Interleukin 10 (IL-10) gene variants and haplotypes in Tunisian women with polycystic ovary syndrome (PCOS): a case-control study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder that affects women in their child-bearing age, and is associated with insulin resistance and type 2 diabetes. The etiology of PCOS involves multiple factors including genetic, metabolic and immunological factors. Interleukin - 10 (IL-10), as an anti-inflammatory cytokine, plays a critical role in this regard. We investigated the potential role of IL-10 gene variants in the development of PCOS in Tunisian population. METHODS AND RESULTS: 115 cases and 120 controls were recruited in the current case control study. Rotterdam consensus criteria were used to diagnose PCOS patients. Genotyping for IL-10, rs1800896, rs1800871 and rs1800872 variants, was performed by real time PCR. The results obtained showed that the minor allele frequency of rs1800896, rs1800871and rs1800872 were comparable between PCOS cases and control subjects (P = 0.30, P = 0.71, and P = 0.57 respectively). The distribution analysis revealed an unsignificant association of the three tested variants, in all genetic models. Haplotype analysis identified one haplotype CCA with a protective role in PCOS development (P = 0.05; OR (95% CI) = 0.56 (0.32 - 0.99)). This association did not persist after adjustment for multiples covariates (Pc = 0.154). CONCLUSIONS: Our study is the first to show how ethnicity influences the association of IL-10 gene variants with PCOS susceptibility. No allelic nor genetic association were observed between the tested variants and PCOS in Tunisian women, however, a particular IL-10 haplotype with a protective effect for PCOS was identified.

Association of matrix metalloproteinase-2 gene variants with diabetic nephropathy risk.

BACKGROUND: Diabetic nephropathy is a highly destructive microvascular complication of diabetes. Genetic predisposition is involved in the pathogenesis of diabetic nephropathy, with multiple allelic polymorphisms associated with the development and progression of the disease, thereby increasing the overall risk. To date, no study is available that shows the association of matrix metalloproteinase-2 (MMP-2) gene polymorphisms with diabetic nephropathy risk. Thus, we investigated the potential genetic influence of MMP-2 promoter variants in the development of diabetic nephropathy in type 2 diabetic patients. METHODS: In total, 726 type 2 diabetic patients and 310 healthy controls were included in the study and genotyped for MMP-2, -1306C/T, -790T/G, -1575G/T and -735C/T by real-time PCR. The analysis of the outcomes was performed assuming three genetic models. The threshold for statistical significance was set at 0.05. RESULTS: The results showed that the minor allele frequency of the -790T/G variant was significantly higher in patients with and without nephropathy compared to controls. Furthermore, the distribution analysis revealed a significant association of the -790T/G variant, in all genetic models, with increased risk of diabetic nephropathy that persisted after adjusting for key covariates. No significant associations between MMP-2, -1306C/T, -1575G/T, -735C/T and the risk of diabetic nephropathy were detected. Haplotype analysis identified two risk haplotypes GCGC and GTAC associated with diabetic nephropathy. CONCLUSIONS: The present study is the first to demonstrate the allelic and genotypic association of the MMP-2-790T/G variant and two haplotypes with an increased risk of diabetic nephropathy in a Tunisian population with type 2 diabetes.

Clinical Implications of Krüpple-like Transcription Factor KLF-14 and Certain Micro-RNA (miR-27a, miR-196a2, miR-423) Gene Variations as a Risk Factor in the Genetic Predisposition to PCOS.

Polycystic ovary syndrome (PCOS) is a disorder with a symptomatic manifestation of an array of metabolic and endocrine impairments. PCOS has a relatively high prevalence rate among young women of reproductive age and is a risk factor for some severe metabolic diseases such as T2DM, insulin insensitivity, and obesity, while the most dominant endocrine malfunction is an excess of testosterone showing hyperandrogenism and hirsutism. MicroRNAs have been implicated as mediators of metabolic diseases including obesity and insulin resistance, as these can regulate multiple cellular pathways such as insulin signaling and adipogenesis. Genome-wide association studies during the last few years have also linked the Krüpple-like family of transcription factors such as KLF14, which contribute in mechanisms of mammalian gene regulation, with certain altered metabolic traits and risk of atherosclerosis and type-2 DM. This study has characterized the biochemical and endocrine parameters in PCOS patients with a comprehensive serum profiling in comparison to healthy controls and further examined the influence of allelic variations for miRNAs 27a (rs895819 A > G), 196a2 (rs11614913 C > T), 423 (rs6505162C > A), and transcription factor KLF14 (rs972283 A > G) gene polymorphism on the risk and susceptibility to PCOS. The experimental protocol included amplification refractory mutation-specific (ARMS)-PCR to detect and determine the presence of these polymorphic variants in the study subjects. The results in this case−control study showed that most of the serum biomarkers, both biochemical and endocrine, that were analyzed in the study demonstrated statistically significant alterations in PCOS patients, including lipids (LDL, HDL, cholesterol), T2DM markers (fasting glucose, free insulin, HOMA-IR), and hormones (FSH, LH, testosterone, and progesterone). The distribution of Krüppel-like factor 14 rs972283 G > A, miR-27a rs895819 A > G, and miR-196a-2 rs11614913 C > T genotypes analyzed within PCOS patients and healthy controls in the considered population was significant (p < 0.05), except for miR-423 rs6505162 C > A genotypes (p > 0.05). The study found that in the codominant model, KLF14-AA was strongly associated with greater PCOS susceptibility (OR 2.35, 95% CI = 1.128 to 4.893, p < 0.022), miR-27a-GA was linked to an enhanced PCOS susceptibility (OR 2.06, 95% CI = 1.165 to 3.650, p < 0.012), and miR-196a-CT was associated with higher PCOS susceptibility (OR 2.06, 95% CI = 1.191 to 3.58, p < 0.009). Moreover, allele A of KLF-14 and allele T of miR-196a2 were strongly associated with PCOS susceptibility in the considered population.

Biochemical Characterization and Molecular Determination of Estrogen Receptor-α (ESR1 PvuII-rs2234693 T>C) and MiRNA-146a (rs2910164 C>G) Polymorphic Gene Variations and Their Association with the Risk of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is regarded as one of the most frequently encountered endocrine disorders and affects millions of young women worldwide, resulting in an array of complex metabolic alterations and reproductive failure. PCOS is a risk factor for diabetes mellitus, obstructive sleep apnea, obesity and depression in patients. Estrogen receptors (ESRs) are significant candidates in endocrine function and ovarian response in women. Moreover, microRNAs and long non-coding RNAs are emerging as principal mediators of gene expression and epigenetic pathways in various disease states. This study has characterized the clinical parameters in PCOS patients with comprehensive biochemical profiling compared to healthy controls and further examined the influence of allelic variations for estrogen receptor-α (ESR1 PvuII-rs2234693 T>C) and miRNA-146a (rs2910164 C>G) gene polymorphism on the risk of and susceptibility to PCOS. In this case-control study, we have used amplification refractory mutation specific (ARMS)-PCR to detect and determine the presence of these polymorphic variants in the study subjects. Our results demonstrated that most of the biochemical markers, which were analyzed in the study, show statistically significant alterations in PCOS patients, including fasting glucose, free insulin, HOMA-IR, LDL, HDL, cholesterol and hormones such as FSH, LH, testosterone and progesterone, which correlate with the established biochemical alterations in the disorder. Further, it is reported that for estrogen receptor-α (ESR1 PvuII-rs2234693 T>C), the frequency of the T allele (fT) was significantly higher among patients (0.64 vs. 0.44) compared to controls, while the frequency of the C allele (fC) was lower in patients (0.36 vs. 0.56) compared to controls. However, it was found that there was no association of an increased risk of PCOS with the ESR1 PvuII-rs2234693 C>T gene polymorphism. On the contrary, the study found strong association of miRNA-146a (rs2910164 C>G) gene polymorphism with an enhanced risk of PCOS. The frequency of the C allele (fC) was significantly higher among patients (0.52 vs. 0.36) compared to controls. The frequency of the G allele (fG) was found to be lower in patients (0.48 vs. 0.64) compared to controls. The codominant, dominant and recessive models display a statistically significant association of polymorphic variations with PCOS. Moreover, the G allele was associated strongly with PCOS susceptibility with an OR = 1.92 (95%) CI = (1.300−2.859), RR = 1.38 (1.130−1.691) p-value < 0.001.

Molecular determination of progesterone receptor's PROGINS allele (Alu insertion) and its association with the predisposition and susceptibility to polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome, previously known as Stein-Leventhal syndrome, is associated with altered reproductive endocrinology, predisposing a young woman towards the risk of PCOS. It has a prevalence of 6-20% among the reproductive-age women. Progesterone is a key hormone in the pathophysiology of PCOS and patients show diminished response (progesterone resistance), implicating the role of progesterone receptor (PR) as a factor in the disease etiology and prognosis. In this case-control study, we have used mutation-specific PCR (confirmed by Sanger sequencing) to detect the presence of a pathologically significant PR polymorphic variant called as PROGINS. The variant has an Alu insertion in intron G and has two SNPs in exon 4 and exon 5, with all the three aberrations in complete disequilibrium. Our results demonstrated a statistically significant difference in the frequencies of PROGINS between the PCOS patients and healthy controls (p = 0.047). The frequencies of the genotypes CC (A1/A1), CT (A1/A2), and TT (A2/A2) in patients were 74.50%, 20.58%, and 4.90%, and in healthy controls they were 87.28%, 11%, and 1.69%, respectively. Our results put forward two determining factors with regard to PCOS: (i) the frequency of PROGINS allele was significantly higher among PCOS patients compared to the healthy matched controls (0.15 vs 0.07) in the studied population, (ii) the PROGIN allele was significantly associated with the lower levels of serum progesterone in PCOS patients (p < 0.003). The findings are conspicuous as these relate the PROGINS variant to the increased susceptibility of PCOS and might explain the progesterone resistance in patients.

Adiponectin (ADIPOQ) gene variants and haplotypes in Saudi Arabian women with polycystic ovary syndrome (PCOS): a case-control study.

The aim of this study is to assess the association of nine SNPs on ADIPOQ on the PCOS risk among Saudi Arabian Women. A case-control study, including 162 cases and 162 controls in Saudi Arabia, was enrolled. Genotyping was carried out by the allelic discrimination method. Estimated haplotype frequencies were assessed using the maximum likelihood method. Results showed that ADIPOQ SNPs were not associated with PCOS for allelic and genotypic frequencies (p > .05). In haplotype estimation analysis, a significant positive association was detected between 21211 haplotype (rs2241766/rs1501299/rs2241767/rs3774261/rs17366743) in additive model with increased risk of PCOS (p = .009, OR = 2.16 [1.22-3.82] CI 95%). None of the nine SNPs illustrated significant association with the quantitative traits after multiple test corrections. These results support a significant association of 21211 haplotype (rs2241766/rs1501299/rs2241767/rs3774261/rs17366743) of ADIPOQ gene in Saudi women with polycystic ovary syndrome.

Impact of variants on type-2 diabetes risk genes identified through genomewide association studies in polycystic ovary syndrome: a case-control study.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in females, and is associated with altered metabolic processes in particular insulin resistance and diabetes mellitus. PCOS shares with type-2 diabetes (T2D) a number of features, including beta cell dysfunction, impaired glucose tolerance and dyslipidaemia. Recently, genomewide association studies (GWAS) have reported a number of genes with reproducible associations and susceptibilities to T2D. To address this, we examined the association between the T2D GWAS candidate genes (CDKAL1, CDKN2B, COL8A1, HHEX, IGF2BP2, KCNJ1, KCNQ1 and SLC30A8) and PCOS in Saudi women. A case-control study, includes 162 cases and 162 controls was enrolled. Genotyping was carried out by the allelicdiscrimination method. Our results showed that the variants including rs792837 of COL8A1, rs61873498 of KCNQ1 and rs13266634 of SLC30A8 genes to be significantly more frequent in PCOS patients than in controls. Our results suggest that COL8A1, KCNQ1 and SLC30A8, which are previously identified through GWAS as T2D-associated genes, are associated with PCOS.

Haplotyping strategy highlights the specificity of FTO gene association with polycystic ovary syndrome in Tunisian women population.

The FTO (fat mass and obesity associated) gene was associated with different metabolic disorders in populations from different origins but with great difference between African and non-African populations. North-African populations combine many genetic backgrounds, among which African, Berber and Caucasian components, which makes North-Africans a good model for studying the genetic association of FTO. In the present investigation we explored the association of FTO gene with polycystic ovary syndrome (PCOS) in a population from Tunisia (n=278). Single nucleotide polymorphisms (SNPs) used in this study were previously associated in non-African populations: rs8050136 (A/C), rs9939609 (A/T), rs9930506 (G/A), or in both African and non-African populations: rs8057044 (A/G). Genotyping was performed by allelic discrimination method on StepOne real-time PCR system or KASPar technology. Linkage disequilibrium (LD) pattern was assessed by HAPLOVIEW and reconstruction of haplotypes was performed by PHASE, while statistical analyses were performed using StatView and GoldenHelix programs. Among the 13 haplotypes in the population, three (h1, h7 and h13) were strongly associated with PCOS notably h13 (P<0.0001, OR95%CI=0.040 [0.005-0.294]) while SNPs display weaker association. Moreover the LD pattern in FTO in the Tunisian population (r(2) index) was intermediary between those of Caucasian and Africans. This highlights the need for studying the genetics of complex disorders in the North-African populations taking into-account the haplotype structure of candidate loci more than SNPs taken alone.

Polymorphisms in genes coding for the NK-cell receptor NKG2D and its ligand MICA in recurrent miscarriage.

PROBLEM: To investigate the possible association of Natural Killer Group (NKG) receptors gene polymorphisms and MHC class I chain-related protein A (MICA) gene polymorphism with recurrent miscarriage (RM). METHODS: Seven SNPs in NKG2D gene (rs1049174, rs2255336, rs2617160, rs2617161, rs2246809, rs2617169, and rs2617170), one SNP in NKG2A gene (rs1983526), and one SNP in MICA gene (MICA129) were assessed by allelic discrimination (real-time PCR) in both patients and control women. RESULTS: The rs2617170 T/T genotype significantly protected against RM [OR (95%) = 0.63 (0.40-0.98)]. The NKG2D haplotypes analysis on the basis of pairwise LD revealed two haplotype blocks. In block1, we found an increased frequency of CAT (Pc = 0.007; OR = 2.13; 95% CI = 1.24-3.68) and GGA haplotypes (Pc = 0.041; OR = 2.02; 95%CI = 1.03-3.96) and reduced frequency of CAA haplotype (Pc = 0.027; OR = 0.72; 95% CI = 0.54-0.96) in patients. In block2, increased frequency of GATG haplotype (Pc = 10(-4) ; OR = 9.25; 95% CI = 3.04-28.12) and reduced frequency of ATTC haplotype (Pc = 0.035; OR = 0.69; 95%CI = 0.50-0.97) were seen in patients. CONCLUSION: The NKG2D gene polymorphisms may influence the success of pregnancy in Tunisian women.

Common polymorphisms of calpain-10 and the risk of polycystic ovary syndrome in Tunisian population: a case-control study.

Recent studies have suggested that calpain-10 (CAPN10) gene polymorphisms play a role in the susceptibility to polycystic ovary syndrome (PCOS). The aim of the present study was to evaluate the possible association between three single nucleotide polymorphisms (SNPs) in CAPN10 gene: UCSNP-43 (rs3792267), UCSNP-19 (rs3842570), and UCSNP-63 (rs5030952) and PCOS in Tunisian cases and control women. Study subjects included 127 women with PCOS (mean age 29.8 ± 4.7 year) and 150 healthy women (mean age 33.5 ± 5.6 year). CAPN10 genotyping was carried-out by direct PCR and PCR-RFLP. Linkage disequilibrium pattern in the genomic region explored was determined by HAPLOVIEW 4.2 while reconstruction of haplotypes was done using PHASE 2.1. The phylogenetic distribution of haplotypes in the population was determined by ARLEQUIN 2.000. Six haplotypes were observed. None of SNPs associated with PCOS or its components while the haplotype H4 associated with the phenotype PCOS-obese (P < 0.025). Moreover the pair of haplotypes H1/H4 strongly associated with high blood-pressure (OR = 14.4, P < 0.012). This work confirms the association of CAPN10 gene with metabolic components in PCOS and highlights the role of haplotypes as strong and efficient genetic markers.

Association of POL1, MALT1, MC4R, PHLPP and DSEL single nucleotide polymorphisms in chromosome 18q region with type 2 diabetes in Tunisians.

Previous studies and replication analyses have linked chromosome 18q21.1-23 with type 2 diabetes (T2DM) and its complications, including diabetic nephropathy (DN). Here we investigated the association of POL1-nearby variant rs488846, MALT1-nearby variant rs2874116, MC4R-nearby variant rs1942872, PHLPP rs9958800 and DSEL-nearby variant rs9966483 single nucleotide polymorphisms (SNPs) in the 18q region, previously linked with DN in African-Americans, with T2DM in (North African) Tunisian subjects, followed by their association with DN, which was performed subsequent to the analysis of the association with T2DM. Study subjects comprised 900 T2DM cases and 748 normoglycemic control, and genotyping was carried out by PCR-RFLP analysis. Of the 5 SNPs analyzed, POL1-nearby variant rs488846 [P=0.044], and MC4R-nearby variant rs1942872 [P=0.012] were associated with moderate risk of T2DM. However, there was a lack of consistency in the association of the 5 tested SNPs with DN. As such, it appears that the three chromosome 18q region variants appear to play a role in T2DM pathogenesis, but not with DN in North African Tunisian Arabs.

Transcription factor-7-like 2 gene variants are strongly associated with type 2 diabetes in Tunisian Arab subjects.

Genome-wide association studies validated transcription factor 7-like 2 (TCF7L2) gene as confirmed type 2 diabetes (T2DM) susceptibility locus, and an ethnic contribution of TCF7L2 variants to T2DM risk was indicated. The aim of this study was to replicate in a Tunisian Arab population identified associations of common TCF7L2 variants with T2DM. We tested the association of TCF7L2 variants: rs4506565, rs7903146, rs12243326, and rs12255372, with T2DM in 900 Tunisian patients and 875 control subjects. TCF7L2 genotyping was done by allelic discrimination/real-time PCR method. Minor allele frequencies of rs4506565 (P=2.4×10(-8)), rs7903146 (P=1.2×10(-6)), rs12243326 (P=8.4×10(-8)) and rs12255372 (P=1.1×10(-5)) were significantly higher in cases. The four tested TCF7L2 variants were in linkage disequilibrium, and 4-locus (rs4506565, rs7903146, rs12243326, rs12255372) haplotype analysis demonstrated that haplotype 1111 was negatively associated (Pc<0.001), while haplotypes 2222 (Pc=0.008) and 2211 (Pc=0.020) were positively associated with T2DM risk, after controlling for a number of covariates. The strong contribution of TCF7L2 gene variants to T2DM among Tunisians is in line with similar findings in other ethnic groups, confirming TCF7L2 as a common T2DM candidate gene.

Lack of association between genetic polymorphisms within KCNQ1 locus and type 2 diabetes in Tunisian Arabs.

AIMS: Polymorphisms of KCNQ1 were previously associated with type 2 diabetes (T2DM) in select Caucasian and non-Caucasian populations. We investigated the association of rs231361, rs231359, rs151290, rs2237892, rs2283228, rs2237895, and rs2237896 KCNQ1 polymorphisms with T2DM in Tunisian Arabs. SUBJECTS AND METHODS: Subjects comprised 900 T2DM patients and 600 normoglycemic controls. KCNQ1 genotyping was done by allelic discrimination (real-time PCR) and PCR-RFLP methods; the contribution of KCNQ1 polymorphisms to T2DM were analyzed by Haploview and regression analysis. RESULTS: Minor allele frequency (MAF) of the 7 tested KCNQ1 variants was comparable between T2DM cases and controls. Mild association of rs2237892 genotypes with T2DM was seen (P=0.014), highlighted by the significant association of the C/T genotype with increased T2DM risk (OR, 2.11; 95%CI, 1.25-3.53), after adjusting for BMI, gender, systolic and diastolic blood pressure, and serum lipid profile. Heterogeneity in linkage disequilibrium pattern between tested KCNQ1 variants analyzed was seen. Two-locus (rs231361 and rs231359) and 5-locus (remaining 5 SNPs) haplotype analysis did not reveal any significant association with any of the haplotypes contained in either block 1 or block 2. CONCLUSION: These results indicate that there was no evidence for an association of KCNQ1 polymorphisms with T2DM in Tunisian Arabs.

Cytochrome P450 (CYP3A4*18) and glutathione-S-transferase (GSTP1) polymorphisms in a healthy Tunisian population.

AIM: Human cytochrome P450 3A and glutathione S-transferase (GST) enzymes evolved to catalyze the metabolism of numerous common therapy drugs and endogenous molecules. Members of the CYP3A are the majority expressed in human liver and intestine. The genetic factors play an important role in the interindividual variability in CYP3A and GST activity. Detection of CYP3A4 and GST variant alleles and knowledge about their allelic frequency in specific ethnic groups are important to lead to individualized drug dosing and improved therapeutics. METHODS: We determined the allelic frequency of the CYP3A4*18 and GSTP1 in a group of 138 healthy Tunisian subjects using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. It is based on a specific PCR product cut by restriction endonucleases. This method offers the advantage of cutting DNA by the appropriate restriction enzyme at the correct mutation site hence enhancing its reliability. Electrophoretic separation demonstrates the presence (or absence) of restriction sites. RESULTS: In the group of 138 unrelated individuals, the frequencies of the CYP3A4*18 and GSTP1 variant allele in this Tunisian population were 0.02 and 0.01, respectively. CONCLUSIONS: The present study describes polymorphisms of Cyp3A4 and GST among Tunisian subjects. We developed a simple assay for the detection of the CYP3A4*18 and GSTP1 polymorphisms and we compared our allelic frequencies to other populations. No significant difference was obtained. This study provides the first analysis of CYP3A4*18 and GSTP1 mutant allele frequencies in the Tunisian population.

Single-nucleotide polymorphisms and haplotypes in the adiponectin gene contribute to the genetic risk for type 2 diabetes in Tunisian Arabs.

Adiponectin is an adipocyte-produced protein involved in regulating glucose, lipid, and energy metabolism, and is encoded by ADIPOQ (APM1) gene. ADIPOQ polymorphisms were previously associated with type 2 diabetes (T2DM) in Caucasian and non-Caucasian populations. We investigated the contribution of 13 polymorphisms in the promoter, coding regions, and 3'untranslated region of ADIPOQ gene to T2DM in 917 patients and 748 normoglycemic control subjects. ADIPOQ genotyping was done by allelic discrimination method. Of the 13 ADIPOQ variants analyzed, higher minor allele frequency of rs16861194 (P<0.001), rs17300539 (P<0.001), rs266729 (P<0.001), rs822396 (P=0.02), rs2241767 (P=0.03), and rs1063538 (P=0.02) were seen in T2DM cases. Varied association of ADIPOQ genotypes with T2DM was seen according to the genetic model used: rs17300539 and rs266729 were significantly associated with T2DM under the three models, while rs16861194 was association with T2DM under additive and dominant models, and rs822396, rs2241766, and rs1063538 were associated with T2DM under the dominant models only. Haploview analysis revealed low linkage disequilibrium between the ADIPOQ variants, resulting in high haplotype diversity, and two blocks were identified, each differentially associated with T2DM. These results support a significant association of ADIPOQ gene polymorphism with T2DM in Tunisian Arabs.

Human Alu insertion polymorphisms in North African populations.

Several features make Alu insertions a powerful tool used in population genetic studies: the polymorphic nature of many Alu insertions, the stability of an Alu insertion event and, furthermore, the ancestral state of an Alu insertion is known to be the absence of the Alu element at a particular locus and the presence of an Alu insertion at the site that forward mutational change. This study analyses seven Alu insertion polymorphisms in a sample of 297 individuals from the autochthonous population of Tunisia (Thala, Smar, Zarzis, and Bou Salem) and Libya with the aim of studying their genetic structure with respect to the populations of North Africa, Western, Eastern and Central Europe. The comparative analyses carried out using the MDS and AMOVA methods reveal the existence of spatial heterogeneity, and identify four population groups. Study populations (Libya, Smar, Zarzis, and Bou Salem) are closest to North African populations whereas Thala is isolated and is closest to Western European populations. In conclusion, Results of the present study support the important role that migratory movements have played in the North African gene pool, at least since the Neolithic period.

Renin-angiotensin-aldosterone system genotypes and haplotypes affect the susceptibility to nephropathy in type 2 diabetes patients.

BACKGROUND: The association between renin C-4063T and angiotensinogen (AGT) T174M, M235T, and A-6G polymorphisms with diabetic nephropathy (DN) was investigated in Tunisian type 2 diabetes (T2DM) patients. METHODS: Study subjects comprised 917 T2DM patients (405 normoalbuminuric, 329 microalbuminuric and 185 macroalbuminuric). Genotyping was done by PCR-RFLP. RESULTS: Renin C-4063T allele and genotype frequencies were comparable between DN cases and normoalbuminuric controls. Although AGT 235T and -6G allele, and 235T/T and -6G/G genotype frequencies were higher in DN compared to normoalbuminuric patients, they were comparable between microalbuminuric or macroalbuminuric patients. Three-locus AGT haplotype analysis (A-6G/T174M/M235T) identified DN-protective (ATM, AMM, GTM) and DN-susceptible (GTM, ATT, GMT and AMT) haplotypes, and demonstrated enrichment of GTT haplotype in macroalbuminuric compared to microalbuminuric or normoalbuminuric patients. Regression analysis confirmed negative (AMM) and positive (GTM, ATT, GMT, AMT) association of AGT haplotypes with microalbuminuria, and negative (AMM) and positive (GTM and ATT) association of AGT haplotypes with macroalbuminuria. None of the AGT haplotypes was associated with DN severity. CONCLUSIONS: Genetic variation at the AGT gene influences the risk of nephropathy in T2DM patients but not extent of DN severity, and thus represents a potential DN genetic susceptibility locus worthy of replication.

Common polymorphisms of calpain-10 and the risk of Type 2 Diabetes in a Tunisian Arab population: a case-control study.

BACKGROUND: Genetic variations in the calpain-10 gene (CAPN10), in particular the at-risk diplotype (112/121), were previously implicated with increased risk of type 2 diabetes (T2D). METHODS: We examined the association of CAPN10 UCSNP-43 (rs3792267), UCSNP-19 (rs3842570), and UCSNP-63 (rs5030952) SNPs with T2D in 917 Tunisian T2D patients and 748 non-diabetic controls. CAPN10 genotyping was done by PCR-RFLP. RESULTS: Enrichment of UCSNP-19 2R (minor) allele and 2R/2R genotype was found in T2D patients; the allele and genotype distribution of UCSNP-43 and UCSNP-63 alleles and genotypes were not significantly different between patient groups and non-diabetic control subjects. Regression analysis demonstrated progressive increases in T2D risk in 3R/2R [OR (95% CI) = 1.35 (1.08 - 1.68)] and 2R/2R [OR (95% CI) = 1.61 (1.20 - 2.18)] genotypes. Of the six haplotypes detected, enrichment of haplotype 111 (UCSNP-43/UCSNP-19/UCSNP-63) was seen in patients (Pc = 0.034); the distribution of the other haplotypes was comparable between patients and control subjects; neither haplotype 211 nor haplotype 212 was observed. Furthermore, the frequency of all CAPN10 diplotypes identified, including the "high-risk diplotype (112/121) reported for Mexican-Americans and Northern Europeans, were comparable between patients and controls. CONCLUSIONS: CAPN10 UCSNP-19 variant, and the 111 haplotype contribute to the risk of T2D in Tunisian subjects; no significant associations between CAPN10 diplotypes and T2D were demonstrated for Tunisians.

Identification of specific angiotensin-converting enzyme variants and haplotypes that confer risk and protection against type 2 diabetic nephropathy.

BACKGROUND: Cross-sectional and family studies identified angiotensin-converting enzyme (ACE) gene as a risk factor for diabetic nephropathy (DN). The contribution of ACE gene variants to DN development and progression is controversial and varies among different ethnic/racial groups. METHODS: We investigated the association of three ACE gene variants with DN, rs1799752 insertion/deletion (I/D), rs1800764T/C and rs12449782A/G in 917 Tunisian type 2 diabetic (T2DM) patients: 515 with (DN) and 402 without (DWN) nephropathy. ACE genotyping was done by PCR-based assays; haplotype estimation was performed using H-Plus software (chi(2)-test based). RESULTS: Genotype frequency distributions of the three studied variants were in Hardy-Weinberg equilibrium. Minor allele frequency of rs1800764 was higher in DN patients than DWN patients or healthy controls, and minor allele frequency of rs1799752 was higher in DN than DWN patients. Higher frequency of rs1799752 and rs1800764 homozygous mutant genotypes was seen in DN compared to DWN patients. Of the three variants, only rs1799752 deletion/deletion (D/D) genotype was associated with a significant increase in albumin to creatinine ratios levels, and D/D carriers had elevated low-density lipoprotein, total cholesterol and urea. Three locus haplotype [rs1799752(I/D)/rs1800764(T/C)/rs12449782(A/G)] analysis revealed that the frequency of DCG haplotype was higher, while that of ITG and ICA haplotypes were lower among unselected type 2 diabetic patients. Taking ITA haplotype as reference, multivariate regression analysis confirmed the negative (ITG), and positive (DCG, DTG, DCA and DTA) association of specific ACE haplotypes with DN, after adjusting for potential nephropathy-linked covariates. CONCLUSIONS: Our results support the involvement of specific ACE variants in DN pathogenesis and demonstrate the presence of DN-specific haplotypes at the ACE locus.

Contribution of type 2 diabetes associated loci in the Arabic population from Tunisia: a case-control study.

BACKGROUND: Candidate gene and genome-wide association studies have both reproducibly identified several common Single Nucleotide Polymorphisms (SNPs) that confer type 2 diabetes (T2D) risk in European populations. Our aim was to evaluate the contribution to T2D of five of these established T2D-associated loci in the Arabic population from Tunisia. METHODS: A case-control design comprising 884 type 2 diabetic patients and 513 control subjects living in the East-Center of Tunisia was used to analyze the contribution to T2D of the following SNPs: E23K in KCNJ11/Kir6.2, K121Q in ENPP1, the -30G/A variant in the pancreatic beta-cell specific promoter of Glucokinase, rs7903146 in TCF7L2 encoding transcription factor 7-like2, and rs7923837 in HHEX encoding the homeobox, hematopoietically expressed transcription factor. RESULTS: TCF7L2-rs7903146 T allele increased susceptibility to T2D (OR = 1.25 [1.06-1.47], P = 0.006) in our study population. This risk was 56% higher among subjects carrying the TT genotype in comparison to those carrying the CC genotype (OR = 1.56 [1.13-2.16], P = 0.002). No allelic or genotypic association with T2D was detected for the other studied polymorphisms. CONCLUSION: In the Tunisian population, TCF7L2-rs7903146 T allele confers an increased risk of developing T2D as previously reported in the European population and many other ethnic groups. In contrast, none of the other tested SNPs that influence T2D risk in the European population was associated with T2D in the Tunisian Arabic population. An insufficient power to detect minor allelic contributions or genetic heterogeneity of T2D between different ethnic groups can explain these findings.