miRNA implication in the pathogenesis and the outcome of Tunisian endemic pemphigus foliaceus.

Pemphigus foliaceus (PF) is a bullous autoimmune skin disease diagnosed through sera and skin analyses. PF severity is associated with maintained anti-Dsg1 sera levels and its prognosis is unpredictable. MicroRNA (miRNA), dynamic regulators of immune function, have been identified as potential biomarkers for some autoimmune diseases. This study aimed to assess the miRNA expression of miR-17-5p, miR-21-5p, miR-146a-5p, miR-155-5p and miR-338-3p using quantitative real-time PCR in peripheral blood mononuclear cells (PBMC) and lesional skin samples from untreated and treated PF patients (both remittent and chronic) over 3 months. Overall, miRNA expression was significantly higher in PBMC than in biopsy samples. Blood miR-21 expression was increased in untreated patients compared to controls and had a diagnostic value with an AUC of 0.78. After 6 weeks, it decreased significantly, similar to anti-Dsg1 antibodies and the PDAI score. In addition, a positive correlation was observed between cutaneous miR-21 expression and the disease activity score. Conversely, cutaneous expressions of miR-17, miR-146a and miR-155 were significantly higher in treated chronic patients compared to remittent ones. The cutaneous level of miR-155 positively correlated with pemphigus activity, making it a potential predictive marker for patients' clinical stratification with an AUC of 0.86.These findings suggest that blood miR-21 and cutaneous miR-155 can be used as supplemental markers for PF diagnosis and activity, respectively in addition to classical parameters.

Characterization of a novel ABCC2 mutation in infantile Dubin Johnson syndrome.

BACKGROUND AND AIMS: The Dubin Johnson Syndrome (DJS) occurs mostly in young adults but an early-onset of the disease has been reported in less common forms (Neonatal DJS and Infantile DJS). In this case, the clinical findings are of limit for the DJS diagnosis. Hence, the genetic testing remains the method of choice to provide an accurate diagnosis. In our study, we aimed to perform a genetic analysis for two siblings presented with an intrahepatic cholestasis before the age of 1 year to provide a molecular explanation for the developed phenotype. PATIENTS & METHODS: A Tunisian family, having two siblings, manifesting signs of a hepatopathy, was enrolled in our study. A molecular analysis was performed, using a panel-based next generation sequencing, supplying results that were the subject of computational analysis. Then, a clinical follow-up was carried out to assess the evolution of the disease. RESULTS: The genetic analysis revealed the presence of a novel missense c.4179G > T, (p.M1393I) mutation in ABCC2 gene associated with a substitution c.2789G > A (R930Q) in ATP8B1 gene. Predictive results consolidated the pathogenic effect of both variants. These results confirmed the DJS diagnosis in the studied patients. The clinical course of both patients fit well with the benign nature of DJS. CONCLUSION: We described here a novel ABCC2 mutation associated with a putative ATP8B1 modifier variant. This finding constituted the first report of a complex genotype in DJS. Hence, genetic analysis by a panel-based next generation sequencing permits an accurate diagnosis and the identification of putative variants that could influence the developed phenotype.

A homozygous ABCB4 mutation causing an LPAC syndrome evolves into cholangiocarcinoma.

Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. In the present study, we reported a case of a 63-year-old woman, who presented a biliary pain beginning at the age of 30, recurrent after cholecystectomy, along with "comet-tail shadows" revealed by ultrasonography thus, fulfilling the diagnosis of LPAC. This disease evolved into a cholangiocarcinoma. To understand the molecular basis of this phenotype, we performed the ABCB4 gene sequencing, followed by in silico analysis and Q-RT-PCR assay. The results displayed a homozygous missense sequence variation (c.140G > A, p.Arg47Gln), predicted as pathogenic according to MutPred. Accordingly, this gave rise to a decreased hepatic ABCB4 mRNA level and structural alterations of the mutated protein. Eventually, we reported, here, the first description of an ABCB4 missense mutation (p.Arg47Gln) at homozygous state in a Tunisian LPAC syndrome. An elucidation of its functional consequences was performed. Besides, this case suggests that the delayed diagnosis of LPAC syndrome and the lack of UDCA treatment may contribute in the development of complications, such as cholangiocarcinoma.

Comparison of in silico prediction and experimental assessment of ABCB4 variants identified in patients with biliary diseases.

Genetic variations of the phosphatidylcholine transporter, ABCB4 cause several biliary diseases. The large number of reported variations makes it difficult to foresee a comprehensive study of each variation. To appreciate the reliability of in silico prediction programs, 1) we confronted them with the assessment in cell models of two ABCB4 variations (E528D and P1161S) identified in patients with low phospholipid-associated cholelithiasis (LPAC); 2) we extended the confrontation to 19 variations that we had previously characterized in cellulo. Four programs (Provean, Polyphen-2, PhD-SNP and MutPred) were used to predict the degree of pathogenicity. The E528D and P1161S variants were studied in transfected HEK293 and HepG2 cells by immunofluorescence, immunoblotting and measurement of phosphatidylcholine secretion. All prediction tools qualified the P1161S variation as deleterious, but provided conflicting results for E528D. In cell models, both mutants were expressed and localized as the wild type but their activity was significantly reduced, by 48% (P1161S) and 33% (E528D). These functional defects best correlated with MutPred predictions. MutPred program also proved the most accurate to predict the pathogenicity of the 19 ABCB4 variants that we previously characterized in cell models, and the most sensitive to predict the pathogenicity of 65 additional mutations of the Human Gene Mutation Database. These results confirm the pathogenicity of E528D and P1161S variations and suggest that even a moderate decrease (by less than 50%) of phosphatidylcholine secretion can cause LPAC syndrome. They highlight the reliability of in silico prediction tools, most notably MutPred, as a first approach to predict the pathogenicity of ABCB4 variants.