Novel Missense and Splice Site Mutations in USH2A, CDH23, PCDH15, and ADGRV1 Are Associated With Usher Syndrome in Lebanon.

The purpose of this study was to expand the mutation spectrum by searching the causative mutations in nine Lebanese families with Usher syndrome (USH) using whole-exome sequencing. The pathogenicity of candidate mutations was first evaluated according to their frequency, conservation, and in silico prediction tools. Then, it was confirmed via Sanger sequencing, followed by segregation analysis. Finally, a meta-analysis was conducted to calculate the prevalence of USH genes in the Lebanese population. Three missense mutations, two splice site mutations, and one insertion/deletion were detected in eight of the families. Four of these variants were novel: c.5535C > A; p.(Asn1845Lys) in exon 41 of CDH23, c.7130G > A; p.(Arg2377Gln) in exon 32 of ADGRV1, c.11390-1G > A in USH2A, and c.3999-6A > G in PCDH15. All the identified mutations were shown to be likely disease-causing through our bioinformatics analysis and co-segregated with the USH phenotype. The mutations were classified according to the ACMG standards. Finally, our meta-analysis showed that the mutations in ADGRV1, USH2A, and CLRN1 are the most prevalent and responsible for approximately 75% of USH cases in Lebanon. Of note, the frequency USH type 3 showed a relatively high incidence (23%) compared to the worldwide prevalence, which is around 2-4%. In conclusion, our study has broadened the mutational spectrum of USH and showed a high heterogeneity of this disease in the Lebanese population.

Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes.

Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are therapeutic compounds widely administered in the clinic for their multiple uses (autoimmune diseases, post-transplant immunosuppression and cancer). Despite these advantages, their therapeutic potential is limited by occasional adverse effects (myelotoxicity and hepatotoxicity) and by a relatively frequent lack of efficacy. Previous studies have demonstrated that azathioprine decreased the viability of rat hepatocytes. In order to investigate cytotoxic effects of thiopurines in human liver, we used primary human hepatocytes and a highly differentiated human hepatoma cell line, HepaRG, treated or not with azathioprine, 6-mercaptopurine and 6-thioguanine. In parallel, expression of the genes involved in the metabolism of thiopurines, glutathione synthesis and antioxidant defences was measured by quantitative PCR. We clearly demonstrate that human liver parenchymal cells were much less sensitive than rat hepatocytes to thiopurine treatments. The toxic effects appeared after 96 h of treatment while ATP depletion was observed after a 24 h incubation with azathioprine and 6-mercaptopurine. Toxic effects were more pronounced for azathioprine and 6-mercaptopurine, when compared to 6-thioguanine, and might explain glutathione synthesis and antioxidant enzyme induction only by these two drugs. Finally, we also demonstrate for the first time an up-regulation by azathioprine and 6-mercaptopurine of inosine monophosphate dehydrogenase which might have consequences on the de novo biosynthesis of guanine nucleotides and thiopurines metabolism.

The rs3957357C>T SNP in GSTA1 Is Associated with a Higher Risk of Occurrence of Hepatocellular Carcinoma in European Individuals.

Glutathione S-transferases (GSTs) detoxify toxic molecules by conjugation with reduced glutathione and regulate cell signaling. Single nucleotide polymorphisms (SNPs) of GST genes have been suggested to affect GST functions and thus to increase the risk of human hepatocellular carcinoma (HCC). As GSTA1 is expressed in hepatocytes and the rs3957357C>T (TT) SNP is known to downregulate GSTA1 mRNA expression, the aims of this study were: (i) to explore the relationship between the TT SNP in GSTA1 and the occurrence of HCC; (ii) to measure GSTA1 mRNA expression in HCCs. For that purpose, we genotyped non-tumor-tissue-derived DNA from 48 HCC patients and white-blood-cell-derived DNA from 37 healthy individuals by restriction fragment length polymorphism (RFLP). In addition, expression of GSTA1 mRNA was assessed by real-time PCR in 18 matching pairs of HCCs and non-tumor livers. Survival analysis was performed on an annotated microarray dataset containing 247 HCC patients (GSE14520). The GSTA1 TT genotype was more frequent in HCC than in non-HCC patients (27% versus 5%, respectively), suggesting that individuals carrying this genotype could be associated with 2-fold higher risk of developing HCCs (odds ratio = 2.1; p = 0.02). Also, we found that GSTA1 mRNA expression was lower in HCCs than in non-tumor livers. HCCs expressing the highest GSTA1 mRNA levels were the smallest in size (R = -0.67; p = 0.007), expressed the highest levels of liver-enriched genes such as ALB (albumin, R = -0.67; p = 0.007) and COL18A1 (procollagen type XVIII, R = -0.50; p = 0.03) and showed the most favorable disease-free (OR = 0.54; p<0.001) and overall (OR = 0.56; p = 0.006) outcomes. Moreover, GSTA1 was found within a 263-gene network involved in well-differentiated hepatocyte functions. In conclusion, HCCs are characterized by two GSTA1 features: the TT SNP and reduced GSTA1 gene expression in a context of hepatocyte de-differentiation.

Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells.

Acquisition of drug resistance by cancer cells is attributed to various factors including alterations in apoptotic pathways, enhanced expression of multidrug resistance-associated proteins, altered drug metabolism or uptake and/or overexpression of cytoprotective genes. Thus, potential induction of defence pathways by anticancer drugs might have a marked incidence on cancer cell resistance. 5-Fluorouracil (5-FU) remains the most commonly used anticancer drug for the treatment of colorectal cancer, although objective response rates are as low as 20%. The aim of our study was to investigate the effects of 5-FU on cytoprotective systems in human colon HT-29 cells. Our results demonstrate that 5-FU induced the expression of mRNAs encoding glutathione transferases and antioxidant enzymes. To further determine the mechanisms involved in 5-FU effects, we investigated whether it activates the Nrf2/antioxidant response element pathway which is implicated in the regulation of several genes involved in cytoprotection. Translocation of Nrf2 into the nucleus after 5-FU exposure was demonstrated by immunocytochemistry and western blotting. Using an ARE-driven reporter gene assay, activation of the luciferase activity by 5-FU was also evidenced. Moreover, transfection of HT-29 cells with siRNA directed against Nrf2 inhibited induction of Nrf2 target genes and increased 5-FU cytotoxicity. In conclusion, we demonstrate for the first time that 5-FU activates the Nrf2/ARE pathway which in turn induces cytoprotective genes and modulates chemosensitivity of HT-29 colon cancer cells. Therefore, we postulate that Nrf2 might represent a potential therapeutic target in 5-FU treatment of colon cancer.