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With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Large prospective studies established a link between obesity and breast cancer (BC) development. Yet, the mechanisms underlying this association are not fully understood. Among the diverse adipocytokine secreted by hypertrophic adipose tissue, leptin is emerging as a key candidate molecule linking obesity and cancer, since it promotes proliferation and invasiveness of tumors. However, the potential implication of leptin on tumor escape mechanisms remains unknown. This study aims to explore the effect of leptin on tumor resistance to NK lysis and the underlying mechanism. We found that leptin promotes both BC resistance to NK92-mediated lysis and β oxidation on MCF-7, by the up-regulation of a master regulator of mitochondrial biogenesis, the peroxisome proliferator activated receptor coactivator-1 α (PGC1A). Using adenoviral approaches, we show that acute elevation of PGC1A enhances the fatty acid oxidation pathway and decreases the susceptibility of BC cells to NK92-mediated lysis. Importantly, we identified the involvement of PGC1A and leptin in the regulation of hypoxia inducible factor-1 alpha (HIF1A) expression by tumor cells. We further demonstrate that basal BC cells MDA-MB-231 and BT-20 exhibit an increased PGC1A mRNA level and an enhanced oxidative phosphorylation activity; in comparison with luminal BC cells MCF7 and MDA-361, which are associated with more resistance NK92 lysis. Altogether, our results demonstrate for the first time how leptin could promote tumor resistance to immune attacks. Reagents blocking leptin or PGC1A activity might aid in developing new therapeutic strategies to limit tumor development in obese BC patients.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that lack antigen-specific receptors and exhibit innate effector functions such as cytokine production that play an important role in immediate responses to pathogens especially at mucosal sites. Mouse and human ILC subsets have been extensively characterized in various tissues and in blood. In this study, we present the first characterization of ILCs and ILC subsets in rat gut and secondary lymphoid organs using flow cytometry and single cell RNA sequencing. Our results show that phenotype and function of rat ILC subsets are conserved as compared to human and mouse ILCs. However, and in contrast to human and mouse, our study unexpectedly revealed that ILC2 and not ILC3 was the dominant ILC subset in the rat intestinal lamina propria. ILC2 predominance in the gut was independent of rat strain, sex or housing facility. In contrast, ILC3 was the predominant ILC subset in mesenteric lymph nodes and Peyer patches. In conclusion, our study demonstrates that in spite of highly conserved phenotype and function between mice, rat and humans, the distribution of ILC subsets in the intestinal mucosa is dependent on the species likely in response to both genetic and environmental factors., (Copyright © 2020 Abidi, Laurent, Bériou, Bouchet-Delbos, Fourgeux, Louvet, Triki-Marrakchi, Poschmann, Josien and Martin.)

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4β7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4β7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.

Chemokines and their receptors (members of the GPCR super-family) are involved in a wide variety of physiological processes and diseases; thus, understanding the specificity of the chemokine receptor family could help develop new receptor specific drugs. Here, we explore the evolutionary mechanisms that led to the emergence of the chemokine receptors. Based on GPCR hierarchical classification, we analyzed nested GPCR sets with an eigen decomposition approach of the sequence covariation matrix and determined three key residues whose mutation was crucial for the emergence of the chemokine receptors and their subsequent divergence into homeostatic and inflammatory receptors. These residues are part of the allosteric sodium binding site. Their structural and functional roles were investigated by molecular dynamics simulations of CXCR4 and CCR5 as prototypes of homeostatic and inflammatory chemokine receptors, respectively. This study indicates that the three mutations crucial for the evolution of the chemokine receptors dramatically altered the sodium binding mode. In CXCR4, the sodium ion is tightly bound by four protein atoms and one water molecule. In CCR5, the sodium ion is mobile within the binding pocket and moves between different sites involving from one to three protein atoms and two to five water molecules. Analysis of chemokine receptor evolution reveals that a highly constrained sodium binding site characterized most ancient receptors, and that the constraints were subsequently loosened during the divergence of this receptor family. We discuss the implications of these findings for the evolution of the chemokine receptor functions and mechanisms of action., Competing Interests: The authors have declared that no competing interests exist.

Background: The activation of Fas/Fas ligand (FasL) and DR4-DR5/tumor necrosis factor-related-apoptosis-inducing ligand (TRAIL) pathways in cancer cells triggers apoptosis. The objective of this study was to investigate the prognostic value of soluble FasL (sFasL) and soluble (sTRAIL) in the serum of patients with bladder cancer., Methods: The sFasL and sTRAIL levels in the sera of patients with bladder cancer or healthy donors were determined using the enzyme-linked immunosorbent assay. Micro-culture tetrazolium viability assay and Western blot were used to analyze cell cytotoxicity and death receptors protein expression respectively., Results: Whether no difference in sTRAIL levels was seen between patients and controls, the level of sFasL was higher in patients than that in healthy donors. According to, sFasL level was the highest in the serum of patients with superficial stage or low- and medium-grade cancer. Moreover, sFasL in patients with superficial noninvasive bladder tumors or low- and medium-grade cancers was higher than that in patients with invasive carcinomas and high-grade cancers. Patients with high levels of sFasL survive longer than those with low levels, probably related to the cytotoxic potential of FasL preserved in its soluble form., Conclusion: The data suggest that monitoring the level of sFasL and its cytotoxic activity could be a prognostic marker in the follow-up of patients with bladder cancer., (© 2018 S. Karger AG, Basel.)

Various studies in western countries found Akt amplification to be a frequent event in human cancers, including bladder, but the correlation with clinicopathological features is controversial. Such studies have not been reported in African populations, including Tunisians. The purpose of this study was to assess expression of the phosphorylated/activated forms of Akt in tumors from Tunisian patients with bladder cancer and to correlate its expression with pathological and clinical parameters of the disease. The study included 72 patients of whom 34 were diagnosed as low- to medium-grade and 35 as high-grade; 30 were muscle stage and 39 non-muscle stage. Primary tumors from these patients, normal adjacent tissues, or bladder cancer cell-lines were analyzed for Ser473 phosphorylated Akt expression by Western blot. Seventy-two percent of primary tumors from patients with bladder cancer had increased levels of p-Akt. The p-Akt levels in patients with high-grade bladder cancer were significantly elevated compared to patients with low- or medium-grade bladder cancer. In invasive carcinoma, the p-Akt level was significantly higher than in superficial non-invasive bladder tumors. Concerning the influence of tobacco on Akt activation, no significant differences of p-Akt expression were found between non-smoker and smoker patients. Altogether, our results suggest that Akt activation can provide useful prognostic information and that tobacco represents a serious risk factor for recurrence in a cohort of Tunisian patients.

Background: The aim of this study was to investigate the clinical significance of three immune cell-related transcription factors, T-bet, GATA-3 and Bcl-6 in bladder cancer in Tunisian patients., Methods: Expression of T-bet, GATA-3 and Bcl-6 genes was assessed using RT-qPCR in 65 bladder cancers from patients: 32 being diagnosed as low- and medium-grade, 31 as high-grade, 25 as muscle invasive stage and 39 as non-muscle invasive stage. Gene expression was statistically correlated according to the grade, the stage, tobacco consumption, the BCG response and disease severity., Results: T-bet levels in patients with high-grade bladder cancer were significantly elevated compared to patients with low- or medium-grade bladder cancer (p = 0.005). In invasive carcinoma (T2-T4), the T-bet levels were significantly higher than in superficial non-invasive bladder tumors (Tis, Ta, and T1) (p = 0.02). However, T-bet is predictive of the response to BCG. Its expression is high in good responders to BCG (p = 0.02). In contrast, the expression of GATA-3 and Bcl-6 in non-invasive carcinoma (p = 0.008 and p = 0.0003) and in patients with low- and medium-grade cancers (p = 0.001 and p < 0.0001) is significantly higher than in invasive bladder tumors and in patients with high-grade bladder carcinoma, respectively. In addition, heavy smokers, whose tumors express low levels of GATA-3 and Bcl-6, are poor responders to BCG (p = 0.01 and p = 0.03). Finally, better patient survival correlated with GATA-3 (p = 0.04) and Bcl-6 (p = 0.04) but not T-bet expression., Conclusions: Our results suggest that T-bet expression in bladder tumors could be a positive prognostic indicator of BCG therapy, even if high levels are found in high-grade and stage of the disease. However, GATA-3 and Bcl-6 expression could be considered as predictive factors for good patient survival.

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mammalian thyroid hormone receptors (TRs) have multiple isoforms, including the bona fide receptors that bind T3 (TRα1, TRβ1 and TRβ2) and a non-hormone-binding variant, TRα2. Intriguingly, TRα2 is strongly expressed in the brain, where its mRNA levels exceed those of functional TRs. Ablation of TRα2 in mice results in over-expression of TRα1, and a complex phenotype with low levels of free T3 and T4, without elevated TSH levels, suggesting an alteration in the negative feedback at the hypothalamic-pituitary level. As the hypothesis of a potential TRH response defect has never been tested, we explored the functional role of TRα2 in negative feedback on transcription of hypothalamic thyrotropin, Trh. The in vivo transcriptional effects of TRα2 on hypothalamic Trh were analysed using an in vivo reporter gene approach. Effects on Trh-luc expression were examined to that of two, T3 positively regulated genes used as controls. Applying in vivo gene transfer showed that TRα2 over-expression in the mouse hypothαlamus abrogates T3-dependent repression of Trh and T3 activation of positively regulated promoters, blocking their physiological regulation. Surprisingly, loss of function studies carried out by introducing a shTRα2 construct in the hypothalamus also blocked physiological T3 dependent regulation. Thus, modulating hypothalamic TRα2 expression by either gain or loss of function abrogated T3 dependent regulation of Trh transcription, producing constant transcriptional levels insensitive to feedback. This loss of physiological regulation was reflected at the level of the endogenous Trh gene, were gain or loss of function held mRNA levels constant. These results reveal the as yet undescribed dominant negative role of TRα2 over TRα1 effect on hypothalamic Trh transcription.

The geostrategic location of North Africa as a crossroad between three continents and as a stepping-stone outside Africa has evoked anthropological and genetic interest in this region. Numerous studies have described the genetic landscape of the human population in North Africa employing paternal, maternal, and biparental molecular markers. However, information from these markers which have different inheritance patterns has been mostly assessed independently, resulting in an incomplete description of the region. In this study, we analyze uniparental and genome-wide markers examining similarities or contrasts in the results and consequently provide a comprehensive description of the evolutionary history of North Africa populations. Our results show that both males and females in North Africa underwent a similar admixture history with slight differences in the proportions of admixture components. Consequently, genome-wide diversity show similar patterns with admixture tests suggesting North Africans are a mixture of ancestral populations related to current Africans and Eurasians with more affinity towards the out-of-Africa populations than to sub-Saharan Africans. We estimate from the paternal lineages that most North Africans emerged ∼15,000 years ago during the last glacial warming and that population splits started after the desiccation of the Sahara. Although most North Africans share a common admixture history, the Tunisian Berbers show long periods of genetic isolation and appear to have diverged from surrounding populations without subsequent mixture. On the other hand, continuous gene flow from the Middle East made Egyptians genetically closer to Eurasians than to other North Africans. We show that genetic diversity of today's North Africans mostly captures patterns from migrations post Last Glacial Maximum and therefore may be insufficient to inform on the initial population of the region during the Middle Paleolithic period.

Cellular resistance to insulin caused by reduced glucose transport and metabolism is a primary defect leading to the development of metabolic disease. While the etiology of insulin resistance is multifactorial, reduced insulin action is associated with impaired activity of the glucose transporter GLUT4 in insulin-sensitive tissues. Yet, the role of adipose tissue GLUT4 deregulation in the pathogenesis of insulin resistance, obesity, and diabetes is still unclear. In this study, we assessed the relative GLUT4 level in human subcutaneous adipose tissue from obese, diabetic, and diabetic obese versus control subjects, using a real-time PCR method. GLUT4 mRNA levels were considerably decreased among type 2 diabetic patients compared with those of the controls (P < 0.01), whereas no such difference was found between obese and normal-weight controls. Multiple linear regressions analysis in both diabetic non-obese and diabetic obese groups showed a negative correlation between GLUT4 mRNA expression and both markers of obesity or insulin resistance (P < 0.01). However, in obese group, GLUT4 was inversely associated only with HOMA-IR (P < 0.01). Our findings showed that adipose GLUT4 gene expression changes were more related to insulin resistance and type 2 diabetes rather than to obesity.

Objectives: Although a relationship between obesity and metabolic consequences with thyroid function has been reported, the underlying pathogenesis is not completely known. In the current study, we evaluated the thyroid function in obese and/or diabetic patients compared to healthy normal weight peers, exploring the possible association between components of metabolic syndrome and thyroid function parameters., Methods: We recruited 108 subjects (56 male and 52 female). In all subjects, thyroid stimulating hormone (TSH), free thyroxine (FT4), fasting plasma levels of insulin and glucose, homeostasis model assessment for insulin resistance, and obesity parameters were assessed., Results: We found that circulating levels of TSH and FT4 were significantly increased in overweight and obese subjects. However, the data do not reveal any change of these hormones in diabetics. Multivariate linear regression analysis showed that TSH was directly associated with both obesity and insulin resistance parameters (p < 0.05). FT4 was negatively associated only with obesity parameters (p < 0.05)., Conclusions: Our data strongly support that the changes of thyroid hormones may be influenced by adiposity and its metabolic consequences, such as insulin resistance. This relationship can be explained by a cross talk between adipose tissue release and thyroid function. Nevertheless, metformin treatment seems to affect thyroid function in diabetic patients by maintaining plasma thyrotropin levels to subnormal levels.

Background: The incidence of obesity has dramatically increased in overall the world. It is a consequence of imbalance between energy intake and energy expenditure. Leptin is a fat derived adipokine that has emerged over the past decade as a key hormone in the regulation of food intake and energy expenditure. Elevated leptin levels are found in obese humans, suggesting a role of leptin in regulating body weight and adiposity., Aim: The aim of this study was to investigate the change of leptin mRNA expression level and its correlation with obesity and several metabolic variables in Tunisian patients., Methods: Real time quantitative polymerase chain reaction (QPCR) analysis was carried out among two groups who underwent an abdominal surgery: controls (n = 9) and obese patients (n = 7)., Results: Leptin mRNA expression in subcutaneous adipose tissue was markedly increased in obese patients (p < 0.01). It was positively correlated with measures of obesity waist circumference (WC) (r = 0, 71, p < 0.01) and body mass index (BMI) (r = 0, 68, p < 0.01). Interestingly, leptin gene expression was also correlated to insulin resistance index (r = 0, 72, p < 0.01)., Conclusion: The present study is the first investigation of leptin regulation in subcutaneous adipose tissue of Tunisian population. Our data showed that leptin levels are higher in obese subjects than in control subjects. This indicates that the subcutaneous adipose plays an important role in impaired adipokine regulation, and consequently in developing metabolic disorder.

Previous studies have shown the expression WISP3 and RhoC in cell lines of inflammatory breast cancer (IBC). The aim in the current study was to compare the expression of both genes, in biopsy samples collected from Tunisian patients with localized or metastatic breast cancer and patients with IBC. We investigated 127 patients enrolled in Salah Azaiez Institute in Tunis. Using the RT-PCR, we showed the phenotype (WISP3-, RhoC+) is significantly associated with IBC tumors, while the (WISP3+, RhoC-)phenotype is mostly associated to non-IBC tumors. The frequencies of these tumor phenotypes are significantly different between these tumor groups (p = 10(- 7); relative risk or RR = 3.25; confidential interval or CI 95% = 1.90-5.53). Immunohistochemical test revealing the presence of WISP3 and RhoC proteins correlates with the expression in the biopsy of their encoding genes as detected by RT-PCR. In conclusion, it appears that WISP3 and RhoC genes expression status defines a molecular signature of IBC.

Thyroid hormone receptor (TR) and peroxisome proliferator-activated receptor gamma (PPARgamma) co-regulate numerous peripheral metabolic responses. To examine potential crosstalk between PPARgamma and TRbeta in the hypothalamus, thyrotropin-releasing hormone (Trh) regulation in the newborn mouse hypothalamus was followed. QPCR showed PPARgamma to be expressed in the hypothalamus at this developmental stage. Intracerebral injection of PPARgamma agonists modified transcription from a TRH-luc construct introduced into the hypothalamus and increased serum thyroxine levels. Furthermore, shRNA-based in vivo PPARgamma knockdown amplified T(3)-independent transcription and PPARgamma overexpression dose-dependently abrogated T(3)-dependent Trh repression. Overexpression of retinoid X receptor-alpha (RXRalpha), the common heterodimeric partner of PPARgamma and TRbeta, rescued PPARgamma abrogation of T(3)-dependent repression. Thus, competition for RXR could represent one mechanism underlying this hypothalamic crosstalk between PPARgamma and TRbeta. These demonstrations of PPARgamma effects on hypothalamic Trh transcription in vivo consolidate the role of the TRH neuron as a central integrator of energy homeostasis.

The Kirsten Rat Sarcoma (KRAS) oncogene has been introduced recently as a genetic biomarker for metastatic sporadic colorectal cancer prior to anti-EGFR treatment. Identifying patients with KRAS mutations that not respond to EGFR targeted therapies require sensitive, rapid and efficacious routine technique. We have attempted to evaluate the efficiency of three conventional methods: direct sequencing, HRM and DHPLC, to detect mutations in codon 12 and 13 of the KRAS exon2 gene. For this first Tunisian study on KRAS, we detected 45.83% of altered KRAS gene among 48 formalin-fixed paraffin-embedded sporadic colorectal adenocarcinoma patients. The use of HRM-sequencing allowed as enlarging the detected KRAS exon 2 mutations (22/48) in comparison with direct sequencing (17/48). DHPLC was used to confirm results when consensus was not observed between HRM and direct sequencing. This study brings an interesting data concerning an inter-method validation between sequencing and HRM in the investigation of sporadic colorectal cancer biomarker. It also shows that KRAS mutations occur at similar frequencies in Tunisian patients as in other populations; and suggests that the same genes are at play in sporadic CRC cancer, despite ethnic, geographical and environmental differences between countries.

Background: Tumor cells express surface structures different from normal cells. These structures may be recognized by the immune system, which ensure anti-tumoral surveillance. Antigenic presentation requires HLA molecules role. Since, these molecules are encoded by a high polymorphic system, immune response can be modulated according to HLA genotype. So, HLA polymorphism could be correlated with tumor escape from anti-tumor immunosurveillance., Aim: We have aimed to search for possible associations between HLA DQB1 alleles and the histoprognostical parameters in breast cancer in the Tunisian population., Methods: DQB1 alleles were determined by PCR-SSO molecular typing in 100 healthy matched and unrelated Tunisian female and 87 Tunisian women with breast cancer., Results: Allelic distribution between the two studied groups showed no significant associations between this locus and the occurrence, the EE grade and the lymph node invasion of breast cancer in the Tunisian population., Conclusion: This result may be explained by the fact that cancer is a multifactoral disease due to several interacting factors that might change from one population to another.

Objectives: Sporadic colorectal cancer is influenced by numerous single nucleotide polymorphisms (SNPs), each with minor effects on the cancer risk. This study seeks to determine whether there is any association of the I1307K, E1317Q and D1822V variants within the Adenomatous polyposis coli gene (APC) and risk to develop colorectal cancer in Tunisian population., Methods: Direct sequencing was used to investigate three SNPs in the APC in 48 Tunisian sporadic colorectal cancer cases and 63 controls., Results: There was no statistically significant association between the I1307K, E1317Q and D1822V variants investigated and colorectal cancer risk., Conclusion: The lack of association may show that these variants selected for this study are not involved in the colorectal carcinogenic process. Otherwise, the eventual biological effect is so little to go undetected, unless increasing the sample size.

Objective: Our aim is to investigate the relation between CARD15 3020insC mutation, anti-Saccharomyces cerevisiae antibodies (ASCA) and disease phenotype, in Tunisian inflammatory bowel disease (IBD) patients., Materials: A hundred Tunisian patients with IBD (75 Crohn's disease CD and 25 ulcerative colitis UC) and 60 matched healthy controls were studied., Methods: CARD15 mutation was analysed by using an allele-specific polymerase chain reaction and sequencing. Assessment of ASCA in serum was performed by ELISA., Results: The frequency of the mutation was significantly higher in Crohn's disease than in control (p = 0,0005; OR = 20.45; CI 95% = 2.86-413.85) and did not differ statistically in UC group (p = 0, 05) from control. ASCAs were present in 60% of CD and 20, 8% of UC., Conclusion: This study suggests that in northern Tunisian population, 3020insC mutation in NOD2/CARD15 gene is a prevalent mutation leading to the typical Crohn's disease including ileal location, stricturing and penetrating clinical types and ASCA expression.

Objective: To test whether IL-10 promoter region polymorphisms are associated with susceptibility to inflammatory bowel disease, we examined the contribution of interleukin- 10 (IL-10) gene polymorphisms to Crohn's disease (CD) and Ulcerative colitis disease (UC) occurrence and also to CD phenotype. MATERIELS AND METHODS: SNPs at positions -627 (C > A) and -1117 (G > A) in the IL-10 promoter were determined in a sample of 105 Tunisian patients with IBD (75 CD and 30 UC) and 90 matched healthy controls., Results: The 627 CA genotype is associated with ileal location (p = 0.015) and with stricturing (p = 510-3) and penetrating (p = 310-3) presentation of CD. An additive effect between IL10 variants and CARD15 3020 insC mutation (p = 0,006) on severe forms of CD was shown., Conclusions: In Tunisian population, the 3020insC insertion in NOD2/CARD15 gene is a marker of susceptibility to CD, while the A allele at position -627 in the IL-10 promoter increases the risk of CD ileal location and severe disease presentation. A genetic epistasis between IL-10 gene polymorphisms and CARD15/NOD2 gene mutation was suggested.

Aim: The aim of this work was to study the correlation between the mitochondrial microsatellite, situated between the nucleotides 303 and 315 of the mitochondrial genome and the breast cancer in Tunisia., Materials and Methods: We have analyzed, by PCR-sequencing, the polymorphism of a mitochondrial microsatellite in 40 Tunisian patients and 39 healthy Tunisian donors. Comparisons of epidemiologic and sequences data were performed by chi-2 test., Results: We have revealed, for this mitochondrial microsatellite, seven different haplotypes in patients and five different haplotypes in controls. The haplotypic distribution was not significant between patients and controls but a negative association between one of these haplotypes (309+C 315+C) and the lymph node invasion was found., Conclusion: The haplotype 309+C 315+C is negatively correlated with lymph node invasion of breast cancer in Tunisia.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the development of hundreds to thousands of adenomatous polyps in colon and rectum. The APC gene (adenomatous polyposis coli) is considered as the major mutated gene in FAP. It has been shown that biallelic germline mutations in the base-excision-repair gene MYH can be responsible for a recessive inheritance of adenomatous polyposis (AP). This study is the first Tunisian genetic analysis on AP patients. Multiplex ligation-dependent probe amplification (MLPA) was used to screen the APC gene for large genomic rearrangements. The total APC and MYH exon sequences and exon-intron edges were sequenced in an effort to detect germline mutations, four were explored. Mutations were detected in four patients that fulfil the clinical criteria of AP. Three mutations were found in the APC gene, of which two were novel (c.1636&#95;1639delAGTG and c.2514 G>T) and all gave rise to a truncated APC protein. The missense G382D mutation, already described in north and south European populations was found in the MYH gene at the homozygous state in the fourth patient with moderate AP. Our preliminary study provides a basis for implementation of genetic counselling for AP.

CYP2D6 enzyme is implicated in the metabolism of drugs and nicotine. Genetic variability within CYP2D6, results in different CYP2D6 phenotypes. Inheritance of polymorphic CYP2D6 metabolizing enzyme is likely to be an important determinant of inter-individual variations in susceptibility to cancer. In this work, we have conducted a case control study in order to assess the role of CYP2D6*4 variant in bladder cancer development in a Tunisian cohort. A total of 80 patients with TCC of bladder cancer and 109 healthy controls were included in the present study. The frequency of CYP2D6*4 allele, characterized by loss of BstNI site, was observed in 8.25% of healthy volunteers and in 10.62% of patients. The CYP2D6*4/CYP2D6*4 genotype was observed in only 2.75% of controls and was absent in cases. In all group of patients, the CYP2D6*4 allele did not appear to influence bladder cancer susceptibility (p > 0.05). A similar result was obtained when we stratified cases group according to tobacco status. Conversely, patients carrying the BstNI site at the homozygous state, mostly combined as homozygous wild genotype, could be at more risk of bladder cancer invasiveness than those having the heterozygous genotype.

Polymorphisms in some chemokine receptor genes are associated with susceptibility to and progression of human immunodeficiency virus-1 (HIV-1) infection. Most mutations detected in the CC-chemokine receptor 5 (CCR5) gene are specific to different populations. In this study, we focused on polymorphisms of the CCR5 coding region in three healthy populations from Tunisia, corresponding to a cosmopolitan population from Tunis, and two isolated Berber populations. In addition to the CCR5-Delta32 deletion, eleven single nucleotide polymorphisms were detected. Some of these point mutations were associated with the same genotype and even the same haplotype. The (L55Q-C101X), I124, V131F, T143N, A159V, I237, T239A and G301R alleles have not been described previously, whereas the CCR5-Delta32, L55Q, A335V and Y339F variants have already been reported in the literature. The distribution and frequency of these variants were different among the three groups studied, a result in agreement with the mosaic genetic structure of the Tunisian population. To determine whether these alleles affect HIV-1 transmission, we compared allele frequencies between healthy and HIV-1 infected individuals from Tunis. The frequency of the CCR5-Delta32 variant was significantly different between the two groups, leading us to conclude that this mutation might confer protection against HIV infection in Tunisian populations.

Familial Adenomatous Polyposis (FAP) and Attenuated FAP (AFAP) are caused by a germline mutation in the Adenomatous polyposis coli (APC) gene. Recently, a new pathway characterized by a biallelic mutation in the MYH gene, with a recessive model of inheritance was discovered for this inherited syndrome. This report describes a Tunisian patient with an attenuated FAP phenotype, presenting seven colon polyps and an adenocarcinoma but no detectable germline mutations in the FAP target genes. A well known somatic mutation was found in the APC mutation cluster region (MCR). This case shows that further studies are needed to fully understand all the pathways of the FAP syndrome.

The MutYH gene is an adenine-specific DNA glycosylase that prevents G/T transversions. Germline mutation in this gene causes MYH-associated polyposis (MAP) that predispose to hereditary colorectal cancer (CRC). This study describes for the first time the association of the MutYH mutation with sporadic CRC. From the 48 Tunisian sporadic CRC cases analyzed, two patients showed somatic mutation of the MutYH gene. In addition, the two hotspot germline mutations MutYH Y165C and G382D seem to be infrequent in sporadic CRC.