The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10⁻¹¹), chr5p15.3 (SLC9A3; P=6.8 × 10⁻¹²), chr6p21.3 (HLA Class II; P=1.2 × 10⁻⁸) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10⁻⁹) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10⁻¹⁰), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.

Rationale: Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations. Objectives: To define a statistically valid combination of systematically defined clinical features that strongly associates with PCD in children and adolescents. Methods: Investigators at seven North American sites in the Genetic Disorders of Mucociliary Clearance Consortium prospectively and systematically assessed individuals (aged 0–18 yr) referred due to high suspicion for PCD. The investigators defined specific clinical questions for the clinical report form based on expert opinion. Diagnostic testing was performed using standardized protocols and included nasal nitric oxide measurement, ciliary biopsy for ultrastructural analysis of cilia, and molecular genetic testing for PCD-associated genes. Final diagnoses were assigned as “definite PCD” (hallmark ultrastructural defects and/or two mutations in a PCD-associated gene), “probable/possible PCD” (no ultrastructural defect or genetic diagnosis, but compatible clinical features and nasal nitric oxide level in PCD range), and “other diagnosis or undefined.” Criteria were developed to define early childhood clinical features on the basis of responses to multiple specific queries. Each defined feature was tested by logistic regression. Sensitivity and specificity analyses were conducted to define the most robust set of clinical features associated with PCD. Measurements and Main Results: From 534 participants 18 years of age and younger, 205 were identified as having “definite PCD” (including 164 with two mutations in a PCD-associated gene), 187 were categorized as “other diagnosis or undefined,” and 142 were defined as having “probable/possible PCD.” Participants with “definite PCD” were compared with the “other diagnosis or undefined” group. Four criteria-defined clinical features were statistically predictive of PCD: laterality defect; unexplained neonatal respiratory distress; early-onset, year-round nasal congestion; and early-onset, year-round wet cough (adjusted odds ratios of 7.7, 6.6, 3.4, and 3.1, respectively). The sensitivity and specificity based on the number of criteria-defined clinical features were four features, 0.21 and 0.99, respectively; three features, 0.50 and 0.96, respectively; and two features, 0.80 and 0.72, respectively. Conclusions: Systematically defined early clinical features could help identify children, including infants, likely to have PCD. Clinical trial registered with ClinicalTrials.gov (NCT00323167).

Cystic Fibrosis (CF) exhibits morbidity in several organs, including progressive lung disease in all patients and intestinal obstruction at birth (meconium ileus) in ~15%. Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes. With >6,500 participants from the International CF Gene Modifier Consortium, genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 (min p = 3.83x10^-10); replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1, (min p<2.2x10^-16, 2.81x10⁻¹¹, respectively); and replicated a suggestive locus on chromosome 7 near PRSS1 (min p = 2.55x10^-7). PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression. We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ. We developed and applied a colocalization framework called the Simple Sum (SS) that integrates regulatory and genetic association information, and also contrasts colocalization evidence across tissues or genes. The associated modifier loci colocalized with expression quantitative trait loci (eQTLs) for ATP12A (p = 3.35x10^-8), SLC6A14 (p = 1.12x10^-10) and SLC26A9 (p = 4.48x10^-5) in the pancreas, even though meconium ileus manifests in the intestine. The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity. Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 (p = 2.3x10^-4). Cystic Fibrosis is realizing the promise of personalized medicine, and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies. Cystic Fibrosis (CF) impacts the normal functioning of several organs including the pancreas, intestines and lungs. CF is caused by mutations in the CF transmembrane conductance regulator, but individuals with the same mutations have different disease severity. For example, only ~15% of individuals with CF are born with intestinal obstruction at birth (meconium ileus) while all have progressive lung disease with varying severity. Modifier genes contribute to variation across individuals with CF. In this study we identified modifiers of meconium ileus susceptibility, encompassing the previously identified SLC26A9 and SLC6A14, and two new loci ATP12A and PRSS1. To understand the mechanism by which these modifier genes impact CF severity we developed a new statistical method that assesses colocalization with regulatory information. Using this methodology we show, surprisingly, that all the meconium ileus association signals colocalize with gene expression in the pancreas rather than in intestinal tissues. A distinct and neighbouring locus near SLC6A14 also contributes to CF lung disease. Aided by transcriptomics of nasal epithelia from CF patients we found that each locus impacts variation in gene expression of SLC6A14 with tissue specificity. Understanding the contributing tissue and responsible gene are necessary to prioritize modifiers as alternative therapeutic targets.

Technological advances in genetics have made feasible and affordable large studies to identify genetic variants that cause or modify a trait. Genetic studies have been carried out to assess variants in candidate genes, as well as polymorphisms throughout the genome, for their associations with heritable clinical outcomes of cystic fibrosis (CF), such as lung disease, meconium ileus, and CF-related diabetes. The candidate gene approach has identified some predicted relationships, while genome-wide surveys have identified several genes that would not have been obvious disease-modifying candidates, such as a methionine sulfoxide transferase gene that influences intestinal obstruction, or a region on chromosome 11 proximate to genes encoding a transcription factor and an apoptosis controller that associates with lung function. These unforeseen associations thus provide novel insight into disease pathophysiology, as well as suggesting new therapeutic strategies for CF.

In many respects, genetic studies in cystic fibrosis (CF) serve as a paradigm for a human Mendelian genetic success story. From recognition of the condition as a heritable pathological entity to implementation of personalized treatments based on genetic findings, this multistep pathway of progress has focused on the genetic underpinnings of CF clinical disease. Along this path was the recognition that not all CFTR gene mutations produce the same disease and the recognition of the complex, multifactorial nature of CF genotype–phenotype relationships. The non- CFTR genetic components (gene modifiers) that contribute to variation in phenotype are the focus of this review. A multifaceted approach involving candidate gene studies, genome-wide association studies, and gene expression studies has revealed significant gene modifiers for multiple CF phenotypes. The bold challenges for the future are to integrate the findings into our understanding of CF pathogenesis and to use the knowledge to develop novel therapies.