Impact of toll-like receptor 4 variations on nasopharyngeal carcinoma risk and survival in tunisian population.

INTRODUCTION: The Toll-like receptor 4 (TLR4), an important member of the host's innate immune response, is coded by a polymorphic gene. This polymorphism could be a predisposing factor for NasoPharyngeal Carcinoma (NPC). AIM: To determine the association between TLR4 gene polymorphisms and the susceptibility to NPC in a cohort of Tunisian affected patients. METHODS: Genomic DNAs from 245 unrelated patients affected by undifferentiated carcinoma type (UCNT) and 264 unrelated healthy controls were genotyped for the five single nucleotides polymorphisms (SNPs) of TLR4 locus (4434 A>G (rs1927914),7263 G>C (rs10759932), 6134 A>G(rs4986790), 8851C>T (rs 4986791), 5272 T>C(rs11536889), +8469 T>C (rs11536891)) by Taqman® 5'-nuclease assay. RESULTS: Among all polymorphisms studied, only the rs4986790 G and rs4986791 T alleles were significantly more prevalent in patients' group than controls (45% vs. 38%; p=0.03; pc=0.06) and increased the risk of the NPC (OR=1.3, 95% CI=1.01-1.69). Also, we found that the frequency of the rs4986790 AA and rs4986791 TT genotypes was significantly higher in controls than in patients (25.7% vs 37%; p=0.006, pc=0.02) and conferred a protector factor in NPC (OR= 0.59, 95% CI= 0.39-0.87). Further, based on the Kaplan-Meier survival curve we observed also the positive effect ofrs1927914 AA genotype on a prognostic of NPC (p=0.006; pc=0.01). CONCLUSION: Our study demonstrated that impaired production of TLR4 seems to be a risk factor of NPC development but functional studies are needed to confirm these findings. As to rs1927914 AA appears to be a good biomarker for better survival in a patient with NPC.

BRCA1/BRCA2 Mutations Shaped by Ancient Consanguinity Practice in Southern Mediterranean Populations

The aim of this study is to investigate the involvement of consanguinity on BRCA1/2 mutation incidence in Southern Mediterranean populations and to confirm their low penetrance by comparison of their recurrence in sporadic and familial breast cancer in a context of ancient consanguinity practice. Our study comprises of two parts: First, a comparison of the consanguinity rates of the South Mediterranean countries in a relationship with the frequency of BRCA1 deleterious mutations in breast cancer families and the recurrence of these mutations. Second, we investigated 23patients with a family history of breast cancer, 51 patients without a family history of breast cancer using next-generation sequencing of BRCA2 and then confirmed by Sanger sequencing for the novel mutation. As results, we clearly show a strong relationship between the frequency of BRCA1 deleterious mutations in breast cancer families and rate of consanguinity, since they are significantly inversely correlated. Four deleterious mutations were found in BRCA2 gene including a novel frame-shift mutationc.9382_9383dup in a patient with familial breast cancer and three other frame-shift mutations c.6591_6592del, c.1310_1313del and c.7654dup in patients with sporadic breast cancer.These results are discussed in a context of selective pressure of ancient consanguinity practice. In conclusion, the study of BRCA1/2 gene in Southern Mediterranean countries revealed low penetrance recurrent mutations in sporadic and familial breast cancer. These mutations have been selected in a context of ancient consanguinity practice along with protective genetic and environmental factors.

Involvement of genetic factors and lifestyle on the occurrence of colorectal and gastric cancer.

Gastrointestinal cancers are diseases due to genetic and environmental factors. In this present work we are interested in the influence of environmental factors on the occurrence of gastrointestinal cancers in Tunisian population. We found that the MTHFR C677T polymorphism was associated with colorectal cancer (P<0.04) but not with gastric cancer. In addition, we have shown that alcohol is associated with an increased risk of colorectal cancer, but the consumption of cheese is protective. Furthermore, we studied tymidylate synthase gene involved in folate metabolism. Indeed, we observed that the 5'UTR repeat polymorphism, is associated with risk of colorectal cancer, and the LL genotype (3R/3R) was significantly frequent in patients with colorectal cancer compared to controls (p=0.002; OR=2.7, 95% CI=1.4-5.2). While we found that SL genotype (2R/3R) was associated with risk of gastric cancer (p=0.015; OR=4.46, 95% CI=1.08-19-64). This polymorphism was also shown to be a predictor of response to chemotherapy based 5'-fluorouracil. However, we are interested in studying the GPX -1 gene involved in phase I metabolism of xenobiotics. We therefore evaluated the risk of TT genotype in GPX-1 C599T polymorphism with the onset of gastric cancer (P=0.0001; OR=5.41, 95% CI 1.98 to 15.58) and colorectal cancer (P=0.00008; OR=4.40, 95% CI 1.93 to 10.27). To clarify the possible relationship between environmental factors and the occurrence of the disease, we studied the additive effect of risk genotype and behavior in order to highlight the interaction of gene-environment factors.

Involvement of IL17A, IL17F and IL23R Polymorphisms in Colorectal Cancer Therapy.

IL23/IL17 pathway plays an important role in the development of inflammatory bowel diseases (IBD). In general, the genes encoding the cytokines are genetically polymorphic and polymorphisms in genes IL23R and IL17 have been proved to be associated with its susceptibility to inflammatory diseases as well as cancer including colorectal cancer. Moreover, it has been shown that these interleukins are involved in anti-tumor or pro-tumor effects of various cancers. Previously, we showed that there is a significant association between IL17A, IL17F and IL23R polymorphisms as well as the occurrence of colorectal cancer and the clinical features of the disease. The purpose of the present work is to investigate an association between IL17A, IL17F and IL23R polymorphisms in 102 Tunisian patients with colorectal cancer treatment. The association was analyzed by statistical tools. We found that patients with mutated genotypes of IL17A G197A SNP could be a risk factor for the inefficiency of chemotherapy and radiotherapy. Unlike IL17F variant, patients with wild type genotypes require surgery and adjuvant chemotherapy. On the one hand, we found no evidence that supports a significant association between IL23R polymorphism and the combined genotypes of these three genes and the colorectal cancer treatment. On the other hand, we showed that there is an important interaction between IL17A/IL17F polymorphisms and the stage of the disease as well as its treatment. Finally, patients with IL17F wild type genotype highlighted that there is a valid longer OS without all treatments and with radiotherapy and a neoadjuvant chemotherapy. In contrast, we observed that there are no relationships between IL17A, IL23R and the survival of these patients neither with nor without the treatment. Our results suggest that polymorphisms in IL17A and IL17F genes may be a predictive source of colorectal cancer therapy type. Therefore, IL17F may serve as an independent prognostic factor for overall survival in patients with colorectal cancer.

Wild-type genotypes of BRCA1 gene SNPs combined with micro-RNA over-expression in mammary tissue leading to familial breast cancer with an increased risk of distant metastases' occurrence.

Germ line deleterious mutations of BRCA1 gene are not the unique factor that could inactivate BRCA1 protein which leads to familial breast cancer onset with distant metastases' occurrence. The present research explores the role that could be assigned to BRCA1 SNPs to inactivate BRCA1 protein and therefore to the occurrence of familial breast cancer with an increased risk of distant metastases' occurrence. The presence or the absence of BRCA1 protein was first analyzed by applying the immunohistochemistry technique to the tumors with sporadic and familial breast cancer. Then, a case-control study was conducted including 40 patients with familial breast cancer, 46 ones with sporadic breast cancer and 34 healthy controls based on the genotyping of nine BRCA1 SNPs (c.442.58delT, c.2082C>T, c.2311T>C, c.2612C>T, c.3113A>G, c.3119G>A, c.3548A>G, c.4308T>C and 4837A>G) via direct sequencing. Finally, the functional role that could be assigned to these SNPs was focused upon. miRbase site was used as a bioinformatics tool to predict potential micro-RNAs (miRs) targeting SNPs that are associated with familial breast cancer according to the results of this research. These predicted miRs were confirmed by Q-PCR analysis and correlated with BRCA1 protein expression among patients along with potential distant metastases. Clinical outcome showed that distant metastasis concerned 45 % of familial breast cancer patients and 19.5 % with sporadic breast cancer. Analysis of BRCA1 protein expression revealed a negative staining among 46.6 % of familial breast cancer patients and only 16.6 % within sporadic breast cancer ones. The association of four variants was identified within BRCA1 gene (c.442.58 delT, c.2311T>C, c.2612C>T and c.4308T>C) to familial breast cancer across their wild genotypes. miR-1179 was selected as potential miR that targets the region of BRCA1 mRNA containing the c.2311T>C variant within the TT genotype. The expression of miR-1179 was significantly associated with familial breast cancer patients without BRCA1 deleterious mutations compared to those with sporadic breast cancer according to TT genotype along with BRCA1 negative staining and according to the occurrence of distant metastases. Combination between TT genotype of c.2311T>C and miR-1179 over-expression could generate a lack of BRCA1 protein leading to a high risk of familial breast cancer with distant metastases.

MicroRNAs 146a and 147b biomarkers for colorectal tumor's localization.

The recently identified class of microRNAs (miRs) provided a new insight into cancer research, since abnormalities of members of microRNAs family have been found in various types of cancer. However, the relationship between five miRNAs (miR146a, miR155, miR21, miR135a, and miR147b) and colorectal cancer remains unclear. In the present study, we examined expression of these miRNAs in 25 pair-matched colon cancer tissues and normal colon mucosa. The expression levels of miR146a, miR155, miR21, miR135a, and miR147b were quantified by real-time PCR. We found that miR21, miR146a, and miR135a were all expressed at higher levels in colon tumors. On the other hand, miR146a and miR147b expressions are significantly higher in left colon compared to right colon. These two miRs, especially miR146a, seemed to be markers for the left colon tumors. Moreover, significant proportional and inverse correlations were found between miR expressions in tumor and healthy tissue, and the correlations profiles were different depending on cancer localization. Taken together, these results lead us to suggest the presence of different mechanisms regulating miRs expression and consequently their target genes in left and right colon. So the pathway of colorectal carcinogenesis would be different according to the site of the tumor.

Genetic testing in Tunisian families with heritable retinoblastoma using a low cost approach permits accurate risk prediction in relatives and reveals incomplete penetrance in adults.

Heritable retinoblastoma is caused by oncogenic mutations in the RB1 tumor suppressor gene. Identification of these mutations in patients is important for genetic counseling and clinical management of relatives at risk. In order to lower analytical efforts, we designed a stepwise mutation detection strategy that was adapted to the spectrum of oncogenic RB1 gene mutations. We applied this strategy on 20 unrelated patients with familial and/or de novo bilateral retinoblastoma from Tunisia. In 19 (95%) patients, we detected oncogenic mutations including base substitutions, small length mutations, and large deletions. Further analyses on the origin of the mutations showed mutational mosaicism in one unilaterally affected father of a bilateral proband and incomplete penetrance in two mothers. In a large family with several retinoblastoma patients, the mutation identified in the index patient was also detected in several non-penetrant relatives. RNA analyses showed that this mutation results in an in-frame loss of exon 9. In summary, our strategy can serve as a model for RB1 mutation identification with high analytical sensitivity. Our results point out that genetic testing is needed to reveal or exclude incomplete penetrance specifically in parents of patients with sporadic disease.

3020insC NOD2/CARD15 polymorphism associated with treatment of colorectal cancer.

Chronic inflammation is closely linked to cancer. The risk of damage by colorectal cancer (CRC) may increase due to autoimmune disease and cryptogenic inflammation. Therefore, genetic factors implicated in the chronic irritation in inflammatory bowel disease such as NOD2/CARD15 may predispose to CRC. In this report, we shed the light on the possible contribution of the NOD2 3020insC variant to CRC risk in a series of 246 Tunisian subjects including 101 patients with CRC and 145 healthy controls. NOD2/CARD15 polymorphism was genotyped by sizing fluorescently labeled PCR products and automated sequencers. We analyzed the association between the molecular features at this gene in relation to tumor and patient characteristics and treatments. Through this qualitative analysis, we found that CRC patients with mutant allele of NOD2/CARD15 were suffering from Crohn's disease (CD) with canonic presentation. We also observed a positive association between 3020insC polymorphism and surgery and chemotherapy. We suppose that around 3% of colorectal cases which happen at an age older than 50 years are associated with the canonic form of CD along with the 3020insC mutation and that these patients are in need for chemotherapy.

Significant association between interleukin-17A polymorphism and colorectal cancer.

Interleukin (IL) 17A is an inflammatory cytokine expressed by Th 17 cells and plays a role in tissue inflammation by inducing release of proinflammatory and neutrophil-mobilizing cytokines. We have investigated the association between colorectal cancer and polymorphisms of IL17A (rs2275913. G197A). The study was performed in 241 subjects (102 with colorectal cancer and 139 healthy controls). Genotypes were determined by fluorescent-based restriction fragment length polymorphism method. The association between the molecular features at the gene in relation to tumor and patient clinical characteristics was analyzed. There was a significant difference between the genotype frequencies of IL17A G197A of control subjects (GG 68.34 % and GA + AA 31.65 %) and patients with colorectal cancer (GG 47.05 % and GA + AA 52.94 %) (p = 0.001 with odds ratio (OR) 2.45 (1.43-4.11)). IL17A G197A polymorphism is particularly associated with colon cancer. Indeed, the IL17A GG genotype could be considered as a protective factor against colon cancer (p = 0.00001) with OR 3.77 (2.04-6.99). We have noted a significant association of IL17A G197A polymorphism not only with tumor localization (p = 0.003) but also with tumor differentiation (p = 0.0005) in CRC patients. We have also showed a significant association of G197A variant with an increased risk of advanced stage (p = 0.005). Our result suggests that the A allele of IL17A gene is involved in susceptibility to colorectal cancer and is associated with clinical features as tumor location, tumor differentiation, and TNM stage. IL17A polymorphism may serve as biomarker of disease location and progression.

Impact of lifestyle factors and nutrients intake on occurrence of gastrointestinal cancer in Tunisian population.

This study aims to show the relationship between lifestyle and risk of colorectal and gastric cancers in Tunisian population. The food frequency survey method was used to obtain information about the dietary intake and way of life. Nutrients intake was calculated according to the food composition database. According to our results, the consumption of vegetables, fruits, fish, as well as coffee seems to be protective against digestive cancer, while the consumption of citrus and olive oil is protective against gastric cancer. Tobacco, alcohol, and tea represent a risk against gastrointestinal cancer. Highly educated people are more conscious of the crucial role of prevention. In addition, nutrients were significantly associated with colorectal and gastric cancer. The findings suggest that lifestyle is associated with a risk of gastrointestinal cancer. Moreover, higher intake of nutrients from foods was observed more in cases with colorectal and gastric cancer than controls.

Thymidylate synthase polymorphism in sporadic colorectal and gastric cancer in Tunisian population: a predictive role in 5-fluorouracil based chemotherapy treatment.

In our study, we investigate the possible association of thymidylate synthase polymorphism, 28 bp tandem repeat in 5'-UTR (transcription enhancer element) with susceptibility of colorectal and gastric cancer in Tunisian population. Because thymidylate synthase provides an effective prediction of chemotherapy treatment based on 5-fluorouracil, our interest in this study was focused on finding an eventual interaction between thymidylate synthase polymorphism and treatment of sporadic colorectal and gastric cancer. Whole blood was collected into EDTA tube, after centrifugation for 15 min, the buffy coat was isolated, and genotyping of TS 5'-UTR polymorphism was carried by polymerase chain reaction method using appropriate primers. Determination of the different genotypes was done directly on the stained agarose gel. Our finding showed that the 5'tandem repeat polymorphism of the thymidylate synthase gene is associated with risk of colorectal cancer; thus, LL (3R/3R) genotype is significantly high in patients with colorectal cancer compared to controls (P = 0.002; OR 2.7; 95 % CI 1.4-5.2). In addition, we found a positive association between SL (2R/3R) genotype in the thymidylate synthase 5'-UTR and gastric cancer risk (P = 0.015; OR 4.46; 95 % CI 1.08-19.64). Furthermore, we found a correlation of thymidylate synthase polymorphism with the fluorouracil-based therapy regimes and also with preoperatory radiation in patients with colorectal cancer. Thymidylate synthase is associated with risk of colorectal cancer but not with gastric cancer; however, heterozygous SL (2R/3R) polymorphism is associated with risk of gastric cancer; moreover, the 5' tandem repeat polymorphism of thymidylate synthase gene was an independent predictor of the clinical treatment.

Significant association between IL23R and IL17F polymorphisms and clinical features of colorectal cancer.

Th17cells are involved in inflammatory and autoimmune diseases. These cells may be involved in pathological processes mainly producing pro-inflammatory cytokines. Recently, it was shown that the IL23/IL17 pathway plays an important role in the development of inflammatory bowel disease. In general, genes encoding cytokines are genetically polymorphic and polymorphisms in genes IL23R el IL17F were shown associated with susceptibility to Crohn's disease and ulcerative colitis which in their turn are considered as risk factors for developing colorectal cancer (CRC). Our approach is to study IL17F and IL23R polymorphisms as risk factor associated with CRC in the Tunisian population in patients and healthy controls. Interesting, we noted a significant association between IL17F and IL23R polymorphisms and tumor location (p=0.0001 and p=0.049, respectively), tumor histology (p=0.007 and p=0.049, respectively) and tumor architecture (p=0.0000000001 and p=0.07, respectively) in CRC patients. We also showed a significant association of IL17F variant with an increased risk of TNM stage III/IV (p=0.007), showing an increased risk of advanced stage. Finally, we observed a positive link between IL17F polymorphism and CRC patients with lymph nodes (p=0.0000000001) and metastasis (p=0.00000009). However, we found no evidence to support a significant association between IL17F and IL23R polymorphisms and colorectal cancer susceptibility. Our findings suggest that IL17F and IL23R polymorphisms were significantly associated with clinical features variables. The IL17F cytokine appear to be involved in the control of tumor growth and invasion of gastrointestinal tumors. IL17 and IL23 polymorphisms or those of their receptors as important determinants of susceptibility to colorectal cancer are still subject to questioning.

miRNAs in Sera of Tunisian patients discriminate between inflammatory breast cancer and non-inflammatory breast cancer.

In recent years, circulating miRNAs have attracted interest as stable, non-invasive biomarkers for various pathological conditions. Here, we investigated their potential to serve as minimally invasive, early detection markers for inflammatory breast cancer (IBC) and non-inflammatory breast cancer (non-IBC) in serum. miRNA profiling was performed on serum from 20 patients with non-IBC, 20 with IBC, and 20 normal control subjects. Real-time reverse transcription-polymerase chain reaction (qRT-PCR) was applied to measure the level of 12 candidate miRNAs previously identified in other research(miR-342-5p, miR-342--3p, miR-320, miR-30b, miR-29a, miR-24, miR-15a, miR-548d-5p, miR-486-3p, miR-451, miR-337-5p, miR-335).We found that 4 miRNAs (miR-24, miR-342-3p, miR-337-5p and miR-451) were differentially expressed in serum of IBC patients compared to non-IBC, and 3 miRNAs (miR-337-5p ,miR-451and miR-30b) were differentially expressed in IBC and non-IBC patients combined compared to healthy controls. miR-24, miR-342-3p, miR-337-5p and miR-451 were found to be significantly down-regulated in IBC patients compared to non-IBC. Likewise, the expression level of mir-451 showed significant down-regulation in IBC serum, while mir-30b and miR-337-5p were up-regulated in non-IBC serum comparatively to normal controls. Using receiver operational curve (ROC) analysis, we show that dysregulated miRNAs can discriminate patients with IBC and non-IBC from healthy controls with sensitivity ranging from 76 to 81% and specificity from 66 to 80%, for three separate miRNAs. In conclusion, our data suggest that circulating miRNAs are potential biomarkers for classifying IBC and non-IBC, and may also be candidates for early detection of breast cancer.

Positive link between variant Toll-like receptor 4 (Asp299Gly and Thr399Ile) and colorectal cancer patients with advanced stage and lymph node metastasis.

Toll-like receptors (TLRs) are considered as major endotoxin-signaling receptor and as crucial sensors of innate immunity. TLRs recognize pathogen-associated molecular patterns; induce effectors genes involving inflammatory cytokines and therefore initiation of adaptative immune responses against pathogens. Recently, it has been shown that TLRs are involved in tumor progression. In fact, increased level of TLR4 is associated with progression of colon malignancies. Even, TLR4 polymorphism has been shown associated with susceptibility to have colorectal cancer. Our study aimed to investigate an association between TLR4 Asp299Gly (D299G) and Thr399Ile (T399I) polymorphisms in Tunisian patients with colorectal cancer. Using a primer extension method (SNaPshot), we genotyped two variants of TLR4 D299G and T399I in 100 patients with colorectal cancer and 140 healthy controls in Tunisian population. Interesting, we noted a significant association between T399I polymorphism and tumor differentiation (p = 0.027) and tumor architecture (p = 0.02) in colorectal cancer (CRC) patients. We also showed a significant association of D299G with an increased risk of advanced stage (p = 0.03). Finally, we observed a positive link between D299G and T399I polymorphisms and CRC patients with lymph node (p = 0.00024; p = 0.0005, respectively) and metastasis (p = 0.001; p = 0.002, respectively). However, we found no evidence to support a significant association between TLR4 D299G and T399I polymorphisms and colorectal cancer susceptibility. Our findings suggest that TLR4 D299G and T399I polymorphisms are significantly associated with clinical features variables. TLR4 polymorphisms may serve as biomarker of disease progression. Therefore, our results need confirmation in even larger studies.

Status of antioxidant enzyme: glutathione peroxidase and total polyphenol level in plasma of Tunisian patients suffering from colorectal and gastric cancer: interaction with clinical outcome.

In our case-control study, we measure the antioxidant status by dosing enzymes involved in oxidant stress in plasma of patients with colorectal and gastric cancer, and in the second step, we investigate the impact of chemotherapy before and after surgery on plasma antioxidant status and polyphenols in patients. Blood serum was collected from patients with stomach and colorectal cancer before conventional treatment, and glutathione peroxidase (GSHPX) enzyme activities and total polyphenols were determined by spectrophotometric methods. In our study, we found a significant decrease in glutathione peroxidase activity in plasma of patients compared with controls (P = 0.02), although we did not find a significant association between total polyphenols and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) or ABTS in plasma of colorectal and stomach cancer compared with control; furthermore, we observed no significant difference in the average plasma polyphenols in patients treated with chemotherapy before and after surgery. We have shown the decrease in GSHPX activity in plasma of cases with colorectal and gastric cancer, and this decrease reflects that the oxidative stress is associated with tumor tract and related to oxidative metabolism; however, no association was found between total polyphenols and ABTS in our study.