MC3R modifies CF lung disease by increasing the level of CFTR. J. Park1, J. Pilewski2, D. Belchis3, S. Blackman4, G. R. Cutting1 1) Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD; 2) Departments of Medicine and of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA; 3) Pathology, Johns Hopkins School of Medicine, Baltimore, MD; 4) Pediatric Endocrinology, Johns Hopkins University, Baltimore, MD.

   Lung disease accounts for 90% of the mortality in cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the CFTR gene affecting 70,000 individual worldwide. Heritability of lung disease severity estimated from affected twin analysis (H2=0.54) indicates a prominent role for genetic modifiers. Linkage analysis of affected siblings identified a region on 20q13.2 (LOD=5.03) harboring a modifier of lung function. The 1.3 Mb region of maximum linkage (1 LOD drop) contains only 5 genes, one of which is the melanocortin-3-receptor (MC3R). MC3R is a compelling candidate for modifying CF lung disease as an empiric screen for new CF therapies using siRNAs revealed that reduction of MC3R resulted in restoration of chloride channel activity to cells expressing CFTR bearing the common CF mutation F508del. To assess whether MC3R is a modifier of CF lung disease, we searched for mutations, analyzed its tissue expression and evaluated its effect on CFTR. Sequencing of MC3R in 150 sibpairs with CF identified 4 common and 4 rare variants. Three rare coding variants showed evidence of association with lung disease severity (as measured by survival-adjusted CF-specific FEV1 Z-score) [g.54824368A>C (Z-score=+1.6, p<0.05), rs41274722 (Z-score=-1.2, p<0.03), rs61735259 (Z-score=+1.5, p<0.01)]. MC3R mRNA was detected by RT-PCR in normal human bronchial epithelial (HBE) cells and CF bronchial epithelial (CFBE) cells homozygous for F508del-CFTR. MC3R was detected in the lysates of primary airway epithelial cells by Western blot (n=5). IHC staining with 2 anti-MC3R antibodies (Abcam & LS Bio) revealed a signal in surface airway epithelial cells of normal and CF lung. A reduction in MC3R expression by 40-79% in K562 cells using siRNAs increased the quantity of heterologously expressed GFP-WT-CFTR (298%, p<0.0001), GFP-F508del-CFTR (479%, p<0.03) and F508del-CFTR (336%, p<0.04). Similarly, transfection of CFBE cells with MC3R siRNAs resulted in a dramatic increase in the amount of endogenously expressed F508del-CFTR. Together, these results suggest that MC3R modifies CF lung disease by increasing the level of mutant forms of CFTR, such as F508del, that retain residual function, which enables recovery of CFTR-mediated chloride transport to airway epithelia. Multiple synthetic ligands have been developed that selectively target melanocortin receptors suggesting a novel therapeutic approach to CF lung disease based on targeting of a genetic modifier.

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