Cystic fibrosis genetics: from molecular understanding to clinical application

Abstract

The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

Key points

• Investigation of disease-causing variants such as F508del is resolving the mechanisms underlying cystic fibrosis transmembrane conductance regulator (CFTR) folding and will inform rational design of compounds to correct the folding of mutant CFTR.
• New tissue culture methods will facilitate the evaluation of molecular targeted therapy for a wide array of CFTR genotypes, and new animal models should enable assessment of treatment at the earliest stages of the disease.
• Analyses of affected twin and sibling pairs have quantified the contribution of genetic and non-genetic modifiers to variation in key features of cystic fibrosis.
• Candidate and genome-wide approaches have identified biologically plausible gene modifiers of lung disease severity, neonatal intestinal obstruction and diabetes in cystic fibrosis.
• Annotation of variants in CFTR will increase the utility of genetic testing in newborn screening, carrier testing and diagnostic settings. Assignment of variants as disease-causing will validate efforts to target variants for molecular therapies.
• Small-molecule therapy for cystic fibrosis has been successful for patients carrying a subset of CFTR variants. Grouping of variants according to responses in cell-based assays (that is, theratypes) could expedite treatment of affected individuals with rare CFTR genotypes.

Introduction

Cystic fibrosis (OMIM 219700) is a life-limiting autosomal recessive disorder that affects ~70,000 individuals worldwide. The condition affects primarily those of European descent, although cystic fibrosis has been reported in all races and ethnicities. Abnormally viscous secretions in the airways of the lungs and in the ducts of the pancreas in individuals with cystic fibrosis cause obstructions that lead to inflammation, tissue damage and destruction of both organ systems (Fig. 1). Other organ systems containing epithelia — such as the sweat gland, biliary duct of the liver, the male reproductive tract and the intestine — are also affected. Loss of pancreatic exocrine function results in malnutrition and poor growth, which leads to death in the first decade of life for most untreated individuals. Replacement of pancreatic enzymes and intensive therapy guided by multidisciplinary teams have revolutionized the treatment of cystic fibrosis, resulting in progressive improvements in survival to a median predicted age of ~37 years for children born with cystic fibrosis today¹. Obstructive lung disease is currently the primary cause of morbidity and is responsible for ~80% of mortality².

 

Figure 1: Cardinal features of cystic fibrosis and relative contribution of genetic modifiers to variation in select cystic fibrosis traits.

       

 

A diagnosis of cystic fibrosis is based on the presence of clinical findings shown on the left, along with an elevated sweat chloride concentration (>60 mM). The degree of organ system dysfunction varies considerably among affected individuals. Genetic modifiers and non-genetic factors both contribute to airway obstruction and infection with Pseudomonas aeruginosa — two traits that define lung disease in cystic fibrosis. Cystic fibrosis transmembrane conductance regulator (CFTR) genotype is the primary determinant of the degree of pancreatic exocrine dysfunction. The presence of CFTR variants associated with severe pancreatic exocrine dysfunction is essentially a pre-requisite for the development of diabetes and intestinal obstruction. In the setting of severe endocrine dysfunction, genetic modifiers determine when, and if, diabetes occurs and whether neonatal intestinal obstruction occurs. Genetic variation plays the predominant part in nutritional status as assessed by body mass index (BMI)70.

 

Twenty-five years ago, a variant (p.Phe508del; also known as F508del in legacy nomenclature) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was found to be the most common cause of cystic fibrosis^{3, 4, 5}. Demonstration that CFTR functions as a chloride channel regulated by cyclic AMP (cAMP)-dependent phosphorylation⁶ was consistent with the ion transport disturbances documented in cystic fibrosis tissues^{7, 8}. Key insights into cystic fibrosis pathophysiology were derived from the study of CFTR mutants⁹, correlation of CFTR dysfunction with the cellular manifestations of cystic fibrosis¹⁰, and elucidation of protein partners involved in biogenesis and membrane function¹¹. Identification of disease-causing variants in CFTR contributed a tool for both the diagnosis of cystic fibrosis and the identification of cystic fibrosis carriers¹², demonstrated the degree to which CFTR dysfunction correlates with clinical features¹³, and revealed that CFTR dysfunction can create phenotypes other than cystic fibrosis¹⁴. Over the past 5 years, there has been remarkable progress in the development of small-molecule therapy targeting CFTR bearing select disease-causing variants^{15, 16}.

 

The purpose of this Review is to highlight advances over the past decade in our understanding and treatment of cystic fibrosis that were informed by genetics. Given the breadth of the cystic fibrosis field, not all of the important contributions and publications relevant to the topic can be included. Examples have been chosen to illustrate that genetics continues to have a role in the research of Mendelian disorders long after the causative variants and the responsible gene have been discovered. This Review covers new insights into the processing defect caused by the F508del variant, advances in stem cell technology that can enable testing of therapeutics for a wide range of CFTR genotypes and the development of new animal models that are informing our understanding of organ pathology in cystic fibrosis. I also summarize progress in parsing genetic and non-genetic contributions to variability in cystic fibrosis and in the identification of modifier loci. The final section describes efforts to determine the molecular and phenotypic consequences of the majority of cystic fibrosis-causing variants and to develop molecular treatments for every defect in CFTR.

 

Figure 2: Molecular consequences of variants in CFTR.

       

 

The degree to which epithelial ion transport is altered in an individual with cystic fibrosis is determined by the effect of each disease-causing variant on the quantity and the function of cystic fibrosis transmembrane conductance regulator (CFTR). The key steps of CFTR biogenesis in an epithelial cell are depicted. The membrane-spanning domains of CFTR are shown as red boxes, the two nucleotide-binding domains as yellow circles, and the regulatory domain as a blue circle. The quantity of CFTR protein in the apical cell membrane is a product of the amount of RNA transcribed, the efficiency of RNA splicing, the fraction of protein correctly folded and the stability of the protein in the membrane. The level and/or content of CFTR transcripts can be affected by disease-causing variants in the promoter (for example, c.−234TA (also known as −102TA in legacy nomenclature))¹⁴⁰ and splice sites (for example, c.3717 + 12191 CT (legacy 3849 + 10 kb CT))¹⁴¹, or by variants that introduce a premature termination codon (PTC) and that lead to RNA decay (for example, p.Gly542X; (legacy G542X)¹⁴². The processing of CFTR can be altered by variants that cause aberrant folding of the protein, leading to degradation (for example, p.Phe508del (legacy F508del))¹⁸, or by variants that cause reduced membrane stability as a result of increased rates of endocytosis (for example, p.Asn287Tyr (legacy N287Y))¹⁴³. The function of CFTR is dependent on activity of the ion channel and on the efficiency of conductance of ions through the channel. Disease-causing variants cause reduction in activity (for example, p.Gly551Asp (legacy G551D)¹⁴⁴ or changes in the conduction properties of the chloride channel (for example, p.Arg334Trp (legacy R334W))¹⁴⁴. cAMP, cyclic AMP; ER, endoplasmic reticulum.

 

Figure 3: Molecular treatments for cystic fibrosis.

       

 

Conclusions

The discovery of CFTR 25 years ago was a triumph for genetics and a potent demonstration of its ability to deliver the molecular culprit in a Mendelian disorder. Cystic fibrosis is now positioned to reap the dividends of personalized medicine as variant-specific therapy is deployed, and a growing understanding of the genetic and environmental modifiers of cystic fibrosis enables targeting of individual risk factors. The development of new genetic models of cystic fibrosis in pigs, ferrets, rats and zebrafish provides opportunities to investigate pathophysiology and to explore therapies at the earliest stages of disease. Newborn and population screening enables prospective management of affected individuals from birth, and genomic variation will provide information on the trajectories that individual patients are likely to follow. Genetics has played and will continue to play a key part in achieving a normal lifespan for individuals with cystic fibrosis.

 

Nature Reviews Genetics | Review

Article series: Disease mechanisms

 

Garry R. Cutting^1, 

 

Published online 18 November 2014

Corrected online 20 November 2014