Abstract
Cystic fibrosis (CF) is a fatal, autosomal and recessive genetic disease that is mainly due to inactivating mutations in the chloride channel CF transmembrane conductance regulator (CFTR). Sodium hyperabsorption by the airways, mediated by the epithelial Na+ channel (ENaC), profound lung inflammation and dysregulation of the calcium homeostasis are presumably causally related to loss of CFTR-dependent chloride function in CF patients. One strategy for development of CF therapeutics is the identification of pharmacological agents that correct processing defect of F508del-CFTR and/or stimulate the channel activity of mutated proteins. The protein-repair therapy is based on several observations showing that in the presence of a pharmacological corrector, tailored to the specific F508del genotype, the misfolded protein escapes the ER and targeted to the plasma membrane. We have identified numerous F508del correctors with a CF drug discovery program using a robotic cell-based assay combined to molecular, biochemical and electrophysiological approaches. Among them, we identified miglustat, an orally bioavailable N-alkylated imino sugar (N-butyldeoxynojirimycin, Zavesca?). We investigated effects of high concentration (100 ?M) of miglustat on several CF characteristics and demonstrated after short-term (2-4 h) treatment of CF cells, a partial rescue of the defective F508del-CFTR trafficking and function [1], an improvement of the altered Ca2+ homeostasis [2], a down-regulation of ENaC-dependent Na+ hyperabsorption [3] and an anti-inflammatory effect of miglustat [4]. We suggested that the mechanism by which miglustat corrects the defective F508del-CFTR trafficking is correlated to a disturbance of the ER quality control system in CF cells. In support of that, miglustat is an ?1,2-glucosidase inhibitor preventing the interaction between F508del-CFTR and calnexin in the ER. More recently, we explored the concentration- and time-dependence of miglustat-induced correction of ionic transports in the human respiratory CF epithelial cells. The most salient result is the demonstration that a daily treatment for 2 months with low concentration of miglustat (3?M) resulted in progressive, stable, reversible and sustained correction of the F508del-CFTR deficient trafficking [5]. Then by investigating different biological and cellular aspects of cystic fibrosis such as Na+ hyperabsorption and dysregulation of the Ca2+ homeostasis, we were able to show paralleled normalization of these parameters correlated with the restoration of the F508del-CFTR function. In conclusion, we provided the first evidence that a respiratory CF cell can acquire a non-CF like phenotype when chronically treated with low-concentration of a pharmacological drug resulting in progressive, stable, reversible and sustained correction of F508del-CFTR trafficking, down-regulation of sodium hyperabsorption and regulation of the calcium homeostasis. This body of information makes the use of miglustat attractive as a potential pharmacologic therapy for those CF patients who have at least one F508del-CFTR allele. Miglustat, a medicament already prescribed in another orphan disease, is now evaluated in CF patients within a pilot phase 2a clinical trial.

Abstract
Ca2+-activated Cl- channels (CaCCs) play important roles in various cellular mechanisms, including fluid secretion in epithelia, sensory transduction, and regulation of neuronal and smooth muscle excitability. Molecular identity of this type of channels was controversial. Our group has recently identified TMEM16A as a possible CaCC. The aim of our present study is to characterize the properties of the Cl- currents associated with TMEM16A expression and to compare them with those of classical CaCCs described in several previous studies. For this purpose, we have used the patch clamp technique in the whole-cell configuration on FRT cells stable-transfected with the TMEM16A(abc) isoform. To analyse the ion channel selectivity, we substituted Cl- in the extracellular solution with other anions (I-, Br-, SCN-, and gluconate) and we measured the resulting shift in the reversal potential of membrane currents. Our data indicate that TMEM16A-dependent channels have ion selectivity properties similar to those of native CaCCs (Hartzell, Putzier and Arreola, Annu. Rev. Physiol. 2005. 67:719-58). We also studied the Ca2+-dependence of TMEM16A channels by changing the cytosolic free Ca2+-concentration in the 0.017 - 1.35 ?M range. By plotting the maximal current elicited at + 100 mV versus the cytosolic Ca2+ concentration, and fitting the data with a Hill function, we found a Kd = 91.8 nM and a Hill coefficient nH = 2.32. These values are close to those published previously for CaCCs (Kd = 61 nM, nH = 2.7; Arreola, Melvin and Begenisish, J. Gen. Physiol. 1996. 108:35-47). In conclusion, our results confirm that TMEM16A is a membrane protein involved in Ca2+-dependent Cl- transport. This remark evidences that TMEM16A may represent an important pharmacological target to treat cystic fibrosis in which activation of an alternative Cl- channel may compensate for the defective CFTR activity

Abstract
Geographically isolated populations have been successfully used to localize genes for recessive inherited diseases, including non-syndromic sensorineural recessive hearing loss (NSRHL). To date, 67 loci for NSRHL have been localized on human chromosomes (DFNB loci), and 22 of the corresponding genes have been identified; eight of those loci were first mapped in Palestinian families. We mapped recessive, severe to profound, prelingual NSHL in a four-generation consanguineous Palestinian Family K. The maximum LOD score was 4.19 at the 6.1MB interval on chromosome 22q13 bounded by recombinants in D22S1045 and D22S282. The DFNB28 region is distal to MYH9 and to the region deleted in Velo-cardio-facial Syndrome. Other Palestinian families were found to be linked to the same region, suing homozygosity mapping across all linked families, the causative gene was finally cloned. Deaf mouse mutants in conjunction with linkage analysis of families with deafness have been instrumental in the identification of human genes. Nevertheless, a great number of human deafness loci do not have a corresponding ?mouse model?; and on the other hand there are a large number of deaf mouse mutants with no human homologue. Part of the deficit we hope to complete using mouse models generated form the ENU mutagenesis program. In our lab, we have cloned a number of those ENU mutants including Doarad, Beethoven, headturner, and recently Headchuk which is the second ENU mutant mouse in our lab that has a mutation in Jagged1. More work is now being done on other ENU mutants in the lab, to determine their causative genes and to characterize their ear phenotypes.