Novel pharmacologic therapies for cystic fibrosis.

The Journal of Clinical Investigation | February 1999 | Volume 103 | Number 4 447 It has been nearly 10 years since the identification and cloning of the cystic fibrosis (CF) gene was announced (1). Despite rapid advances in our understanding of the molecular determinants of the disease, CF remains one of the most common lethal inherited autosomal recessive disorders in the Caucasian population worldwide. Seventy percent of patients carry at least one copy of the most common mutation — ∆F508 — but over 800 unique mutations have been described (http://www.genet. sickkids.on.ca/cftr/). Based on the molecular outcome, genotypes can be assigned to one of five classes of mutations. It is hoped that by designing novel therapeutic molecules whose activity is specialized to correct specific mRNA or protein defects in CF transmembrane conductance regulator (CFTR), the morbidity and mortality of CF could be dramatically improved. Several prototype therapies directed at restoration of CFTR function by targeting the underlying molecular defect are already in Phase I clinical trials. These designer drugs require that genotype be determined to guide the choice of therapy. Spectrum of defects in CFTR. The CFTR protein is a 1480 amino acid, glycosylated membrane glycoprotein that functions as a cAMP-regulated chloride channel in exocrine glands and secretory epithelia. The nucleotide sequence predicts a structure with two sets of six transmembrane domains (TMD), two nucleotide binding domains (NBDs), and a regulatory (R) domain (1). The NBDs are involved in the gating of the CFTR chloride channel and portions of the TMDs form the pore. The R domain is intracellular and also is involved in gating the channel (2) by a mechanism dependent on its phosphorylation state (3). CFTR is required for normal activity of the outwardly rectifying chloride channel (ORCC), perhaps via CFTR-mediated ATP transport (4). CFTR is also critical to control of the epithelial sodium channel (EnaC) (5). Disruption of CFTR function alters the salt and water content of luminal secretions and impairs airway mucosal defense (6). Most of the reported mutations in CFTR are point mutations involving one or a few nucleotides. They are distributed as follows: missense (40%), nonsense (or termination, 18%), splice-site (18%), frameshift (22%), and other (promoter, in-frame deletions, etc., 2%). Missense and nonsense mutations change a single amino acid or introduce a premature termination site. A splice-site mutation can destroy one or more exons or introduce a novel exon, but because this mutation is leaky, it can permit low levels of the normally spliced mRNA. Frameshift mutations often disrupt the synthesis of the mRNA. Nevertheless, the most useful way to look at genotype in CF is by classification according to the molecular fate. Class I mutations lead to defects in the synthesis of stable CFTR mRNA transcripts resulting in absence of the CFTR protein. About half of all mutations in CFTR (encompassing premature termination, exon skipping, aberrant mRNA splicing, and frameshifts) are thought to fall into this class and result in complete loss of CFTR protein/function. Class II mutations, including ∆F508, complete protein translation but produce an abnormal protein that fails to escape the endoplasmic reticulum. Little or no CFTR reaches the plasma membrane, and the absence of all surface CFTR results in a severe phenotype. It is being increasingly recognized that mutations in unrelated genes can create defective proteins, which fail to traffic properly through the cell. Classically, missense mutations creating an abnormal protein were thought to be relatively benign or less consequential than nonsense mutations (null) or large deletions. This is no longer strictly the case because examples from CF and other inherited disorders demonstrate that a synthesized protein that fails to mature along the normal biosynthetic pathway often becomes quite destructive (7). Class III mutations disrupt activation and regulation of CFTR at the plasma membrane. Thus biosynthesis, trafficking, and processing are undisturbed, but the channel may be defective with respect to ATP binding and hydrolysis, or phosphorylation. Mutations, such as G551D, tend to be associated with a severe phenotype. Class IV mutations affect chloride conductance or channel gating and thus result in reduced chloride current. As might be expected, mutations in this class, such as R117H or P574H, are thought to confer a milder phenotype. Class V mutations reduce the level of normal CFTR protein by alterations in the promoter or by altering splicing. Currently it is thought that a reduction in mRNA to less than 10% of normal results in disease in CF. Examples of Class V mutations include 3849 + 10kb C→T, A455E, and 5T. No classification scheme works perfectly, and the genetics are often complicated. A single missense mutation can be associated with features from two different classes. For example, the ∆F508 CFTR is misprocessed (Class II) but also has a reduced channel open time (Class IV) (8). Many patients have two different mutations in CFTR, a situation termed compound heterozygosity. It is thought that presence of a Class IV or Class V mutation on one allele can ameliorate the consequences of a severe mutation on the other allele, such as ∆F508. Much rarer is the occurrence of two different mutations in a single gene on the same allele. The best example of this is the thymidine tract in intron 8, where 5T and 7T modify disease expression. The Class IV mutaThis series continues from the February 1999, no. 3, issue. See also pages 441–445 in this issue. Perspective