid arthritis (RA) in Tunisians. PATIENTS AND METHODS: Using PCR-based methods, 104 RA patients and 150 healthy controls were investigated. We compared allele and genotype frequencies in RA patients versus controls and analyzed their correlations with erosive form (EF). RESULTS: IL1-RN VNTR A1A3 genotype is associated with higher risk of RA (P=0.012, OR=4.31). Among the cases, males who carry this genotype were more exposed to RA (P=0.044, OR=8, 47). For IL1- ß gene, a significantly higher frequency of the -511C/C genotype was observed in RA patients in comparison to controls (P=0.013, OR=2.45). This higher frequency was especially observed in women (P=0,003, OR=3.42). In contrast, IL10-1082G/G genotype was less common in patients (P=0.046, OR=0.46). According to EF, men carrying IL1-RN VNTR A1A3 (P=0.005 OR=5.28) and IL1-ß-511C/C (P=0.015 OR=2.61) genotypes develop non EF of RA. Moreover, TNFα-308 A allele (P=0.024, OR=1.84) and A/A genotype (P=0.033, OR=3.1) were positively associated to EF of RA. However, G allele (P=0.024, OR=0.31) and GG genotype (P=0.31, OR=0.031) of the TNFα-308 were protectors. CONCLUSION: Our results indicated that IL-1RN VNTR, IL-1ß (-511C>T) and IL-10 (-1082 G>A) are associated with susceptibility to RA, and that IL-1RN VNTR, IL-1ß (-511C>T) and TNFα (-308 G>A) are associated with severity of RA.

IL-10 gene promoter and intron polymorphisms and changes in IL-10 secretion in women with idiopathic recurrent miscarriage.

STUDY QUESTION: Is recurrent pregnancy loss (RPL) associated with polymorphisms in the promoter and intron regions of the interleukin-10 (IL-10) gene? SUMMARY ANSWER: IL-10 rs1518111 was found to be associated with RPL but the commonly studied promoter variants rs1800872, rs1800871 and 1800896 were not. WHAT IS KNOWN ALREADY: Reduced expression of IL-10 is implicated in RPL, due to defective maternal immune tolerance (causing early miscarriages) or placental vascular insufficiency (causing late losses). IL-10 production is in part inherited, and IL-10 gene variants associated with reduced IL-10 expression have been analyzed for their association with RPL, often with inconclusive results. STUDY DESIGN, SIZE, DURATION: A retrospective case-control study was performed between January 2011 and April 2012. The subjects comprised 296 RPL cases and 305 control women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genotyping of the IL-10 intron (rs1878672, rs3024492, rs1554286, rs1518111, rs3024491, rs3024490) and promoter (rs1800872, rs1800871, rs1800896) variants was done by real-time PCR, with defined clusters. MAIN RESULTS AND THE ROLE OF CHANCE: A higher minor allele frequency (MAF) of rs1518111 (P = 0.03) was in seen RPL cases; but the MAFs of the remaining SNPs were comparable between cases and controls. Setting the homozygous major allele genotype (1/1) as the reference, significantly higher frequencies of heterozygous rs1554286 and rs1800872, and homozygous rs1800896 genotype carriers, and a reduced frequency of homozygous rs1518111 genotype carriers, were seen in RPL cases, while the distribution of the remaining genotypes were comparable between cases and controls. Serum IL-10 levels were significantly reduced in RPL cases compared with control women (P = 0.002), and this correlated with rs1518111 and rs1800871 genotypes in both groups, and with the rs1800872 genotype among control women. A nine-locus (rs1878672, rs3024492, rs1554286, rs1518111, rs3024491, rs3024490, rs1800872, rs1800871 and rs1800896) haploview analysis demonstrated an increased frequency of haplotype 112112121 in RPL cases, thus conferring a disease susceptibility nature to this haplotype. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study was that it was limited to Bahraini Arabs, thereby necessitating parallel studies of other ethnic groups. Another limitation is the study design, which prompts speculation on whether it is a cause-effect relationship. WIDER IMPLICATIONS OF THE FINDINGS: While the lack of association of the various IL-10 promoter variants with RPL was in agreement with reports from varied ethnic groups, this is the first study to confirm the association between IL-10 rs151811 intronic variant and RPL. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by grants from the Arabian Gulf University Research Fund. None of the authors report any competing interests.

Polymorphisms of transcription factor-7-like 2 (TCF7L2) gene in Tunisian women with polycystic ovary syndrome (PCOS).

BACKGROUND AND AIMS: Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects women in their child-bearing age, and is often associated with insulin resistance and type 2 diabetes (T2DM). Given the overlap between PCOS and T2DM, we investigated the association of transcription factor-7-like 2 (TCF7L2) variants rs4506565, rs7903146, rs12243326, and rs12255372 with the susceptibility to PCOS. SUBJECTS AND METHODS: Study subjects comprised 119 Tunisian women with PCOS (mean age 29.8±4.7years), and 150 control women (mean age 30.6±5.9years). TCF7L2 genotyping was done by the allelic discrimination/real-time PCR method. RESULTS: Minor allele frequencies (MAFs) of rs4506565 (P=0.61), rs7903146 (P=0.68), rs12243326 (P=0.56), and rs12255372 (P=0.60) were comparable between PCOS cases and control subjects. As the four tested TCF7L2 variants were in linkage disequilibrium, 4-locus (rs4506565, rs7903146, rs12243326, rs12255372) haplotype analysis demonstrated that haplotype 2111 was initially negatively associated with PCOS [P=0.035; OR (95% CI)=0.13 (0.02-0.85)], which was later lost upon correcting for multiple comparisons [Pc=0.248]. CONCLUSION: Our data suggest that there is weak or no contribution of TCF7L2 gene polymorphism to PCOS in Tunisian women. Further studies with larger samples are necessary to confirm this observation.

Endothelial nitric oxide synthase gene variants and haplotypes associated with an increased risk of idiopathic recurrent miscarriage.

We investigated the association of endothelial nitric oxide synthase (NOS3) polymorphisms rs2070744 (-786T> C), 27-bp repeat 4b/4a, rs1799983 (Glu298Asp), rs3918188 (-734C> A), and rs743507 (113G> A) with idiopathic recurrent miscarriage (IRM). This was a case-control study involving women with confirmed IRM (n = 296), and 305 age- and ethnically matched control women. NOS3 rs2070744, rs1799983, rs3918188, and rs743507 genotyping was done by TaqMan assays; NOS3 4b/4a genotyping was done by PCR-ASA. A higher frequency of -786C and 298Asp alleles was seen in IRM cases, which remained associated independently with IRM on multivariate analysis. Allele and genotype distribution of 4b/4a, rs3918188 (-734C> A) and rs743507 (113A> G) were comparable between IRM cases and control women. Taking homozygous wild-type genotype as a reference, regression analysis confirmed the association of Glu298Asp and -786T/C, and rs743507 homozygous carriers with IRM risk. Marked linkage disequilibrium was seen between tested NOS3 variants, thus allowing the construction of 5-locus [-786T> C/4b4a/Glu298Asp/-734C> A/113G> A] haplotypes. Taking the common T4bGCA haplotype as a reference, multivariate analysis confirmed the positive association of C4bTCG haplotype with IRM, after controlling for traditional covariates. Genetic variation at the NOS3 locus represents a genetic risk factor for increased susceptibility to IRM.

Lack of replication of common EXT2 gene variants with susceptibility to type 2 diabetes in Lebanese Arabs.

OBJECTIVE: Recent genome-wide association studies and replication analyses have reported the association of variants of the exostosin-2 (EXT2) gene and risk of type 2 diabetes mellitus (T2DM) in some populations, but not in others. This study investigated the associations of EXT2 variants rs1113132, rs3740878 and rs11037909 with T2DM in a Lebanese Arab population. METHODS: This case-control study involved 995 T2DM patients and 1076 control subjects. Genotyping was done by the allelic exclusion method. RESULTS: While minor allele frequencies (MAFs) of rs11037909 (P=0.028) and rs3740878 (P=0.048), but not rs1113132 (P=0.841), were higher in patients, this was lost after correcting for multiple testing. Apart from EXT2 rs1113132, which was marginally associated with T2DM in the additive model (P=0.054), but not after adjustment for covariates, none of the tested EXT2 SNPs were associated with T2DM in any of the genetic models tested. However, variable associations of EXT2 variants with T2DM were noted according to BMI status. While the three tested EXT2 variants were not associated with T2DM in obese subjects, rs1113132 and rs11037909, but not rs3740878, were associated with T2DM in non-obese subjects. Meta-analysis revealed a significant association of rs11037909 and a marginal association of rs3740878 with T2DM in the fixed model. Using a common (GTA) haplotype as reference, three-locus (rs1113132/rs11037909/rs3740878) haplotype analysis demonstrated no association between any of the EXT2 haplotypes with T2DM, not even before correcting for multiple testing. CONCLUSION: This study demonstrated no association of rs1113132, rs3740878 and rs11037909 EXT2 variants with T2DM.

Genetic variation in TGFB1 gene and risk of idiopathic recurrent pregnancy loss.

Transforming growth factor ß1 plays a significant role in pregnancy outcome. We investigated the association of TGFB1 exon 1 (rs1800471, rs1800470) and promoter region (rs1800469, rs1800468) polymorphisms with recurrent pregnancy loss (RPL) in 675 Tunisian women: 304 women with a history of three consecutive pregnancy losses of unknown etiology with the same partner and 371 age-matched multiparous control women. TGFB1 genotyping was done by TaqMan assays. Higher minor allele frequency for rs1800471 (P< 0.001), but not for rs1800470, rs1800469 or rs1800468 was found in RPL cases compared with controls. A significant difference in the distribution of rs1800471 genotypes was seen between the RPL cases and control women, irrespective of the genetic model used. Increased RPL risk was seen with rs1800471 allele C in the heterozygous state and to a greater degree in the homozygous state, thus establishing a dose-dependent effect. Haploview analysis revealed differential linkage disequilibrium between the TGFB1 single-nucleotide polymorphisms analyzed. TGFB1 haplotype analysis identified eight common haplotypes (rs1800471/rs1800470/rs1800469/rs1800468) with three (GTTG, Pc = 0.02; CCTG, Pc = 0.02 and CTCG, Pc = 0.02) positively associated with RPL and one (GCCG, Pc = 0.009) negatively associated with RPL. This study provides the first evidence that the TGFB1 genotype may influence RPL.

Contribution of common variants of ENPP1, IGF2BP2, KCNJ11, MLXIPL, PPARγ, SLC30A8 and TCF7L2 to the risk of type 2 diabetes in Lebanese and Tunisian Arabs.

BACKGROUND: While several type 2 diabetes mellitus (T2DM) susceptibility loci identified through genome-wide association studies (GWAS) have been replicated in many populations, their association in Arabs has not been reported. For this reason, the present study looked at the contribution of ENNP1 (rs1044498), IGF2BP2 (rs1470579), KCNJ11 (rs5219), MLXIPL (rs7800944), PPARγ (rs1801282), SLC30A8 (rs13266634) and TCF7L2 (rs7903146) SNPs to the risk of T2DM in Lebanese and Tunisian Arabs. METHODS: Study subjects (case/controls) were Lebanese (751/918) and Tunisians (1470/838). Genotyping was carried out by the allelic discrimination method. RESULTS: In Lebanese and Tunisians, neither ENNP1 nor MLXIPL was associated with T2DM, whereas TCF7L2 was significantly associated with an increased risk of T2DM in both the Lebanese [P < 0.001; OR (95% CI): 1.38 (1.20-1.59)] and Tunisians [P < 0.001; OR (95% CI): 1.36 (1.18-1.56)]. Differential associations of IGF2BP2, KCNJ11, PPARγ and SLC30A8 with T2DM were noted in the two populations. IGF2BP2 [P = 1.3 × 10(-5); OR (95% CI): 1.66 (1.42-1.94)] and PPARγ [P = 0.005; OR (95% CI): 1.41 (1.10-1.80)] were associated with T2DM in the Lebanese, but not Tunisians, while KCNJ11 [P = 8.0 × 10(-4); OR (95% CI): 1.27 (1.09-1.47)] and SLC30A8 [P = 1.6 × 10(-5); OR (95% CI): 1.37 (1.15-1.62)] were associated with T2DM in the Tunisians, but not Lebanese, after adjusting for gender and body mass index. CONCLUSION: T2DM susceptibility loci SNPs identified through GWAS showed differential associations with T2DM in two Arab populations, thus further confirming the ethnic contributions of these variants to T2DM susceptibility.

Identification of specific vascular endothelial growth factor susceptible and protective haplotypes associated with recurrent spontaneous miscarriages.

BACKGROUND: We investigated the association of vascular endothelial growth factor (VEGF) gene polymorphism with recurrent spontaneous miscarriage (RSM). METHODS: VEGF -2578C/A, -1154G/A, -634G/C, +936C/T single nucleotide polymorphisms (SNPs) were assessed in 304 RSM patients, and 371 age-and body mass index-matched control subjects using real-time PCR. RESULTS: Higher minor allele frequency of -1154G/A (P < 0.001) and +936C/T (P < 0.001), but not -2578C/A (P = 0.55) or -634G/C (P = 0.87) SNPs, were seen in patients. Significant differences in the distribution of -1154G/A (P = 0.006) and +936C/T (P = 0.015), but not -2578C/A (P = 0.473) or -634G/C (P = 1.000) genotypes, were seen in cases compared with control women. Of the possible 16 VEGF haplotypes, 9 were found to be common, and were included. A significantly lower frequency of C G C C (P = 0.008), and A G G C (P < 0.001) haplotypes, and a higher frequency C G C T (P = 0.020), and C G T (P = 0.004) haplotypes were seen in patients. CONCLUSIONS: These results strongly support that VEGF polymorphisms, in particular-1154G/A and +936C/T, are significantly associated with RSM. Our results confirm, in the largest sample to date, previous works in other populations on VEGF polymorphism in RSM.

Association of the R67X and W303X non-sense polymorphisms in the protein Z-dependent protease inhibitor gene with idiopathic recurrent miscarriage.

Protein Z-dependent protease inhibitor (ZPI) is a 72 kDa single-chain serpin which inhibits the activated coagulation factors X and XI. Two non-sense polymorphisms of ZPI, R67X and W303X, were recently identified, and were linked with a prothrombotic state. Here, we investigated the association of the R67X (728C>T) and W303X (1438G>A) variants in the ZPI gene with recurrent spontaneous miscarriage (RSM). This was a case-control study involving a total of 288 women with a history of two consecutive or ≥3 non-consecutive pregnancy losses between 8 and 12th week of gestation, along with 304 age-matched and ethnically matched multiparous control women, with no personal or family history of pregnancy complications. The minor allele frequency of R67X (P = 0.003) and W303X (P = 0.014) were higher in RSM cases than in control women. Both single-nucleotide polymorphisms were significantly associated with RSM under the dominant genetic association model, and were in moderate linkage disequilibrium (D' = 0.412; P < 0.001). Taking the common (728)C/(1438)G haplotype as reference, multivariate analysis confirmed the positive association of (728)T/(1438)G [P = 0.043; odds ratio (OR) = 2.25; 95% confidence interval (CI) = 1.03-4.90], and (728)T/(1438)A (P = 0.022; OR = 3.93; 95% CI = 1.23-12.59) haplotypes with increased RSM risk. These differences remained significant after controlling for some covariates. These results demonstrate that both ZPI R67X and W303X non-sense variants and specific ZPI haplotypes are significantly associated with RSM.

HLA class I and class II polymorphism in a population from south-eastern Tunisia (Gabes Area).

The gene frequencies of HLA class I and class II alleles were investigated in 95 healthy Tunisian individuals from Gabes. Our aim was to compare the genetic relationship between Gabesians and Mediterraneans and sub-Sahara Africans using genetic distances, Neighbour-Joining dendrograms, correspondence and haplotypes analysis, thereby providing additional information about evolutionary history of modern-day Tunisians. Subjects were unrelated and of both genders, and HLA class I and class II genes were genotyped using the polymerase chain reaction-sequence specific primer (PCR-SSO) technique. Our data show that south-eastern Tunisians (Gabes area) are related to present-day North Africans (Algerians, Moroccans, Tunisians) and Iberians (Spaniards, Basques), and along with other North Africans, appear to be genetically related to Berbers, an indication that the Arab invasion (7th-11th centuries) of North Africa had minimal contribution on the HLA makeup of North Africans. On the other hand, Iberians including Spaniards and Basques show relatedness to (native Tunisian) Berbers, suggesting that the gene flow of 7th century AD invaders was also low in Iberians. In conclusion, the successive invasions of North Africa in general, and Tunisia in particular, did not modify markedly the genetic makeup of present-day Tunisians. With the exception of Greeks who have a sub-Saharan genetic profile, all Mediterranean populations depict a typical mediterranean substratum.

Interleukin-10 microsatellite variants and the risk of acute coronary syndrome among Tunisians.

The objective of the study was to investigate the association of interleukin (IL)-10 promoter microsatellite polymorphisms, linked with altered IL-10 secretion, with the susceptibility to acute coronary syndrome (ACS) in adult Tunisian patients. We genotyped 291 ACS patients and 291 age-, gender- and ethnically matched control subjects for the microsatellites IL-10R [X78437.2g.5325CA(11_15)] and IL-10G [X78437.2g.8134CA(14_29)] by PCR-based assays. Haplotypes were reconstructed using maximum likelihood method. Regression analysis was used in determining the risk imparted by specific IL-10 genotypes and haplotypes. A significant decrease in IL-10G12 (24 CA repeats) (P<0.001; OR=0.465) and IL-10G15 (27 CA repeats) (P=0.043; OR=0.232), and a significant increase in the low IL-10 producer allele, IL-10R3 (14 CA repeats) (P=0.049; OR=1.461), microsatellites were seen in the ACS group compared with controls. Of the possible 14 haplotypes constructed, there was an enrichment of the R2G9 (13CA vs. 21CA) haplotype in controls [P=0.019; adjusted OR (95% CI)=0.67 (0.48-0.94)] and R2G15 (13CA vs. 27CA) haplotype in cases [P=0.042; adjusted OR (95% CI)=5.29 (1.06-26.30)], thus assigning a protective and susceptible nature to these haplotypes respectively. The differential association of IL-10 microsatellite alleles and haplotypes with ACS suggests that IL-10 contributes to ACS pathogenesis. While the functional attributes of these microsatellite markers remain to be seen, it is likely that they have distinct functional properties (altered IL-10 secretion), which in turn affect the susceptibility to ACS development.

HLA class I and class II polymorphisms in Tunisian Berbers.

BACKGROUND: The HLA polymorphism is a powerful genetic tool to study population origins. By analysing allele frequencies and haplotypes in different populations, it is possible to identify ethnic groups and establish the genetic relationships among them. AIM: The Berber (endogenous Tunisians) HLA class I and class II genotypes were analysed and compared with those of Mediterranean and Sub-Saharan African communities using genetic distances, Neighbour-Joining dendrograms, correspondence and haplotype analysis. SUBJECTS AND METHODS: One hundred and five unrelated Berbers were typed for HLA class I (A, B) and class II (DRB1, DQB1) gene alleles using reverse dot-blot hybridization. RESULTS: High frequencies of A*0201 (24.76%), A*3402 (22.38%) and B*44 (32.85%) alleles were recorded for Berbers, the highest recorded for Mediterranean and North African populations. This study shows a close relatedness of Tunisian Berbers to other Tunisians, North Africans and Iberians. CONCLUSION: The apparent relatedness of Tunisian Berbers to present-day (North African) Tunisians, Algerians and Moroccans suggests that the Arab invasion of North Africa (7(th)-11(th) centuries AD) did not significantly impact the genetic makeup of North Africans. Furthermore, Tunisian Berbers appear to be closely related to Iberians (Spaniards and Basques), indicating that the 7(th) century AD gene flow of invaders was low in Iberians and that the main part of their genetic pool came after the Northward Saharan migration, when hyper-arid conditions were established in Sahara (before 6000 BC). Other studied populations belong to the old Mediterranean substratum, which has been present in the area since pre-Neolithic times. This study indicates a higher proportion of Iberian than Arab ancestry in Tunisian Berbers, which is of value in evaluating the evolutionary history of present-day Tunisians. Greeks seem to share genetic HLA features (Chr 6) with Sub-Saharans. The relatedness of Greeks to Sub-Saharans has been confirmed by other studies based on chromosome 7 genetic markers.