Lung disease modifier genes in cystic fibrosis.

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Review

Lung disease modifier genes in cystic fibrosis夽 Loic Guillot a,b,∗,1 , Julie Beucher c,1 , Olivier Tabary a,b , Philippe Le Rouzic a,b , Annick Clement a,b,d , Harriet Corvol a,b,d a

INSERM, UMR S 938, CDR Saint-Antonie , Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR s 938, CDR Saint-Antonie, Paris, France Centre Hospiyalo-Universitaire (CHU), Rennes, France d Hôpital Trousseau, Pediatric Respiratory Department, AP-HP, Paris, France b c

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Cystic fibrosis CFTR Lung Physiopathology Modifier genes

a b s t r a c t Cystic fibrosis (CF) is recognized as a single gene disorder. However, a considerable diversity in its clinical phenotype has been documented since the description of the disease. Identification of additional gene alleles, so called “modifier genes” that directly influence the phenotype of CF disease became a challenge in the late ‘90ies, not only for the insight it provides into the CF pathophysiology, but also for the development of new potential therapeutic targets. One of the most studied phenotype has been the lung disease severity as lung dysfunction is the major cause of morbidity and mortality in CF. This review details the results of two main genetic approaches that have mainly been explored so far: (1) an “a priori” approach, i.e. the candidate gene approach; (2) a “without a priori” approach, analyzing the whole genome by linkage and genome-wide association studies (GWAS), or the whole exome by exome sequencing. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Candidate gene approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Candidate genes involved in the inflammatory response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Others inflammatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Candidate genes involved in the infectious response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Soluble mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Candidate genes involved in epithelial tissue damage and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Glutathion and Glutathion-S-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Nitric oxide synthases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Candidate genes involved in the pharmacogenetic response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. ␤2-adrenergic receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Glucocorticoid receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whole genome and exome studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Linkage and genome wide association studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Exome wide association studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

夽 This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. ∗ Corresponding author at: Inserm U938, Cystic Fibrosis: Physiopathology and Phenogenomics, Hôpital Saint-Antoine, Bâtiment Kourilsky, 34 rue Crozatier, 75012 Paris, France. Tel.: +33 1 49 28 46 82; fax: +33 1 43 40 17 48. E-mail address: [email protected] (L. Guillot). 1 LG and JB should be considered as first-authors. http://dx.doi.org/10.1016/j.biocel.2014.02.011 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Guillot L, et al. Lung disease modifier genes in cystic fibrosis. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.02.011

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1. Introduction Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disease in Caucasian populations. Clinical manifestations are caused by an impaired function of exocrine glands in many organs, mainly the respiratory and the gastrointestinal tracts. This genetic disorder is mostly characterized by recurrent lower respiratory infections, exocrine pancreatic insufficiency, and increased electrolyte concentration in sweat. A subset of patients also presents with a variety of other less common symptoms, such as meconium ileus, liver disease, diabetes, and pancreatitis (Robinson, 2001). A great deal has been learned about the physiopathology of CF since the cloning of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene in 1989 (Riordan et al., 1989). The 230 kb CFTR gene with 27 exons located at 7q31.3 encodes a 1480 amino acid protein that forms a cAMP-regulated chloride channel in lipid bilayers of the apical membrane of epithelial cells lining the respiratory and digestive tracts. Although CFTR is primarily a chloride channel, it also controls the transport of other ions and molecules as well as the function of several membrane channels, such as the epithelial sodium channel, potassium channels and outwardly rectifying chloride channels (Sagel and Accurso, 2002; Sheppard and Welsh, 1999; Wine, 1999). Consequently, CFTR regulates the hydration of the airways’ surface fluid. Other cell functions are modulated by CFTR including intravesicular acidification, activation of lysozymal enzymes, endocytic cycling, apoptosis, gap junction communication, chemokine production and activation of NF-␬B. Over 1900 mutations in the CFTR gene have so far been identified and usually classified in different classes according to their molecular pathology: class 1, altered CFTR production; class 2, defective intracellular trafficking of CFTR resulting in failure of the protein to reach the apical membrane; class 3, altered CFTR regulation, class 4, altered CFTR conduction; class 5, altered CFTR-mRNA stability; class 6, altered stability of the mature-CFTR protein. Class 1–3 mutations are most common and are usually associated with pancreatic insufficiency. The most frequent CFTR mutation, known as p.Phe508del (F508del), accounting for around 66% of the mutations worldwide, is a class 2 mutation caused by a 3-bp deletion (c.1521 1523delCTT) of CFTR. Class 4–6 mutations are usually associated with milder phenotypes (Kerem et al., 1989). These mutations of CFTR lead to excessive and uncontrolled lung inflammation that is sustained by chronic infection and resulting finally in tissue damage. In fact, lung inflammation characterized by high numbers of neutrophils in the airways as well as inflammatory mediators such as IL-8, failed in bacterial clearance (Cohen and Prince, 2012). Although CF is recognized as a single gene disorder, a considerable diversity in its clinical phenotype has been documented since the description of the disease. The hypothesis that variation in phenotype can be explained by allelic heterogeneity has been put forward, and many studies have attempted to link the type of CFTR mutations to the severity of the disease. Good genotype–phenotype correlations have been reported for pancreatic enzyme function (Kerem and Kerem, 1995, 1996). Patients with two “severe” mutant alleles are mostly pancreatic enzyme insufficient (PI) and patients with one or two “mild” mutations are typically pancreatic sufficient (PS). In the absence of CF neonatal screening, PS patients are usually diagnosed at a later age and are found to have lower sweat chloride levels, and better survival. However, analysis of the effects of CFTR mutations on pulmonary function, which represents the major cause of morbidity and mortality in CF, has failed to establish a strong genotype–phenotype correlation (McKone et al., 2003). Indeed, among patients who are homozygous for the F508del mutations, striking variations in the progression of lung disease

are observed, suggesting that other environmental and genetic factors influence the clinical phenotype. Interestingly, the discordant phenotypes observed in siblings with CF carrying the same CFTR mutations argue against a major role of the environment in disease expression, and provide support for the search of CF modifier genes (Bronsveld et al., 1999, 2000, 2001; Cutting, 2010; Mekus et al., 1998, 2000, 2003). It should be pointed out that relatively common genetic variations, with little or no overt phenotypic effect on the general population, can have significant effect in the context of CF. Identification of additional gene alleles that directly influence the phenotype of CF disease became a challenge in the late ‘90ies, not only for the insight it provides into the CF pathophysiology, but also for the development of new potential therapeutic targets. One of the most studied phenotype has been the lung disease severity as lung dysfunction is the major cause of morbidity and mortality in CF. Two genetic approaches have mainly been explored so far: (1) an “a priori” approach, i.e. the candidate gene approach, where the genes selected for investigation are identified from the current understanding of CF lung disease pathophysiology; (2) a “without a priori” approach, analyzing the whole genome by linkage and genome-wide association studies (GWAS), or the whole exome by exome sequencing. This review details the results of these several approaches. Whenever available the authors compare their own results from the French modifier consortium to literature data. 2. Candidate gene approach In the candidate-gene approach, the genes selected for investigation are identified from the current understanding of CF lung disease pathophysiology. A complex network of mechanisms is likely to be involved in the progression of the disease. An exaggerated, sustained, and extended inflammatory response to bacterial and viral pathogens, characterized by neutrophil dominated airway inflammation, is one of the main feature of the CF lung disease. In addition, decreased mucociliary clearance, viscous secretion, and altered ion transport are all thought to contribute to respiratory tract colonization by opportunistic species of bacteria such as Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. Aureus) and Aspergillus fumigatus (A. fumigatus). The overwhelming inflammatory response, together with the chronic infection, lead to the destruction of the airways. Overall, this explains the choice of the candidate genes involved in the inflammatory and infectious responses, the epithelial tissue damage and repair, the pharmacogenetic response, the ion transports and the cytoskeleton (Table 1 and Fig. 1). In Table 1, are listed all the candidate genes studied in association with lung disease severity, meanwhile the most relevant are detailed below. 2.1. Candidate genes involved in the inflammatory response 2.1.1. Cytokines 2.1.1.1. Transforming Growth Factor B1 (TGFB1). The TGF␤1 protein, encoded on chromosome 19q13.1-q13.3, is a member of growth and differentiation factors family. It is a multifunctional cytokine which regulates the proliferation and the differentiation of a wide variety of cell types. High levels of TGF␤1 have been described in BAL fluid from CF patients with severe lung disease (Bonfield et al., 1995; Schwarz et al., 2003). Animal and human studies have shown that high TGF␤1 production was associated with lung fibrosis development in response to various inflammatory mediators (El-Gamel et al., 1999; Liu et al., 2001). Polymorphic variants of TGFB1 have been involved in various respiratory diseases such as pulmonary fibrosis, asthma and chronic obstructive pulmonary disease (COPD) (Celedon et al., 2004; Li et al., 2007). In CF, several TGFB1 variants have been


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Table 1 Candidate genes studied in association with CF lung phenotype. P. a: Pseudomonas aeruginosa. FEV1 : Forced Expiratory Volume in 1 second. Pathways

Genes

Phenotypes

References

Inflammatory response

TGFB1 IL8 IL1B

See Table 2 ↑Airway colonization/↑Lung severity =/increased rate of FEV1 decline

IL10

↑Airway colonization/= FEV1

TNF

=/↓ FEV1

FAS 8.1AH

↑Infection susceptibility ↑Infection susceptibility/↓FEV1

SERPINA1 SERPINA3 MIF

See Table 3 ↓Infection susceptibility/↑ FEV1 ↓Infection susceptibility/slower rate of FEV1 decline ↑Infection susceptibility ↑hyperinflation, small airway dysfunction ↑Lung severity ↑Lung severity ↑Lung severity See Table 4 No association

See Table 2 Corvol et al. (2008) and Hillian et al. (2008) Corvol et al. (2008), Labenski et al. (2011) and Levy et al. (2009) Brouard et al. (2005), Corvol et al. (2008) and Tesse et al. (2008b) Arkwright et al. (2003), Buranawuti et al. (2007), Corvol et al. (2008), Drumm et al. (2005), Schmitt-Grohe et al. (2006), Stanke et al. (2006) and Yarden et al. (2005) Kumar et al. (2008) Aron et al. (1999a,b), Corvol et al. (2012) and Laki et al. (2006) See Table 3 Mahadeva et al. (2001) Adamali et al. (2012) and Plant et al. (2005)

FcRIIA SNAP23

Infectious response

Tissue damage and repair

ACE EDNRA AGER MBL2 TLR4/TLR5 CD14 SFTPA2 DEFB1 CFB, C3 GSTs NOS1/NOS3

Pharmacogenetic response

Ion Transport

Cytoskeleton

HMOX1 FUT2/FUT3/ABO/secretor/lewis PPP2R4 ˇ2 AR GR CLCN2 SLC9A3 ABCC1 KRT8

↑Infection susceptibility ↑FEV1 Chronic colonization No No association/worse overall clinical status/↑ lung severity Slower rate of FEV1 decline, ↑P. a colonization/↓P. a colonization ↑Lung severity No Increased FEV1 decline =/↑Lung severity Increased FEV1 decline No association Earlier acquisition of P. a infection/↓ FEV1 Earlier acquisition of P. a infection ↑CF disease severity

studied with conflicting findings illustrated in Table 2 (Arkwright et al., 2000, 2003; Brazova et al., 2006; Bremer et al., 2008; Corvol et al., 2008; Drumm et al., 2005; Faria et al., 2009). The main variants that have been investigated were within the promoter (−509T/C) and within the exon 1: 869T/C (Leu10 /Pro10 ) and 915G/C (Arg25 /Pro25 ). It has also been shown that the variations related to TGFB1 genotypes and lung CF phenotype could be influenced by environmental factor such as second hand smoke exposure (Collaco et al., 2008). Regarding these conflicting results, it appears that TGFB1 might be an important modifier of the CF lung phenotype with variations that might be linked to population ancestry and environmental factors (Weiler and Drumm, 2013).

De Rose et al. (2005) Gisler et al. (2013) Marson et al. (2012a) Darrah et al. (2010) Beucher et al. (2012) See Table 4 Park et al. (2011), Urquhart et al. (2006)/Blohmke et al. (2010) Faria et al. (2009) and Martin et al. (2005) Choi et al. (2006) Tesse et al. (2008a) Park et al. (2011) Feuillet-Fieux et al. (2009), Flamant et al. (2004), Hull and Thomson (1998) and McKone et al. (2006) Texereau et al. (2004), Grasemann et al. (2000)/Grasemann et al. (2003a,b) Park et al. (2011) Taylor-Cousar et al. (2009) Gisler et al. (2013) Buscher et al. (2002), Marson et al. (2012b) and Steagall et al. (2007) Corvol et al. (2007) Blaisdell et al. (2004) Dorfman et al. (2011) Mafficini et al. (2011) Stanke et al. (2011)

2.1.1.2. Interleukine 8 (IL8). IL8 (CXCL8) is a member of the CXC chemokine family, encoded on 4q13.3. It is a powerful neutrophilic chemoattractant, well-known to perpetuate an exuberant neutrophilic inflammation in the CF lung. IL8 gene is located on chromosome 4q13-21. Hillian et al. have shown that three polymorphisms within this gene were associated with CF pulmonary disease severity: rs4073 (IL8 −251T/A), rs2227306 (IL8 781C/T), and rs2227307 (IL8 396T/G) (Hillian et al., 2008). We further observed an association between these 3 IL8 variants and chronic P. aeruginosa airway colonization, although statistical significance was not maintained after correction for multiple testing (Corvol et al., 2008).

Table 2 TGFB1 gene variants associated with CF lung phenotype. FEV1 : Forced Expiratory Volume in 1 second. TGFB1 variants

CF patients (CFTR genotype)

Phenotypes

References

c.869T c.915C c.869C and −509T c.869C and c.915C Haplotype [−509C; intron 5 C; 869T]: 869TC heterozygotes: 869TC heterozygotes:

171 (F508del) 261 (various) 808 (F508del) 118 (various) 472 trios (various) 329 (various) 105 (various)

Increased rate of FEV1 decline More severe lung disease More severe lung disease No association with lung disease severity ↑FEV1 Increased rate of FEV1 decline ↑FEV1

Arkwright et al. (2000) Arkwright et al. (2003) Drumm et al. (2005) Brazova et al. (2006) Bremer et al. (2008) Corvol et al. (2008) de Faria et al. (2009)


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Fig. 1. Candidate modifier genes related to CF-associated physiopathological mechanisms. Genes are clustered in the following boxes: Inflammatory response, Infectious response, Tissue damage and repair, Pharmacogenetic response, and Ion transport. TLR, Toll-Like Receptor; MBL, Manose-Binding Lectin; IL, Interleukin; TNF, Tumor Necrosis factor; TGF, Transforming Growth Factor; AAT, alpha-1 antitrypsin; ACE, Angiotensin Converting Enzyme; MIF, Macrophage Migratory Inhibitory Factor; EDNRA, endothelin receptor type A; GC-RC; Glucocorticoid (GC) receptor; GSH: Glutathion; NOS, Nitric Oxyde synthases; CLCN2: Chloride Channel 2; ABCC1: ATP-Binding Cassette, Subfamily C, Member.

2.1.1.3. Interleukine 1 B (IL1B). IL1B gene, located on 2q13, encodes for a cytokine mainly produced by blood monocytes, which mediates the acute phase response host reactions. We studied two IL1B SNPs: rs1143627 and rs16944 (respectively IL1B −31T/C and IL1B −511C/T) (Corvol et al., 2008), and found no association with CF lung disease severity (Corvol et al., 2008). However, in 2009, Levy et al. reported an association with other IL1B variants in a cohort of 808 CF patients, and replicated their results in a case–control analysis of 126 trios (Levy et al., 2009). They showed that IL1B variants rs1143634 (exon 5) and rs1143639 (intron 6) were overrepresented among patients with a severe CF lung disease course, associated with poorer lung function, but not with P. aeruginosa colonization. These findings were further confirmed in a European family-based study (Labenski et al., 2011). 2.1.1.4. Interleukine 10 (IL10). IL10 gene, located on chromosome 1q32, encodes for an interleukin recognized to be involved in the altered pulmonary inflammatory and infectious response in CF (Noah et al., 1997; Osika et al., 1999). A promoter polymorphism at position −1082G/A (rs1800896) has been reported to modulate IL10 production: IL10 levels were indeed either increased or decreased in the presence of, respectively, the −1082G or the −1082A allele (Turner et al., 1997). We observed that the ‘high producer genotype’ −1082GG was associated with the occurrence of ABPA (allergic bronchopulmonary aspergillosis) and the airway colonization with A. fumigatus (Brouard et al., 2005). Moreover, higher

IL10 serum levels were documented in patients with A. fumigatusrelated lung disease compared to the patients without. However, no influence of IL10 genotypes was observed for colonization with P. aeruginosa. Despite these findings, no association could have been shown with lung function for this variant or for other IL10 variants (Corvol et al., 2008; Tesse et al., 2008b). 2.1.1.5. Tumor Necrosis Factor alpha (TNF-˛). The TNF gene, located on chromosome 6p21.3, encodes for a pro-inflammatory cytokine secreted by macrophages, lymphocytes and airway epithelial cells in response to inflammatory and infectious stimuli. TNF-␣ is a neutrophils’ chemoattractant contributing to the exuberant neutrophilic-dominated inflammation of CF airways. Moreover, lung function and sputum TNF-␣ levels have been shown to be negatively correlated in CF (Greally et al., 1993). TNF polymorphisms have been associated with several respiratory diseases such as asthma and COPD (Castro-Giner et al., 2008). In CF, several variants have been studied by different teams, either within the promoter (−851C/T, −308G/A, −238G/A) or within the intron 1 (691G ins/del), with variable findings. Among those, the −308G/A variant has been extensively explored in several respiratory diseases and has also been shown to modulate TNF-␣ production (Kaluza et al., 2000). Hull et al. provided evidence of a negative association between TNF-308A allele and CF clinical status (Hull and Thomson, 1998), but their results were not further replicated (Arkwright et al., 2003; Corvol et al., 2008; Drumm et al.,




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Table 3 SERPINA1 gene variants associated with CF lung phenotype. P. a: Pseudomonas aeruginosa. FEV1 : Forced Expiratory Volume in 1 second. NS: not studied. Hom: homozygous. Het: heterozygous. SERPINA1 variants


Phenotypes

References

S and Z (deficient alleles)

215 patients (F508del hom/het)

Earlier age of onset of P. a infection No association with FEV1 NS ↑FEV1 NS NS Fewer infective exacerbations Less proportion of P. a colonization No association with age of onset of P. a infection NS NS ↑FEV1 No association No association No association No association

Doring et al. (1994)

1237 G>A (enhancer allele) S and Z 1237 G>A S and Z 1237 G>A S and Z 1237 G>A S and Z 1237 G>A S and Z 1237 G>A S and Z 1237 G>A

157 (various)

124 (various) 269 (F508del hom/het)

320 (various) 716 (various) 808 (F508del)

2005; Schmitt-Grohe et al., 2006). Yarden et al. studied the 4 TNF polymorphisms introduced above in 180 F508del homozygous CF patients (Yarden et al., 2005). They found that the +691Gdel allele was more likely to be associated with better pulmonary function and later onset of infection with P. aeruginosa. They also observed that the genotype −851CC was associated with an increased lung disease severity. Finally, the TNF −238G/A variant has been associated with survival by affecting other life-limiting manifestations (Buranawuti et al., 2007; Stanke et al., 2006). 2.1.2. Others inflammatory genes 2.1.2.1. HLA system. The complex inheritance of lung disease severity in CF may involve block of genes, namely haplotypes. Among them, one conserved haplotype, the 8.1 ancestral haplotype (8.1AH), encoded in the major histocompatibility complex (MHC) region on the short arm of chromosome 6, is highly prevalent in Caucasians, and comprises a cassette of linked alleles that play key roles in the inflammatory response, such as the TNF block. In CF, Laki et al. showed that the 8.1AH was associated with a delayed onset of respiratory colonization with S. aureus and P. aeruginosa (Laki et al., 2006). Recently, we reported that 8.1AH was also associated with CF lung function; the 8.1AH carriers having a significant poorer lung function, with −6.4% of FEV1 compared to the non-carriers, that was a substantial effect given the high prevalence of the haplotype (Corvol et al., 2012). Another haplotype in the MHC complex, the DR7/DQA*0201, has been associated with increased levels of IgE and frequency of P. aeruginosa colonization in CF (Aron et al., 1999b). In CF, as well as in asthma, HLA-DR4 and DR7 alleles were strongly associated with ABPA (Aron et al., 1999a). 2.1.2.2. Plasma serine protease inhibitors Alpha-1 antitrypsine (AAT). The SERPINA1 gene, located on 14q32.13, encodes the alpha-1 antitrypsine (AAT), a major plasma serine protease inhibitor. Its main inhibitory effect concerns the neutrophil elastase, which degrades the alveolar walls’ elastin. In the lung, AAT deficiency results in lung diseases such as emphysema, asthma and/or bronchiectasis (Eriksson, 1965; King et al., 1996). In CF, high levels of neutrophil elastase have been described in the airways, with an imbalance between AAT and elastase (O’Connor et al., 1993). Three SERPINA1 variants have been studied so far in CF: the Z and S deficiency alleles (both resulting in decreased AAT plasma levels) and the 1237 A/G variant in the 3 enhancer region (modifying the AAT regulation mediated by IL6 during infection (Morgan et al., 1997)). Conflicting results have been shown, summarized in Table 3 (Courtney et al., 2006; Doring et al., 1994; Drumm et al., 2005; Frangolias et al., 2003; Henry et al., 2001; Mahadeva et al.,

Mahadeva et al. (1998b) Henry et al. (2001)

Meyer et al. (2002) Courtney et al. (2006) Frangolias et al. (2003) Drumm et al. (2005)

1998b; Meyer et al., 2002). In brief, one study reported an association between Z and S deficiency alleles and age of onset P. aeruginosa lung infection (Doring, 1994), but has not be further replicated (Meyer et al., 2002). This SNP has also been associated with a better lung function (Mahadeva et al., 1998a,b), but these results were also not replicated (Drumm et al., 2005; Frangolias et al., 2003). Similarly, whereas some authors reported that the 1237 A/G variant could have been protective (Courtney et al., 2006; Henry et al., 2001); other did not show any evidence of association (Drumm et al., 2005; Frangolias et al., 2003). Interestingly, the studies that have investigated the largest cohorts did not found any association of these 3 variants with CF pulmonary disease severity (Drumm et al., 2005; Frangolias et al., 2003). 2.1.2.3. AGER. RAGE (receptor for advanced glycation endproducts) is a member of the cell surface receptor immunoglobulin superfamily and a pro-inflammatory mediator highly expressed in the lung. It is encoded by a gene named AGER for which several variants have been described, and among which the promoter AGER −429T/C. We have recently shown in a large cohort of 967 F508del homozygous patients from the French Gene Modifier Genes study that this AGER −429T/C variant was associated with CF lung disease severity and was further able to modulate RAGE expression in vitro (Beucher et al., 2012). 2.2. Candidate genes involved in the infectious response 2.2.1. Soluble mediators 2.2.1.1. Mannose-binding lectin 2. The mannose-binding lectin (MBL) 2 protein, encoded on chromosome 10q11.2-q21, is an innate immune protein produced by the liver that accumulates in the lung during acute inflammation, plays a major role in infectious disorders, and binds to several bacteria among which S. aureus and P. aeruginosa. Its gene displays one particular haplotype that has been extensively studied within the exon 1 (at codons 52, 54 and 57). This haplotype is classified as “MBL2-A” if all 3 variants are wild-type and “MBL2-O” if at least one of these 3 variants is not wild-type. Mutant proteins are not able to form high-order oligomers and possess shortened half-lives. Individuals with two mutations (O/O) have dramatically reduced levels of MBL, whereas intermediate levels are observed in heterozygotes (Garred et al., 2006). In CF, different teams studied the role of MBL2 deficiency alleles on lung disease severity, especially investigating their effects on the infectious status and the lung function (Table 4) (Buranawuti et al., 2007; Davies et al., 2004; Dorfman et al., 2008; Gabolde et al., 1999; Garred et al., 1999; Muhlebach et al., 2006; Yarden et al., 2004). Although conflicting, majority of these studies confirmed




6

Table 4 MBL2 gene variants associated with CF lung phenotype. P. a: Pseudomonas aeruginosa. FEV1 : Forced Expiratory Volume in 1 second. MBL2 variants


Phenotypes

References

rs1800450 (A/A), rs1800451 (A/0), rs5030737 (0/0) rs1800451 rs1800450 rs5030737

149 (various)

Increased rate of FEV1 decline, ↓survival

Garred et al. (1999)

298 (various) 22 (F508del hom) 179 (F508del hom) 149 (various) 254 patients (various) 808 (F508del hom) 112 (various) 1019 (various)

↓FEV1 ↓FEV1 ↓FEV1 especially after 15 years old ↓survival ↓survival No association with FEV1 Earlier P. a infection and ↓FEV1 Earlier P. a acquisition Increased rate of FEV1 decline among adult patient ↑ rate of death or requirement for lung transplantation No association with FEV1 Decrease rate of FEV1 decline No association with FEV1

Davies et al. (2004) Gabolde et al. (1999) Yarden et al. (2004) Muhlebach et al. (2006) Buranawuti et al. (2007) Drumm et al. (2005) Carlsson et al. (2005) Dorfman et al. (2008)

rs5030737 rs1800450

rs7096206 (−221GT SNP on lung function has been observed so far in either pediatric or adult CF patients (Blohmke et al., 2010). 2.2.2.2. CD14. The CD14 gene is located on chromosome 5q31.1. It encodes a protein expressed on white blood cells membranes

Chalmers et al. (2011) Olesen et al. (2006) de Faria et al. (2009)

and involved in the immune and inflammatory responses as it acts as a receptor for lipopolysaccharides, components of the gramnegative bacteria membrane, among which P. aeruginosa. One CD14 promoter gene variant (−159C/T) has been associated with lower circulating CD14 levels in healthy children (Alexis et al., 2001). Martin et al. showed in 45 CF children that the −159CC carriers acquired earlier P. aeruginosa infection (Martin et al., 2005). Moreover, children free of P. aeruginosa had significantly higher plasma CD14 levels, compared to infected children. Further, in a cohort of 105 CF patients compared to controls, higher frequency of the CD14 −159TT genotype were observed among the CF patients; not associated with lung disease severity (de Faria et al., 2009). Other candidate genes involved in the infectious response that have been studied as modifiers in CF are summarized in Table 1.

2.3. Candidate genes involved in epithelial tissue damage and repair 2.3.1. Glutathion and Glutathion-S-transferase Glutathion (GSH) is an ubiquitous tripeptide that plays a key role in the protection of the lung from oxidant-induced lung damage. CFTR deficiency induce decreased GSH transport which leads to a systemic deficiency of GSH in patients with CF (Hudson, 2001). Glutathione-S-transferase (GST) is a supergene family composed of different classes of detoxifying enzymes which catalyze the conjugation of a wide variety of electrophiles and xenobiotics with GSH. Therefore, alterations in GST activities are likely to have a major influence on the ability of cells to resist oxidative stress. GSTs are differentially expressed and active in the various organs; GSTP1 and M3 being the most abundant in the lung, followed by GSTM1 and T1. The largest study investigating the effect of GSTs variants on lung function in CF did not find any association for either GSTM1, GSTP1 or GSTT1 genotypes (Feuillet-Fieux et al., 2009). Hull et al. did however proposed an association of GSTM1 0/0 alleles and a worse overall clinical status in CF (Hull and Thomson, 1998); and our team reported that one GSTM3 variant that consisted on a three-base pair deletion was associated with an increased CF lung disease severity (Flamant et al., 2004). Another GSH down-regulation mechanism is the ␥-glutamylcysteine formation, catalyzed by the glutamatecysteine ligase (GCL). One variant of the GCL, a GAG trinucleotide repeat (TNR), has been shown to be associated with variable GSH levels in vitro: 7 GAG TNR being associated with low GSH levels, 8 GAG TNR with intermediate levels, and 9 GAG TNR with high levels. McKone et al. investigated whether this variant was associated with lung function in a cohort of 440 CF patients with various CFTR mutations; and observed an association only for the CF patients carrying




mild CFTR mutations (McKone et al., 2006). There is indeed a moderate amount of CFTR expression in mild-CFTR mutated patients; so, genetic variation in GCL that leads to improve the oxidant defense via CFTR may lead to a milder CF phenotype. 2.3.2. Nitric oxide synthases Nitric oxide is synthesized from l-arginine by nitric oxide synthases (NOS). The three known NOS isoforms (1, 2 and 3) are expressed in the respiratory tract. Although exhaled levels of airways NO (FENO) are increased in inflammatory lung diseases such as asthma or bronchiectasis, they are decreased in CF (Dotsch et al., 2002). NOS1 gene, located on chromosome 12q24, contains an intronic AAT repeated polymorphism that has been shown to modulate FENO levels (Wechsler et al., 2000). It has been associated with a lower risk of P. aeruginosa and A. fumigatus colonization (Grasemann et al., 2000, 2002) and with milder overall lung disease progression in CF (Texereau et al., 2004). NOS3 gene, located on chromosome 7q35-36, contains a G/T polymorphism at position 894G/T in exon 7 (Marsden et al., 1993). Grasemann et al. did not find any differences in FEV1 or P. aeruginosa colonization frequency between the NOS3 genotypic groups in their overall CF cohort. However, the sub analysis of the females revealed that the 894T carriers had higher FENO, a decreased risk for P. aeruginosa airways colonization, and tend to have better FEV1 (Grasemann et al., 2003b). Other candidate genes involved in tissue damage and repair that have been studied as modifiers are summarized in Table 1. 2.4. Candidate genes involved in the pharmacogenetic response 2.4.1. ˇ2-adrenergic receptors ␤2 -adrenergic receptors (␤2 AR) are expressed on airway smooth muscle cells and colocalized with CFTR at the apical membrane. It was shown in vitro that stimulation of the ␤2 -AR lead to an increased expression of CFTR (Taouil et al., 2003). Binding of endogenous catecholamines or exogenous ␤2 -agonists (such as salbutamol or albuterol) stimulate the ␤2 AR and lead to the bronchodilation (Weiss et al., 2006). ˇ2 AR gene maps to 5q31-q32 and is highly polymorphic with over 55 single-nucleotide polymorphisms (SNPs) recorded in public databases (Reihsaus et al., 1993). Several of these SNPs alter the protein structure by causing a change in amino acid: Arg16Gly, Arg19Cys, Glu27Gln, Val34Met, and Thr164Ile (Hall, 2006). They have been extensively studied in asthma to search for pharmacogenetic associations between genetic variants and the bronchodilator response (Corvol and Burchard, 2008). In CF, several teams investigated the association of both variants Arg16Gly and Glu27Gln and airway responsiveness; observing either a significant association (Buscher et al., 2002; Marson et al., 2012b) or not (Buscher et al., 2002; Steagall et al., 2007). Frequencies of the variants were also shown to be higher than in controls, in an age-dependent manner, higher in adults compared with children (Buscher et al., 2002; Steagall et al., 2007). Recently, Arg16Gly has further been associated with lung disease severity (Marson et al., 2012b). 2.4.2. Glucocorticoid receptor Variations in glucocorticoid sensitivity have been reported to be associated with single nucleotide polymorphisms in the glucocorticoid receptor (GR) gene. Among them, a GR polymorphism in exon 2 (N363S), which alters the N-terminal transactivation domain, was described to be associated with glucocorticoid hypersensitivity. Another polymorphism in exon 2, which comprises 2 point mutations in codons 22 and 23, has been linked to a decreased response to dexamethasone. In the 5 untranslated region, the TthIII polymorphism has been associated with basal cortisol secretion and, in intron 2, the BclI polymorphism, with an increased sensitivity

7

to corticosteroids. We studied these 4 polymorphisms in 255 CF patients and observed that the BclI polymorphism was associated with faster lung disease progression: patients carrying the BclI GG genotype had a more pronounced rate of FEV1 decline (Corvol et al., 2007). For the TthIII, ER22/23EK and N363S variants, analyzes of FEV1 or FVC data revealed no significant difference between the different genotypes.

3. Whole genome and exome studies 3.1. Linkage and genome wide association studies As described above, for more than a decade, gene modifiers in CF were looked for using candidate gene approaches. Early studies were hampered by small sample size and poor study design, yielding mixed results. To overcome these limitations, collaborations were set up in North-America and France. The advent of largescale genomic studies, where several thousand polymorphisms could be measured at once, allows indeed an “uninformed” look at phenotype/genotype correlation. The North-American and the French consortiums for CF gene modifiers have both gone along this direction, typing the genome of several thousand patients. These two separate approaches have proved complementary, allowing (1) large sample size to allow adjusting for the several hundred thousand statistical tests carried out (Sun et al., 2012); and (2) validation in independent cohorts before the results can be accepted. In North-America, 3 large studies have been underway: (1) Johns Hopkins University, Baltimore (G. Cutting: twin/sib, familybased design), (2) UNC/CWRU, North Carolina (M. Knowles, M. Drumm: case–control, extremes-of-phenotype design); and (3) Toronto, Canada (P. Durie: study of a large fraction of all Canadian CF patients). They reported heritability and/or modifiers of lung and other phenotypes using standardized methodologies (Vanscoy et al., 2007). Six years ago, these groups amalgamate to form a large consortium and focused on two genome-wide approaches: linkage and association analyses. The linkage analysis program (G. Cutting) seeks to identify regions of the genome shared among siblings with similar levels of lung disease severity but not shared in siblings with different degrees of lung disease severity. A pilot linkage study in 350 CF sibling pairs and their parents showed evidence of linkage with lung function on chromosomes 5, 12 and 14 (Farrell et al., 2008). The second North-American approach is a GWAS, coordinated by M. Knowles, M. Drumm and P. Durie. They observed interesting signals within 6 DNA regions in a pilot GWAS realized in 320 CF patients homozygous for the F508del CFTR and with extreme pulmonary phenotypes (severe vs mild) (Gu et al., 2009). Herein, IFRD1 (Interferon-Related Developmental Regulator 1) was identified as a potential candidate, as this differentiation factor may have a role in signal transduction. They observed that IFRD1 polymorphisms may be involved in the severity of CF lung disease through the regulation of neutrophil effector function. The ‘CCC’ haplotype composed of 3 IFRD1 SNPs (rs7817, rs807213 and rs6968084) was associated with higher TNF-␣ levels. They also showed that ifrd1 deficient mice had a delayed bacterial clearance from the airway, but also less inflammation. Altogether these pilot studies led to a large linkage and GWAS in ∼3500 pancreatic insufficient North-American CF patients (Wright et al., 2011). They identified 7 regions that might be associated with CF lung disease severity near the following genes: APIP/EHF, AGTR2, HLA-DRA, EEA1, SLC8A3, AHRR and CDH8. Among these loci, the most significant association was the locus that contains EHF and APIP genes (chr11p13, P > 10−9 ). APIP encodes the Apaf-1interacting protein that has been shown to suppress apoptosis in


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the presence of hypoxia (Cho et al., 2007); and EHF is a member of the epithelial-specific Ets transcription factors, participating to the regulation of the epithelial cell differentiation under stress and inflammation (Wright et al., 2011). The linkage analysis (486 sibling pairs from the family based study) identified another significant quantitative trait locus on chromosome 20q13.2. This locus includes several genes among which: MC3R, encoding the melanocortin-3receptor, CBLN4 encoding cerebellin-like4, CASS4, encoding Crk-associated substrate scaffolding 4, and AURKA, encoding Aurora kinase A (Weiler and Drumm, 2013). MC3R is a receptor involved in the metabolic control, but, so far, little is known for the others. Altogether, the linkage and GWAS analysis provided interesting modifiers’ candidates for lung disease, and functional studies are ongoing investigating their putative roles. In France, in 2006, taking advantage of the national CF centers network, we started a national study on CF modifier genes. So far, ∼4200 CF patients have been included with clinical follow-up and DNA samples (that represented ∼75% of the French CF coverage). Along with candidate gene analyses (Beucher et al., 2012), we performed a GWAS in a subset of patients carrying 2 CFTR mutations associated with pancreatic insufficiency, and are currently replicating the results of the lung disease severity association study in an independent North-American cohort. These 2 consortiums are indeed close collaborators, and created 2 years ago the “International CF modifier genes consortium”, which showed last year interesting loci associated with meconium ileus (Sun et al., 2012). This study also identified specific SNPs in three solute carrier family genes SLC26A9, SLC9A3 and SCL6A14. These secondary modifiers of meconium ileus have been recently studied for other early-affecting CF comorbidities to know if they are pleiotropic (Li et al., 2014). SLC26A9 was pleiotropic for meconium ileus and pancreatic damage, SLC9A3 for meconium ileus and lung disease and SLC6A14 was shown to be pleiotropic for meconium ileus and both lung disease and age at first P. aeruginosa infection. The existence of pleiotropy for modifier genes of CF may encourage the development of new therapeutics targets with multi-organ benefits. 3.2. Exome wide association studies So far in CF, exome sequencing analyzes have been focused on the time to chronic P. aeruginosa infection (Emond et al., 2012). Exome sequencing has indeed become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders (Bamshad et al., 2011). However, its use to identify variants associated with complex traits is more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase such power is to compare the exome sequences of individuals with “extreme phenotypes”. As such, Emond et al. compared patients chronically infected with P. aeruginosa very young (before 5 years old, “early P. aeruginosa extreme”, n = 43) versus the patients not chronically infected at 14 years old (“late P. aeruginosa extreme”, n = 48). Applying such an approach, they identified a single gene, DCTN4 (encoding dynactin 4) on chromosome 5q33.1, which was significantly associated with time to chronic P. aeruginosa infection (P = 0.025). They found that 12 out of the 43 individuals in the “early P. aeruginosa extreme” sample carried a missense variant in DCTN4; 9 carried a phe349-to-leu substitution (F349L; rs11954652) and 3 carried a tyr270-to-cys substitution (Y270C; rs35772018). None of the 48 individuals in the late P. aeruginosa extreme sample had missense variants in DCTN4. They validated their results in an independent cohort of 696 CF patients with various CFTR genotypes. They screened DCTN4 gene in those individuals and found: 78 patients heterozygous and 9 homozygous for the F349L mutation; 15 heterozygous for the Y270C mutation; and 1 heterozygous for both mutations. The presence of at least

one DCTN4 missense variant was associated with early age of first P. aeruginosa-positive culture and with early age of onset of chronic P. aeruginosa infection. Dynactin 4 is a subunit of the dynactin complex, involved in microtubule-dependent vesicular transport, spindle assembly, and cell division. The dynein-dependent motor is involved in the autophagy process, as it moves autophagosomes along the microtubules into the lysosomes, for degradation of damaged proteins and microbes. Autophagy is known to have an essential role in the clearance of P. aeruginosa. In CF, the intracellular accumulation of CFTR-F508del is associated with a reduced macroautophagic flux via an inhibition of the autophagosome formation. The authors speculated that, in CF, dynactin 4 isoforms could influence P. aeruginosa infection by reducing the respiratory autophagic clearance of the bacteria. 4. Conclusion The lung disease severity is variable among CF patients, even those with the same CFTR genotype (F508del homozygotes for example) or sharing the same environment (as in siblings). This review details the genetic modifiers that have been shown to contribute to this variability, identified through several approaches, i.e. candidate genes analyses, and whole genome and exome analyses. However, understanding the clinical diversity of CF cannot be limited to the genomic determinants of the sole lung disease. CF affects indeed many organs; most critically the lungs, but also the pancreas, the liver and the intestine. Therefore, the next challenge will be to perform a “phenomics” analysis of CF, i.e. obtaining measurements on numerous CF severity traits to finally analyze these variations according to genotype. The conceptual revolution that characterized GWAS some years ago, whereby several thousand genomic polymorphisms could be correlated at once to an existing condition, is now happening for phenotypes, as the basis to understand to what extent genomic variation may affect a whole range of phenotypes (Houle et al., 2010). Understanding phenotypic variation in relation with variation in genotype offers promise to tailor treatment to the best of each patient’s condition and status: this is the basis of “personalized medicine”. To achieve this goal, it is necessary first to describe the patients’ genome, and then to explore the proper range of phenotypic variation, including not only the intrinsic severity of the disease, but also the response to treatment. Using data from these two approaches, a global map of genomic/phenotypic variation could be obtained, allowing the best choice of care at the patient level. References Adamali H, Armstrong ME, McLaughlin AM, Cooke G, McKone E, Costello CM, et al. Macrophage migration inhibitory factor enzymatic activity, lung inflammation, and cystic fibrosis. Am J Respir Crit Care Med 2012;186:162–9. Alexis N, Eldridge M, Reed W, Bromberg P, Peden DB. CD14-dependent airway neutrophil response to inhaled LPS: role of atopy. J Allergy Clin Immunol 2001;107:31–5. Arkwright PD, Laurie S, Super M, Pravica V, Schwarz MJ, Webb AK, et al. TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 2000;55:459–62. Arkwright PD, Pravica V, Geraghty PJ, Super M, Webb AK, Schwarz M, et al. End-organ dysfunction in cystic fibrosis: association with angiotensin I converting enzyme and cytokine gene polymorphisms. Am J Respir Crit Care Med 2003;167:384–9. Aron Y, Bienvenu T, Hubert D, Dusser D, Dall’Ava J, Polla BS. HLA-DR polymorphism in allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 1999a;104:891–2. Aron Y, Polla BS, Bienvenu T, Dall’ava J, Dusser D, Hubert D. HLA class II polymorphism in cystic fibrosis. A possible modifier of pulmonary phenotype. Am J Respir Crit Care Med 1999b;159:1464–8. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, Nickerson DA, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 2011;12:745–55. Beucher J, Boelle PY, Busson PF, Muselet-Charlier C, Clement A, Corvol H. AGER429T/C is associated with an increased lung disease severity in cystic fibrosis. PLoS One 2012;7:e41913.




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In silico search for modifier genes associated with pancreatic and liver disease in Cystic Fibrosis.

Origins of cystic fibrosis lung disease.

Origins of cystic fibrosis lung disease.

Cystic fibrosis. 2. Lung injury in cystic fibrosis.

Disease-modifying genes and monogenic disorders: experience in cystic fibrosis.

Lung function imaging methods in Cystic Fibrosis pulmonary disease.

Topical cystic fibrosis transmembrane conductance regulator gene replacement for cystic fibrosis-related lung disease.

Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy.

Pathogenesis and management of lung disease in cystic fibrosis.

Polysomnographic Markers in Children With Cystic Fibrosis Lung Disease.

Pulmonary hypertension in cystic fibrosis with advanced lung disease.

Seeing is believing: imaging early lung disease in cystic fibrosis.

Antioxidant supplementation for lung disease in cystic fibrosis.

Microstructural alterations of sputum in cystic fibrosis lung disease.

The role of Lipoxin A4 in Cystic Fibrosis Lung Disease.

Approaches to localizing disease genes as applied to cystic fibrosis.

CXCR4+ granulocytes reflect fungal cystic fibrosis lung disease.

Lung transplantation for cystic fibrosis.

The cystic fibrosis lung microbiome.

Lung Transplantation for Cystic Fibrosis.

Microbiology of lung infection in cystic fibrosis.

Gallbladder disease in cystic fibrosis.

Acute and long-term amiloride inhalation in cystic fibrosis lung disease. A rational approach to cystic fibrosis therapy.

Heart-lung transplantation for cystic fibrosis.