Overexpression of miR-10b in colorectal cancer patients: Correlation with TWIST-1 and E-cadherin expression.

MicroRNAs are emergent players of epigenetics that function as oncogenes or tumor suppressors and that have been implicated in regulating diverse cellular pathways. MiR-10b is an oncogenic microRNA involved in tumor invasion and metastasis in various cancers. Our data have shown that miR-10b is overexpressed in colorectal cancer samples in comparison with non-tumorous adjacent mucosa (p = 0.0025) and that it is associated with severe features such as tumor size >5 cm (p = 0.023), distant metastasis (p = 0.0022), non-differentiated tumors (p = 0.016), and vascular invasion (p = 0.01). Regarding the regulation of its expression, positive correlation between the loss of miR-10b and aberrant DNA methylation (p = 0.02) as well as a loss of TWIST-1 messenger RNA (p = 0.018) have been observed. Furthermore, expression analysis of the downstream miR-10b targets has shown that there are associations between low HOXD10 messenger RNA and E-cadherin protein levels (p < 0.0001, p = 0.0008, respectively) and overexpression of miR-10b. Our data suggests that overexpression of miR-10b results from high levels of TWIST-1 and may induce a decrease of E-cadherin membranous protein levels, thus contributing to the acquisition of metastatic phenotypes in colorectal cancer.

CpG methylation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) and P53 mutation pattern in sporadic colorectal cancer.

The ubiquitin-proteasome system plays an essential regulatory role in various cellular processes. Besides its involvement in normal cellular functions, the alteration of proteasomal activity contributes to the pathological states of several clinical disorders, including cancer. Aberrant methylation of the CpG islands has been reported as an alternative way to inactivate gene expression involved in the ubiquitination process and thus protein degradation in tumor tissues. In this study, we aimed to determine the CpG methylation pattern of the UCHL1 promoter, as well as the mutation spectrum and the expression pattern of P53 in sporadic colorectal cancer (CRC) from Tunisian patients. We found that UCHL1 was methylated in 68.57 % and correlated significantly with lymph node metastasis (P = 0.029) and transcriptional silencing in tumor tissues (P = 0.013). Mutation screening of exons 5-9 of P53 showed that 42.85 % of cases harbor somatic mutation and are positively correlated with the methylated pattern of UCHL1 (P = 0.001). Furthermore, cytoplasmic accumulation of P53 was strongly associated with the unmethylated UCHL1 profile (P = 0.006), supporting the relationship between these two proteins in CRC.

Downregulation of WIF-1 and Wnt5a in patients with colorectal carcinoma: clinical significance.

Activation of the wingless-type (Wnt) signaling pathway is common in various human cancers including colorectal cancer (CRC). Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt ligands and therefore inhibits the Wnt signaling pathway. In this study, we aimed to analyze the expression of two members of Wnt signaling (WIF-1 and Wnt5a) in Tunisian patients with sporadic CRC. WIF-1 was frequently methylated in tumor tissues (87.95 %) compared to normal mucosa (39.54 %) and correlated with distant metastasis and vascular invasion (P = 0.001 and 0.037, respectively). The unmethylated profile of the WIF-1 promoter conferred a benefit to patients in terms of overall survival (P log rank = 0.024). In addition, in the group of patients with methylated WIF-1 promoter, the overall survival rate was significantly prolonged for those with small tumor size (<5 cm) and absence of distant metastasis (P log rank = 0.007 and 0.036, respectively). Aberrant CpG methylation of the WIF-1 promoter leads to transcriptional silencing of this tumor suppressor gene in tumor tissues (P = 0.001). Furthermore, we showed that the level of Wnt5a mRNA was significantly lower in tumor compared to normal tissues (P = 0.031) and lower still in those showing more aggressive behavior (presence of lymph nodes and advanced TNM stage). Our finding supports that WIF-1 is frequently methylated and that Wnt5a acts as a tumor suppressor gene in CRC. Loss of WIF-1 and Wnt5a functions results in more aggressive behavior of the disease.

Case report: Compound heterozygous variants detected by next-generation sequencing in a Tunisian child with ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is an autosomal recessive primary immunodeficiency disorder (PID) caused by biallelic mutations occurring in the serine/threonine protein kinase (ATM) gene. The major role of nuclear ATM is the coordination of cell signaling pathways in response to DNA double-strand breaks, oxidative stress, and cell cycle checkpoints. Defects in ATM functions lead to A-T syndrome with phenotypic heterogeneity. Our study reports the case of a Tunisian girl with A-T syndrome carrying a compound heterozygous mutation c.[3894dupT]; p.(Ala1299Cysfs3;rs587781823), with a splice acceptor variant: c.[5763-2A>C;rs876659489] in the ATM gene that was identified by next-generation sequencing (NGS). Further genetic analysis of the family showed that the mother carried the c.[5763-2A>C] splice acceptor variant, while the father harbored the c.[3894dupT] variant in the heterozygous state. Molecular analysis provides the opportunity for accurate diagnosis and timely management in A-T patients with chronic progressive disease, especially infections and the risk of malignancies. This study characterizes for the first time the identification of compound heterozygous ATM pathogenic variants by NGS in a Tunisian A-T patient. Our study outlines the importance of molecular genetic testing for A-T patients, which is required for earlier detection and reducing the burden of disease in the future, using the patients' families.

Strategies for diagnosis and management of CMMRD in low-resource countries: report of a Tunisian family.

Constitutional Mismatch Repair Deficiency (CMMRD) is a rare childhood cancer predisposition syndrome, caused by biallelic pathogenic germline variants in the mismatch repair genes. Diagnosis and management of this syndrome is challenging, especially in low-resource settings. This study describes a patient diagnosed with colorectal cancer and grade 3 astrocytoma at the age of 11 and 12 respectively. Immunohistochemistry analysis showed a loss of MSH2 and MSH6 protein expression in CRC tissues of the patient. We identified by Targeted Exome Sequencing a homozygous pathogenic germline variant in exon 9 of the MSH6 gene (c.3991 C > T; p.Ala1268Glyfs*6). Genetic investigation of the family showed that the father was heterozygous for the identified pathogenic variant while the brother was wild type for this variant. Our study highlights the importance of a correct and timely diagnosis of CMMRD which can have implications for treatment. It also underlines the imperative need to enhance awareness, diagnostic standards, and surveillance that are crucial for patients and their families.

Mutation in the ß-tubulin gene TUBB4A results in epileptic encephalopathy associated with hypomyelinated leucodystrophy: Unexpected findings reveal genetic mosaicism.

INTRODUCTION: Epileptic encephalopathies (EEs) are a group of heterogeneous epileptic syndromes characterized by early-onset refractory seizures, specific EEG abnormalities, developmental delay or regression and intellectual disability. The genetic spectrum of EE is very wide with mutations in a number of genes having various functions, such as those encoding AMPA ionotropic and glutamate receptors as well as voltage-gated ion channels. However, the list of EE-responsible genes could certainly be enlarged by next-generation sequencing. PATIENTS AND METHODS: The present study reports a clinical investigation and a molecular analysis by the whole exome sequencing (WES) and pyrosequencing of a patient's family affected by epileptic spasms and severe psychomotor delay. RESULTS: Clinical and radiological investigations revealed that the patient presented clinical features of severe and drug-resistant EE-type infantile epileptic spasm syndrome that evolved to Lennox Gastaut syndrome with radiological findings of hypomyelinated leukodystrophy. The results of WES revealed the presence of a novel heterozygous c.466C>T mutation in exon 4 of the TUBB4A gene in the patient. This transition led to the replacement of arginine by cysteine at position 156 (p.R156C) of the conserved helix 4 among the N-terminal domain of the TUBB4A protein. Bioinformatic tools predicted its deleterious effects on the structural arrangement and stability of the protein. The presence of the mutation in the asymptomatic father suggested the hypothesis of somatic mosaicism that was tested by pyrosequencing of DNA from two tissues of the patient and her father. The obtained results showed a lower rate of mutated alleles in the asymptomatic father compared with the affected daughter in both lymphocytes and buccal mucosa cells, confirming the occurrence of paternal mosaicism. The phenotypic features of the patient were also compared with those of previously described patients presenting TUBB4A mutations. CONCLUSIONS: Our study is the first to report a disease-causing variant in the TUBB4A gene in a patient with EE associated with hypomyelinated leucodystrophy. In addition, we expanded the phenotypic spectrum associated with the TUBB4A gene.

Predominance of the Rare EGFR Mutation p.L861Q in Tunisian Patients with Non-Small Cell Lung Carcinoma.

Objectives: Several new cancer therapies targeting signaling pathways involved in the growth and progression of cancer cells were developed as personalized medicine. Our study aimed to identify epidermal growth factor receptor (EGFR) mutations for TKI treatment in non-small-cell lung cancer (NSCLC) Tunisian patients. Methods: Analysis of the TKI sensitivity mutations in exons 18 to 21 of the EGFR gene and exon 15 of the B-raf gene was performed in 79 formalin fixed-paraffin embedded (FFPE) NSCLC samples using pyrosequencing. Results: EGFR mutations were detected in 34 cases among 79 (43%), with the predominance of the L861Q in exon 21 found in 35.3% of the cases (12 out of 34). Deletions in exon 19 were found in 8 cases (23.5%), and only one young male patient had the T790M mutation. Three patients harbored composite EGFR mutations (p.E746_A750del/p.L861R, p.E746_S752>V/p.S768I, and p.G719A/p.L861Q). Furthermore, the EGFR mutated status was significantly more frequent in female patients (p = 0.019), in non-smoker patients (p = 0.008), and in patients with metastasis (p = 0.044). Moreover, the B-raf V600E was identified in 5 EGFR negative patients among 39 analyzed samples (13.15%). Conclusion: The p.L861Q localized in exon 21 of the EGFR gene was the most common mutation identified in our patients (35.3%), whereas the "classic" EGFR mutations such as Del19 and p.L858R were found in 23.5% and 11.7% of the cases, respectively. Interestingly, most of p.L861X mutation-carrying patients showed good response to TKI treatment. Altogether, our findings suggest a particular distribution of the EGFR-TKIs sensitivity mutations in Tunisian NSCLC patients.

Clinical and prognosis value of the CIMP status combined with MLH1 or p16 INK4a methylation in colorectal cancer.

Aberrant DNA methylation of CpG islands occurred frequently in CRC and associated with transcriptional silencing of key genes. In this study, the CIMP combined with MLH1 or p16 INK4a methylation status was determined in CRC patients and correlated with clinicopathological parameters and overall survival. Our data showed that CIMP+ CRCs were identified in 32.9% of cases and that CACNAG1 is the most frequently methylated promoter. When we combined the CIMP with the MLH1 or the p16 INK4a methylation status, we found that CIMP-/MLH1-U (37.8%) and CIMP-/p16 INK4a -U (35.4%) tumors were the most frequent among the four subtypes. Statistical analysis showed that tumor location, lymphovascular invasion, TNM stage, and MSI differed among the group of patients. Kaplan-Meier analyses revealed differences in overall survival according to the CIMP combined with MLH1 or p16 INK4a methylation status. In a multivariate analysis, CIMP/MLH1 and CIMP/p16 INK4a methylation statuses were predictive of prognosis, and the OS was longer for patients with tumors CIMP-/MLH1-M, as well as CIMP-/p16 INK4a -M. Furthermore, DNMT1 is significantly overexpressed in tumors than in normal tissues as well as in CIMP+ than CIMP- tumors. Our results suggest that tumor classification based on the CIMP status combined with MLH1 or p16 INK4a methylation is useful to predict prognosis in CRC patients.

Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli.

Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).

High prevalence of the c.1227_1228dup (p.Glu410GlyfsX43) mutation in Tunisian families affected with MUTYH-associated-polyposis.

Germline mutations in the base excision repair gene MUTYH have been associated with recessive inheritance of multiple colorectal adenomas. Screening of the MUTYH gene was carried on index cases of 10 unrelated Tunisian families and on available DNA samples from some members. Three germline mutations: c.536A > G (p.Y179C), c.1187 G > A (p.G396D) and c.1227_1228dup (p.Glu410GlyfsX43), were identified in the homozygous or compound heterozygous state in 8 out of 10 families. The c.1227_1228dup (p.Glu410GlyfsX43) mutation was the most frequent, since it was found in biallelic homozygous in 7 probands and 2 members of family F1 and in compound heterozygous associated with the c.536 A > G (p.Y179C) or c.1187 G > A (p.G396D) in family F2. Haplotype analysis revealed that the 8 families are unrelated. Moreover, in sporadic colorectal cancer, the c.1227_1228dup (p.Glu410GlyfsX43) mutation was identified in 13 % of patients compared to the p.G396D and p.Y179C found in 1.2 and 2.12 % respectively. Our data shows the high prevalence of the p.Glu410GlyfsX43 mutation in Tunisian families affected with MUTYH associated polyposis as well as in sporadic colorectal carcinoma.

Expression of COX-2 and E-cadherin in Tunisian patients with colorectal adenocarcinoma.

Cyclo-oxygenase 2 (COX-2) and E-cadherin are promising biomarkers for cancer diagnosis and therapy. The aim of this study was to examine the expression of these two proteins in primary colorectal adenocarcinomas and to investigate their association with clinicopathological characteristics including survival of patients. Immunostaining of E-cadherin and COX-2 was assessed in 70 primary colorectal adenocarcinomas from Tunisian patients. Membranous E-cadherin immunostaining and cytoplasmic COX-2 expression were observed in 74.3% and 68.6% of cases respectively. A significant association was found between COX-2 expression and age at diagnosis (P=0.02), and vessel invasion (P=0.037). The expression of E-cadherin correlated with age at diagnosis (P=0.01), and tumor size (P=0.02). In addition, by multivariate analysis, we revealed a significant association with 1-year disease free survival and a tendency with distant metastasis (P=0.017 and P=0.065 respectively). On the other hand, tumors exhibiting COX-2+/E-cadherin-profile were larger (P=0.006), and in an advanced stage (P=0.001). Survival analysis showed that COX-2 over-expression confers a reduced overall survival rate (Plog rank=0.036) and is an independent factor predictive for prognosis.