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.

Abstract
One significant pathophysiological mechanism underlying Cystic Fibrosis (CF) is the hyperabsorption of sodium through ENaC. Further understanding of this misregulation could help develop novel therapeutic targets for treating CF. Channel Activating Proteases (CAPs) have been shown to increase ENaC activity but it is not clear whether CAP activity is physiologically regulated, or whether this regulation is defective in CF airways. We have previously shown that normal primary human bronchial epithelial cultures (HBECs) exhibit a trypsin-sensitive transepithelial voltage (Vt) when airway surface liquie (ASL) has accumulated for 48 hours. Conversely, freshly washed normal and CF HBECs, as well as CF HBECs with accumulated ASLs, remained trypsin-insensitive indicating that ENaC is maximally activated in these cultures. This implies that the CAP inhibitor is soluble and accumulates in the ASL. Additionally, accumulation of this inhibitor is unable to inhibit ENaC conductance in CF cultures. (Tarran et al., 2006). We then searched for soluble CAP inhibitors with trypsin-coated beads, which were added to the ASL. Albumin-coated beads were used as a control for non-specific binding. Using mass spectrometry, we identified sPLUNC1. This protein is secreted, in vivo and in vitro, in the ASL although its function is unknown. To understand of the role of sPLUNC1 in airway physiology, we examined the capacity for ASL regulation in HBECs treated with either anti-sPLUNC1 or anti-luciferase shRNA as a control. qPCR verified a >90% knockdown efficiency, which resulted in significant ASL volume depletion in the sPLUNC1 knockdown HBECs as compared to the controls (ctrl, 9.0 ?1.8 uM; anti-sPLUNC1, 5 ?0.9uM; n=5). This was accompanied by a persistent trypsin-sensitive Vt in sPLUNC1-defecient cultures, indicating that they could no longer regulate ENaC. Our next aim was to examine the effect of sPLUNC1 on ENaC current. To do this, xenopus oocytes were injected with sPLUNC1 and a,?,y ENaC subunits. sPLUNC1 was detected in the media of these oocyte by Western blot and showed a 43% (? 4%, n=40) inhibition of ENaC current when compared to ENaC injected oocytes alone. Western blot analysis revealed that this reduction in current was accompanied by a reduction in cleavage of a, ? and y ENaC subunits suggesting that SPLUNC1 prevents cleavage of ENaC by serine proteases. These findings indicate an important role for sPLUNC1 in airway physiology as it regulates ENaC current and consequently, ASL volume. Understanding the mechanism of sPLUNC1 may provide insight into novel therapies for regulating hyperactive ENaC in CF.

Abstract
Chronic granulomatous disease (CGD) is a rare immunodeficiency disorder characterized by life threatening infections. It results from mutations in any of the four genes CYBB (gp91phox), CYBA (p22phox), NCF-1(p47phox), and NCF-2 (p67phox). The aim of this study was to characterize the molecular basis of CGD in Omani patients. CGD was suspected in 13 patients from 8 families by clinical examination and investigated biochemically (Nitroblue tetrazolium test), and by flowcytometry Dihydrorhodamine test (DHR). Using direct DNA sequencing, GeneScan and allele-specific PCR technologies we analyzed the genetic variations of NCF-1 complex comprising functional and pseudogenes. Only one patient presented Xlinked inheritance pattern, who incidentally also had McLeods Syndrome. He was demonstrated to have a large deletion of approximately 600 kb involving both CYBB gene and the McLeods gene (XK). Amongst the remaining 12 patients of autosomal recessive transmission, we were able to identify mutations in the NCF-1 gene, (the most common form of autosomal recessive CGD) in 6 patients. In four kindred from one family we found the homozygous G784A mutation (Gly262Ser) in Exon 8 of the NCF-1 gene. Additionally, two patients had homozygous ?GT mutations in the functional NCF-1 gene. Thus, amongst 8 families studied, we were able to identify the underlying molecular basis of CGD in four (54%). Despite extensive analysis of all the four major genes of the NADPH-oxidase, we could not define the molecular basis of CGD in 6 patients from the remaining 4 families. The study must be extended towards the analysis of two other genes namely (NCF-4 and Rap1A) in order to decipher the causal locus and associated mutations.

Abstract
Diversity profile of 15 short tandem repeat (STR) loci was evaluated using AmpFISTR Identifier kit (Applied Biosystems) in DNA from 120 healthy native Qataris after informed consent. All the 16 markers (including the Amelogenin gender differenciator) were amplified by PCR using the commercially available ?Identifier primer set? in a multiplexed reaction. Size separation of the PCR products was carried out by electrophoresis on a 3100 genetic analyser (Applied Biosystems) including positive and negative controls as well the size standards. Allele identification and tabulation was performed using Genemapper v3.2 software. Statistical analyses including determination of the matching probability, power of discrimination (PD), polymorphism information content, power of paternity exclusion (PE), and typical paternity index were performed using the software PowerStats (Promega Corporation, Madison, Wisconsin). The discriminating power of a locus, which represents the probability that two randomly chosen subjects do not have the same genotype, was calculated as 1-Pi, and the combined discriminating power of the 15 loci as 1 - (? Pi), where Pi is the sum of the squares of frequencies of all genotypes at a given locus. The higher the discriminating power of a locus, the more efficient it is in discriminating between members of the population. The power of paternity exclusion, defined as the fraction of individuals who would not have the same DNA profile, was calculated from the following formula: PE=h2 (1- 2hH2), where h is heterozygote frequency and H is homozygote frequency. The higher the PE value, more the non-fathers are excluded. The combined power of paternity exclusion was calculated as 1 ? ?(1- PEi). The typical paternity index was calculated as (H+h)/2H. Overall genotype distribution adhered to expectations from Hardy-Weinberg equilibrium by three independent testings for all markers (0.00333). No significant linkage disequilibrium (LD) was noted between any pair of loci after Bonferroni correction (0.000476). We found that the most discriminating locus in the study population was D2S1338 (PD=0.969), while the least informative locus was TPOX (PD=0.821). Individual power of exclusion values ranged from 0.322 (TPOX) to 0.762 (FGA). They indicated low degrees of exclusionary power for the loci when used individually. However, combined power of discrimination and combined power of exclusion were both estimated to be greater than 0.9999 for the Qatar population sample, thereby demonstrating the forensic utility / individual identification of a multilocus-based analysis.

Abstract
One significant pathophysiological mechanism underlying Cystic Fibrosis (CF) is the hyperabsorption of sodium through ENaC. Further understanding of this misregulation could help develop novel therapeutic targets for treating CF. Channel Activating Proteases (CAPs) have been shown to increase ENaC activity but it is not clear whether CAP activity is physiologically regulated, or whether this regulation is defective in CF airways. We have previously shown that normal primary human bronchial epithelial cultures (HBECs) exhibit a trypsin-sensitive transepithelial voltage (Vt) when airway surface liquie (ASL) has accumulated for 48 hours. Conversely, freshly washed normal and CF HBECs, as well as CF HBECs with accumulated ASLs, remained trypsin-insensitive indicating that ENaC is maximally activated in these cultures. This implies that the CAP inhibitor is soluble and accumulates in the ASL. Additionally, accumulation of this inhibitor is unable to inhibit ENaC conductance in CF cultures. (Tarran et al., 2006). We then searched for soluble CAP inhibitors with trypsin-coated beads, which were added to the ASL. Albumin-coated beads were used as a control for non-specific binding. Using mass spectrometry, we identified sPLUNC1. This protein is secreted, in vivo and in vitro, in the ASL although its function is unknown. To understand of the role of sPLUNC1 in airway physiology, we examined the capacity for ASL regulation in HBECs treated with either anti-sPLUNC1 or anti-luciferase shRNA as a control. qPCR verified a >90% knockdown efficiency, which resulted in significant ASL volume depletion in the sPLUNC1 knockdown HBECs as compared to the controls (ctrl, 9.0 ?1.8 uM; anti-sPLUNC1, 5 ?0.9uM; n=5). This was accompanied by a persistent trypsin-sensitive Vt in sPLUNC1-defecient cultures, indicating that they could no longer regulate ENaC. Our next aim was to examine the effect of sPLUNC1 on ENaC current. To do this, xenopus oocytes were injected with sPLUNC1 and ?,?,? ENaC subunits. sPLUNC1 was detected in the media of these oocyte by Western blot and showed a 43% (? 4%, n=40) inhibition of ENaC current when compared to ENaC injected oocytes alone. Western blot analysis revealed that this reduction in current was accompanied by a reduction in cleavage of ?, ? and ? ENaC subunits suggesting that SPLUNC1 prevents cleavage of ENaC by serine proteases. These findings indicate an important role for sPLUNC1 in airway physiology as it regulates ENaC current and consequently, ASL volume. Understanding the mechanism of sPLUNC1 may provide insight into novel therapies for regulating hyperactive ENaC in CF.

Abstract
Thiazolidinediones (TZDs) are synthetic ligands for the nuclear peroxisome-proliferator activated receptor gamma (PPAR?). These agents have potent insulin-sensitizing capabilities and are used clinically for treatment of non-insulin dependent Type II diabetes mellitus. However, their use is limited in patients at risk for cardiovascular disease due to fluid retentive side effects. The side effect etiology is unknown, but the nature of presentation suggests modulation of renal salt and water homeostasis. This contention was strengthened by the creation of renal collecting duct specific PPAR? knockout animals that were resistant to the fluid retentive effects of two clinically used TZDs, rosiglitazone and pioglitazone. This renal site of action suggested a TZD effect on the epithelial Na+ channel (ENaC), a hormone regulated channel involved in fluid-electrolyte balance. However, several anomalies and conflicting data argue against an ENaC-mediated response as the primary target of PPAR? agonist-mediated fluid retention. In several collecting duct cell lines, we have shown that TZDs did not affect ENaC expression or activity. We have used the mpkCCDcl4 (mouse principal cells of the kidney cortical collecting duct, clone 4) cell line to show that PPAR? agonists inhibit vasopressin-stimulated Cl- secretion with agonist dose response relationships that mirror receptor trans-activation profiles. Analyses of the components of the vasopressin-stimulated intracellular signaling pathway indicated no PPAR? agonist-induced changes in basolateral membrane conductances, intracellular cAMP or protein kinase A. The PPAR? agonist-induced decrease in anion secretion is the result of decreased mRNA of the final effector in the pathway, the apically located cystic fibrosis transmembrane regulator (CFTR). These data showing that CFTR is a target for PPAR? agonists provides new insight into the physiology of PPAR? agonist-induced fluid retention. The data, if substantiated by in vivo experiments, indicate that CFTR can play a primary rather than secondary role in renal-mediated fluid balance. In addition, this finding may suggest potential treatment options for diseases in which CFTR activity plays a role. One of the most common of these is polycystic kidney disease (PKD). In PKD, CFTR-mediated Cl- secretion is known to contribute to the growth and maintenance of both renal and hepatic bile duct cysts. TZD inhibition of CFTR should, theoretically, inhibit cyst growth. Animal studies using an orthologous rat model of PKD are in progress. These studies can provide proof-of-principle for TZD-mediated CFTR inhibition as well as providing the basis for a new class of drug therapy for PKD.

Abstract
There are now compelling data to support the hypothesis that CF lung disease results from a failure of innate host defence mechanisms as a consequence of airway mucosal dehydration. The regulation of airway surface liquid (ASL) volume through the coordinated activity of ion channels and transporters in the airway epithelium is crucial to the effectiveness of mucus clearance by both mucociliary and cough clearance mechanisms. In the simplest pathophysiological paradigm for CF the absence or reduction of functional CFTR at the apical membrane of ciliated airway epithelial cells results in an imbalance of CFTR-mediated Cl- secretion and ENaC (epithelial sodium channel)-mediated Na+ absorption resulting in a net loss of fluid from airway surfaces. Approaches to normalize this imbalance and restore fluid to the airway mucosa are predicted to improve mucus clearance and thereby positively impact upon lung health in CF. One approach to restore hydration to the airway mucosa is to attenuate ENaC function, thereby blocking Na+ and fluid reabsorption. Early studies failed to demonstrate a robust clinical benefit of inhaled amiloride, a low potency ENaC blocker, in the CF lung. It has been proposed that the lack of clinical efficacy observed with amiloride was as a consequence of its low potency, short duration of action in the airway and the dose limiting side effect of hyperkalaemia. Novel ENaC blockers that demonstrate a long duration of action in the airway and that are safe, may therefore represent a novel therapeutic opportunity for the treatment of CF lung disease. An approach taken by Novartis, has been to design ENaC blockers specifically for inhaled dosing by dry powder delivery. Drug candidates will be described that exemplify the design strategy adopted to achieve both a potent and sustained activity in the lung together with a reduced potential to induce the mechanism-based side effect of hyperkalaemia, in pre-clinical models.

Abstract
Respiratory virus infection is a major cause of exacerbation of cystic fibrosis (CF) lung disease and CF patients suffer more severe consequences of virus infection. Our goals are to understand the impact of respiratory viruses on the pathogenesis of CF lung disease. To study the effects of virus infection on the human CF ciliated airway epithelium we used an in vitro model of human ciliated airway epithelium (HAE) and respiratory syncytial virus (RSV) as a virus that commonly infects CF patients. Using non-CF HAE, we show that RSV infects only ciliated cells and causes an acute increase in airway surface liquid height (ASL) height as measured by Texas red-dextran staining of ASL followed by confocal microscopy. This effect was maximum at ~3 days post-inoculation (pi) in non-CF HAE (ctrl,7.71?0.5;RSV,14.8?0.9?m, n=4). Parallel experiments with CF HAE revealed that RSV infection of ciliated cells failed to induce an increase in ASL (CF-ctrl,5.9?0.1;CF-RSV,5.9?0.1?m; n=4). These data suggest that non-CF HAE, but not CF HAE, respond to RSV infection by stimulation of fluid secretion into the airway lumen and is likely mediated by epithelial cell ion transport mechanisms. To determine whether the increase in ASL height after RSV was due to active Cl- secretion, we used bumetanide (100?M) which significantly inhibited RSV-induced ASL height (ctrl,7.3?0.6; RSV,14.4?1.3; RSV+bumetanide,9?0.6; ctrl+bumetanide,5.6?0.1?m; n=4). To determine the specific contribution of CFTR-mediated Cl- secretion to this response we used CFTR172 that also significantly inhibited RSV-induced ASL height (ctrl,6.6?0.3;RSV,11.4?1;RSV+CFTR172, 8 ?0.3; ctrl+CFTR172, 6.8?0.5?m; n=4). Since adenosine triphosphate (ATP) and adenosine (ADO) have been previously shown to regulate Cl- secretion and ASL height in non-CF HAE, we determined the ASL concentration of ATP and ADO 3 days after RSV infection in non-CF HAE and found that both were increased by RSV infection (ATP, ctrl,16?5; RSV,26?3nM; ADO, ctrl,151?25; RSV,245?15nM, n=3) suggesting that the increased ASL after RSV infection was due to the release of nucleotides known to regulate ion transport in airway epithelium. We conclude that RSV infection results in a CFTR-dependent increase in ASL height which is mediated by increased nucleotide levels in the ASL and that this cell response to infection is defective in CF airway epithelial cells. We speculate that increased fluid secretion is a host defense mechanism that attempts to clear virus from the airways and this clearance is diminished in CF airways possibly resulting in prolonged RSV infection of the CF airways.