PORTABILITY OF GWAS RESULTS BETWEEN ETHNIC POPULATIONS: GENETIC MARKERS FOR POLYCYSTIC OVARY SYNDROME (PCOS) IN MEDITERRANEAN AREA.

Genome Wide Association Studies (GWAS) are excellent opportunities to define culprit genes in complex disorders such as the polycystic ovary syndrome (PCOS). PCOS is a prevalent disorder characterized by anovulation, hyperandrogenism and polycystic ovaries, which benefitted from several GWASs in Asians and Europeans revealing more than 20 potential culprit genes near associated single nucleotide variations (SNV). Translation of these findings into the clinical practice raises difficulties since positive hits are surrogate SNVs linked with causative mutations by linkage disequilibrium (LD). Studies in Mediterranean populations (e.g. Southern Europe and North Africa) raise supplementary problems because of a different LD-pattern, which may disrupt the link with causative mutations. Our experience in MEDIGENE program between Tunisia and France enforces the necessity of genetic anthropology studies before translating GWAS data. Tunisians are a heterogeneous population with ancestral Berbers, European, Arab and Sub-Saharan African components while South Europeans display a high level of genetic diversity, partially explained by gene flow from North Africa. Human diversity studies require sampling from Middle East and North Africa (MENA) region that will help to understand genetic factors in complex diseases.

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.

Epidemiological survey of vector-borne infections in equids from northern Tunisia.

Leishmaniosis (Leishmania infantum infection) and piroplasmoses (Theileria equi and Babesia caballi infections) are vector-borne diseases with significant economic and public health impacts. Despite their importance, there is a lack of data concerning these infections in equids from Tunisia. The present study was carried out to estimate the prevalence of L. infantum, T. equi and B. caballi in 104 equids from northern Tunisia. The authors reported for the first time on the seroprevalence of anti-Leishmania antibodies in equids in Tunisia (6.7%). The study reported a high infection prevalence of piroplasms (23.1%), revealed for the first time T. equi and B. caballi infections in Tunisian donkeys, and showed that these animals act as reservoirs for the maintenance and dissemination of piroplasms.

La leishmaniose (infection à Leishmania infantum) et les piroplasmoses (infections à Theileria equi et à Babesia caballi) sont des maladies à transmission vectorielle dont les conséquences économiques et l'impact sur la santé publique sont significatifs. Malgré leur importance, en Tunisie la situation des équidés vis-à-vis de ces maladies était jusqu'à présent peu documentée. L'étude décrite par les auteurs avait pour objet d'estimer la prévalence des infections à L. infantum, T. equi et B. caballi chez 104 chevaux du Nord de la Tunisie. La recherche d'anticorps dirigés contre Leishmania a permis de déterminer pour la première fois une prévalence sérologique chez les équidés (6,7 %). L'étude a rapporté une prévalence élevée de piroplasmes (23,1 %) et fait état pour la première fois en Tunisie de l'existence d'infections à T. equi et à B. caballi chez des ânes ; elle montre également que ces derniers font office de réservoirs et jouent un rôle dans le maintien et la dissémination des piroplasmes.

La leishmaniosis (infección por Leishmania infantum) y las piroplasmosis (infecciones por Theileria equi y Babesia caballi) son enfermedades transmitidas por vectores que tienen una importante repercusión en la economía y la salud pública. A pesar de su importancia, existen pocos datos sobre estas infecciones en los équidos tunecinos. Los autores describen un estudio destinado a estimar la prevalencia de L. infantum, T. equi y B. caballi en 104 équidos del norte del país, gracias al cual determinaron por primera vez la seroprevalencia de anticuerpos contra Leishmania en équidos tunecinos (un 6,7%). La investigación puso de manifiesto una elevada prevalencia de la infección por piroplasmas (un 23,1%), reveló por vez primera la infección de asnos tunecinos por T. equi y B. caballi y demostró que estos animales ejercen de reservorio, manteniendo y propagando así los piroplasmas.

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.

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.

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.

European genetic variants associated with type 2 diabetes in North African Arabs.

AIMS: Recent genome-wide association studies (GWAS) and previous approaches have identified many genetic variants associated with type 2 diabetes (T2D) in populations of European descent, but their contribution in Arab populations from North Africa is unknown. Our study aimed to validate these markers and to assess their combined effects, using large case-control studies of Moroccan and Tunisian individuals. METHODS: Overall, 44 polymorphisms, located at 37 validated European loci, were first analyzed in 1055 normoglycaemic controls and 1193 T2D cases from Morocco. Associations and trends were then assessed in 942 normoglycaemic controls and 1446 T2D cases from Tunisia. Finally, their ability to discriminate cases from controls was evaluated. RESULTS: Carrying a genetic variant in BCL11A, ADAMTS9, IGF2BP2, WFS1, CDKAL1, TP53INP1, CDKN2A/B, TCF7L2, KCNQ1, HNF1A, FTO, MC4R and GCK increased the risk of T2D when assessing the Moroccan and Tunisian samples together. Each additional risk allele increased the susceptibility for developing the disease by 12% (P = 9.0 × 10(-9)). Genotype information for 13 polymorphisms slightly improved the classification of North Africans with and without T2D, as assessed by clinical parameters, with an increase in the area under the receiver operating characteristic curve from 0.64 to 0.67 (P = 0.004). CONCLUSION: In addition to TCF7L2, 12 additional loci were found to be shared between Europeans and North African Arabs. As for Europeans, the reliability of genetic testing based on these markers to determine the risk for T2D is low. More genome-wide studies, including next-generation sequencing, in North African populations are needed to identify the genetic variants responsible for ethnic disparities in T2D 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.

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.

Variants within the calpain-10 gene and relationships with type 2 diabetes (T2DM) and T2DM-related traits among Tunisian Arabs.

BACKGROUND: Common variations in the calpain 10 (CAPN10) gene variants UCSNP-43, UCSNP-19 and UCSNP-63, and the 112/121 diplotype, are associated with an increased risk of type 2 diabetes (T2DM) and T2DM-related traits. METHODS: The association of UCSNP-43, -19 and -63 CAPN10 SNPs with T2DM was assessed in 917 Tunisian T2DM patients and 748 ethnically matched non-diabetic controls. CAPN10 genotyping was done by PCR-RFLP. RESULTS: Significant differences in UCSNP-19 MAF, but not UCSNP-43 or -63, and genotype distribution were seen between patients and controls. Heterogeneity in UCSNP-19, but not UCSNP-43 and -63, genotype distribution was noted according to geographical origin. Obesity was associated with UCSNP-19, while raised fasting glucose was associated with UCSNP-63, and increased HDL was associated with UCSNP-43. Enrichment of homozygous UCSNP-19 2/2 was seen in overweight and obese compared with lean patients; logistic-regression analyses demonstrated a positive association of the 2/2 genotype with overweight [P=0.003; OR (95% CI)=2.07 (1.28-3.33)] and obese [P=0.021; OR (95% CI)=1.83 (1.10-3.07)] patients. Of the six CAPN10 haplotypes identified, significant enrichment of only haplotype 111 was seen in T2DM patients [Pc=0.034; OR (95% CI)=1.22 (1.06-1.41)], while the frequency of all identified CAPN10 diplotypes, including the high-risk 112/121, was comparable between patients and controls. CONCLUSION: While CAPN10 UCSNP-19 SNP and haplotype 111 contribute to the risk of T2DM in Tunisian subjects, no significant association between CAPN10 diplotypes and T2DM was demonstrated.

Endothelial nitric oxide synthase Glu298Asp, 4b/a, and -786T>C gene polymorphisms and the risk of ischemic stroke.

BACKGROUND AND PURPOSE: Endothelial nitric oxide synthase (eNOS) gene polymorphisms were associated with reduced NO production, and were evaluated as risk factors for ischemic stroke (IS). We investigated the association between eNOS gene -786T>C (promoter), 27-bp repeat 4b/4a (intron 4), and Glu298Asp (exon 7) polymorphisms with IS in 329 IS patients and 444 controls. MATERIALS AND METHODS: Glu298Asp and -786T>C genotyping was done by PCR-RFLP, 4b/4a was assessed by PCR-ASA. The contribution of eNOS polymorphisms to IS was analyzed by haplotype and multivariate regression analysis. RESULTS: Higher frequency of 298Asp allele was seen in IS patients (P = 1.2 x 10(-10)), which remained independently associated with IS on multivariate analysis after controlling for traditional cerebrovascular risk factors. Allele and genotype distribution of 4b/4a and -786T>C polymorphisms were comparable between patient and controls. Significantly higher prevalence of 298Asp/4b/-786T and 298Asp/4b/-786C haplotypes were seen in IS cases, thus conferring a disease susceptibility nature to these haplotypes. Multivariate regression analysis confirmed the association of 298Asp/4b/-786T and 298Asp/4b/-786C haplotypes, and in addition identified 298Asp/4a/-786T haplotype to be independently associated with IS, after controlling for traditional cerebrovascular risk factors. CONCLUSIONS: Genetic variation at the eNOS locus represent genetic risk factor for increased susceptibility to IS.

Diabetic retinopathy, PAI-1 4G/5G and -844G/A polymorphisms, and changes in circulating PAI-1 levels in Tunisian type 2 diabetes patients.

AIM: The association of altered plasminogen activator inhibitor (PAI)-1 levels and PAI-1 polymorphisms (4G/5G and -844G/A) with diabetic retinopathy (DR) was investigated in 856 type 2 diabetes (T2D) patients, of whom 383 presented with (DR group), and 473 presented without (DWR group), retinopathy. METHODS: PAI-1 4G/5G and -844G/A genotyping were done by PCR-RFLP, and PAI-1 levels were measured by ELISA testing. RESULTS: The genotype distribution of 4G/5G and -844G/A polymorphisms did not deviate from the Hardy-Weinberg equilibrium model among healthy subjects. Higher frequencies of the 4G/4G genotype, and lower frequencies of the -844A allele, -844G/A and -844A/A genotypes, were seen in DR patients, conferring disease susceptibility and protection, respectively. While PAI-1 levels were significantly elevated in the 4G/4G compared with other PAI-1 genotypes, significant differences in PAI-1 levels between DR and DWR patients were seen in the 4G/-844A, 4G/-844G and 5G/-844A haplotype carriers among DR patients. However, comparable distributions of 4G/5G and -844G/A alleles, genotypes and haplotypes, and similar PAI-1 levels, were seen in the proliferative retinopathy (PR) and non-proliferative retinopathy (NPR) patients, indicating that neither PAI-1 variants nor changes in PAI-1 levels were linked to DR severity. Multivariate analyses identified 4G/-844A and 4G/-844G haplotypes as negatively and positively associated, respectively, with DR, but not with DR severity (PR vs NPR) after adjusting for a number of covariates. CONCLUSION: The present study identifies changes in PAI-1 levels and genetic variations at the PAI-1 locus as risk factors for DR, but not DR severity, that may serve as useful markers of increased DR susceptibility.

Lupus anticoagulants and anti-phospholipid antibodies as risk factors for a first episode of ischemic stroke.

BACKGROUND: Antiphospholipid antibodies (aPLA) and lupus anticoagulant (LAC) were shown to precipitate thromboembolic events. Their association with ischemic stroke remains to be seen. OBJECTIVES: We investigated the contribution of LAC, and antibodies directed against the phospholipids cardiolipin (aCL), phosphatidylserine (aPS), and the phospholipid-dependent cofactors beta2-glycoprotein I and annexin V, to the risk for ischemic stroke. PATIENTS/METHODS: LAC and antibody levels were measured in 208 stroke patients and 203 age- and gender-matched control subjects. RESULTS: Positive LAC resulted in an increased risk for stroke [OR (95% CI) = 8.1 (2.4-27.5)]. Significant elevation in aPS IgG, aCL IgM and aCL IgG titers, and increased prevalence of elevated aPS IgG, aCL IgM and aCL IgG (based on P95 cutoff values of healthy individuals) were seen in patients. aPS IgG was associated with cardioembolic, whereas aCL IgG and IgM were elevated in lacunar, atherosclerotic and cardioembolic, and LAC positivity was documented only in lacunar stroke subtypes. The co-presence of LAC with a positive aCL IgM/IgG or aPS IgG did not affect the overall risk for stroke. Multivariate analysis confirmed the association of positive LAC with stroke [aOR (95% CI) = 9.7 (1.8-52.5)], and demonstrated a clear gradation of increasing risk of stroke associated with the four categories of aCL IgG and aPS IgG, and identified aCL IgM P95 as independent predictors of stroke after adjusting for potentially confounding covariates. CONCLUSIONS: Our study demonstrates that the presence of LAC, and elevated aCL IgG and aPS IgG antibodies are risk factors for stroke.

Association between renin-angiotensin-aldosterone system genotypes and haplotypes and risk of ischemic stroke of atherosclerotic etiology.

OBJECTIVE: The association of renin C-4063T and angiotensinogen (AGT) T174M, AGT M235T and AGT A-6G polymorphisms with ischemic stroke of atherosclerotic etiology was investigated in 329 Tunisian patients with stroke and 444 controls. MATERIALS AND METHODS: Genotyping was performed using PCR-RFLP and the contributions of polymorphisms to the risk of stroke were analyzed using haplotype and multivariate regression analysis. RESULTS: AGT 235T and AGT-6G allele and AGT 235T/T, AGT-6A/G and AGT-6G/G genotype frequencies were higher in patients. Linkage disequilibrium (LD) was noted for AGT174T with AGT235M and AGT(-6)A in patients, while AGT235M was in LD with AGT(-6)A in controls and AGT235T was in LD with AGT(-6)G in both groups. The AGT 174T/235T/-6A and AGT 174T/235M/-6G haplotypes were positively and negatively associated with stroke respectively. Multivariate regression analysis identified AGT 174T/235M/-6A, AGT 174T/235T/-6G, AGT 174T/235T/-6A and AGT 174M/235T/-6A haplotypes to be significantly associated with an increased risk of stroke. CONCLUSIONS: Renin-angiotensin-aldosterone system polymorphisms influence the risk of atherosclerotic stroke in Tunisians.

Associations between tumor necrosis factor-alpha and lymphotoxin-alpha polymorphisms and idiopathic recurrent miscarriage.

Heightened expression of tumor necrosis factor (TNF)-alpha and lymphotoxin-alpha (LT-alpha) was associated with pregnancy complications, including idiopathic recurrent miscarriage (RM). Whereas TNF-alpha and LT-alpha gene polymorphisms affect serum cytokine concentrations, their contribution to RM is controversial. The single nucleotide polymorphisms (SNPs) TNF-alpha (-238G/A, -308G/A) and LT-alpha (+252A/G) were investigated in 350 RM women and 200 control women. Higher frequency of the TNF-alpha -238A, but not the TNF-alpha -308A or the LT-alpha+252G, allele was seen in patients, with comparable frequencies of TNF-alpha -238G/A, TNF-alpha -308G/A, and LT-alpha+252A/G genotypes seen between both groups, except for TNF-alpha -238G/G, which was lower in patients. Regression analysis confirmed the association of the TNF-alpha -238G/A SNP with idiopathic RM, and both TNF-alpha -308A/TNF -238G/LT-alpha+252G and TNF-alpha -308G/TNF-alpha -238A/LT-alpha+252G haplotypes played a susceptible role in idiopathic RM. TNF-alpha -238G/A and -238A/A, and LT-alpha+252G/G genotypes were positively associated only with exclusively early RM. This supports the concept of the association of TNF-alpha (-238G/A) and LT-alpha (+252A/G) polymorphic variants in idiopathic RM.

Lupus anticoagulant and antibodies to beta 2-glycoprotein I, annexin V, and cardiolipin as a cause of recurrent spontaneous abortion.

The prevalence of lupus anticoagulant (LAC), anticardiolipin (ACA), anti-beta(2) glycoprotein I (beta(2)GPI), and antiannexin V antibodies were determined in 200 recurrent spontaneous abortion (RSA) patients and 200 age-matched control women. ACA IgG was associated with early, while antiannexin V IgG and LAC were associated with late, and ACA IgG, antiannexin V IgG, and LAC were associated with combined early + late RSA, thereby recommending inclusion of their screening in RSA workout.