ABSTRACT
BACKGROUND: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). METHODS: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. RESULTS: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. CONCLUSIONS: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.
ABSTRACT
BACKGROUND: Cystic fibrosis (CF) is caused by deleterious variants in each CFTR gene. We investigated the utility of whole-gene CFTR sequencing when fewer than two pathogenic or likely pathogenic (P/LP) variants were detected by conventional testing (sequencing of exons and flanking introns) of CFTR. METHODS: Individuals with features of CF and a CF-diagnostic sweat chloride concentration with zero or one P/LP variants identified by conventional testing enrolled in the CF Mutation Analysis Program (MAP) underwent whole-gene CFTR sequencing. Replication was performed on individuals enrolled in the CF Genome Project (CFGP), followed by phenotype review and interrogation of other genes. RESULTS: Whole-gene sequencing identified a second P/LP variant in 20/43 MAP enrollees (47 %) and 10/22 CFGP enrollees (45 %) who had one P/LP variant after conventional testing. No P/LP variants were detected when conventional testing was negative (MAP: n = 43; CFGP: n = 13). Genome-wide analysis was unable to find an alternative etiology in CFGP participants with fewer than two P/LP CFTR variants and CF could not be confirmed in 91 % following phenotype re-review. CONCLUSIONS: Whole-gene CFTR analysis is beneficial in individuals with one previously-identified P/LP variant and a CF-diagnostic sweat chloride. Negative conventional CFTR testing indicates that the phenotype should be re-evaluated.
ABSTRACT
Since genotype imputation was introduced, researchers have been relying on the estimated imputation quality from imputation software to perform post-imputation quality control (QC). However, this quality estimate (denoted as Rsq) performs less well for lower-frequency variants. We recently published MagicalRsq, a machine-learning-based imputation quality calibration, which leverages additional typed markers from the same cohort and outperforms Rsq as a QC metric. In this work, we extended the original MagicalRsq to allow cross-cohort model training and named the new model MagicalRsq-X. We removed the cohort-specific estimated minor allele frequency and included linkage disequilibrium scores and recombination rates as additional features. Leveraging whole-genome sequencing data from TOPMed, specifically participants in the BioMe, JHS, WHI, and MESA studies, we performed comprehensive cross-cohort evaluations for predominantly European and African ancestral individuals based on their inferred global ancestry with the 1000 Genomes and Human Genome Diversity Project data as reference. Our results suggest MagicalRsq-X outperforms Rsq in almost every setting, with 7.3%-14.4% improvement in squared Pearson correlation with true R2, corresponding to 85-218 K variant gains. We further developed a metric to quantify the genetic distances of a target cohort relative to a reference cohort and showed that such metric largely explained the performance of MagicalRsq-X models. Finally, we found MagicalRsq-X saved up to 53 known genome-wide significant variants in one of the largest blood cell trait GWASs that would be missed using the original Rsq for QC. In conclusion, MagicalRsq-X shows superiority for post-imputation QC and benefits genetic studies by distinguishing well and poorly imputed lower-frequency variants.
Subject(s)
Gene Frequency , Genotype , Polymorphism, Single Nucleotide , Software , Humans , Cohort Studies , Linkage Disequilibrium , Genome-Wide Association Study/methods , Genome, Human , Quality Control , Machine Learning , Whole Genome Sequencing/standards , Whole Genome Sequencing/methodsABSTRACT
BACKGROUND AND AIMS: It is not known why severe cystic fibrosis (CF) liver disease (CFLD) with portal hypertension occurs in only ~7% of people with CF. We aimed to identify genetic modifiers for severe CFLD to improve understanding of disease mechanisms. APPROACH AND RESULTS: Whole-genome sequencing was available in 4082 people with CF with pancreatic insufficiency (n = 516 with severe CFLD; n = 3566 without CFLD). We tested ~15.9 million single nucleotide polymorphisms (SNPs) for association with severe CFLD versus no-CFLD, using pre-modulator clinical phenotypes including (1) genetic variant ( SERPINA1 ; Z allele) previously associated with severe CFLD; (2) candidate SNPs (n = 205) associated with non-CF liver diseases; (3) genome-wide association study of common/rare SNPs; (4) transcriptome-wide association; and (5) gene-level and pathway analyses. The Z allele was significantly associated with severe CFLD ( p = 1.1 × 10 -4 ). No significant candidate SNPs were identified. A genome-wide association study identified genome-wide significant SNPs in 2 loci and 2 suggestive loci. These 4 loci contained genes [significant, PKD1 ( p = 8.05 × 10 -10 ) and FNBP1 ( p = 4.74 × 10 -9 ); suggestive, DUSP6 ( p = 1.51 × 10 -7 ) and ANKUB1 ( p = 4.69 × 10 -7 )] relevant to severe CFLD pathophysiology. The transcriptome-wide association identified 3 genes [ CXCR1 ( p = 1.01 × 10 -6 ) , AAMP ( p = 1.07 × 10 -6 ), and TRBV24 ( p = 1.23 × 10 -5 )] involved in hepatic inflammation and innate immunity. Gene-ranked analyses identified pathways enriched in genes linked to multiple liver pathologies. CONCLUSION: These results identify loci/genes associated with severe CFLD that point to disease mechanisms involving hepatic fibrosis, inflammation, innate immune function, vascular pathology, intracellular signaling, actin cytoskeleton and tight junction integrity and mechanisms of hepatic steatosis and insulin resistance. These discoveries will facilitate mechanistic studies and the development of therapeutics for severe CFLD.
ABSTRACT
BACKGROUND: In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. METHODS: Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. RESULTS AND CONCLUSIONS: 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.
ABSTRACT
Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1 mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100 nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1 µM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.
Subject(s)
Cystic Fibrosis , Quinolones , Cricetinae , Animals , Humans , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , CHO Cells , Cricetulus , Amino Acids , Ion Channel Gating , Aminophenols/pharmacology , Adenosine Triphosphate/metabolismABSTRACT
Canonical splice site variants affecting the 5' GT and 3' AG nucleotides of introns result in severe missplicing and account for about 10% of disease-causing genomic alterations. Treatment of such variants has proven challenging due to the unstable mRNA or protein isoforms that typically result from disruption of these sites. Here, we investigate CRISPR-Cas9-mediated adenine base editing for such variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We validate a CFTR expression minigene (EMG) system for testing base editing designs for two different targets. We then use the EMG system to test non-standard single-guide RNAs with either shortened or lengthened protospacers to correct the most common cystic fibrosis-causing variant in individuals of African descent (c.2988+1G>A). Varying the spacer region length allowed placement of the editing window in a more efficient context and enabled use of alternate protospacer adjacent motifs. Using these modifications, we restored clinically significant levels of CFTR function to human airway epithelial cells from two donors bearing the c.2988+1G>A variant.
ABSTRACT
Small molecule drugs known as modulators can treat ~90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23-27 sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Gene Editing , Codon, Nonsense/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , MutationABSTRACT
Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.
Subject(s)
Extracellular Vesicles , Satellite Viruses , Mice , Animals , Humans , Satellite Viruses/genetics , Transduction, Genetic , Dependovirus/genetics , HEK293 CellsABSTRACT
Rationale: Lung disease is the major cause of morbidity and mortality in persons with cystic fibrosis (pwCF). Variability in CF lung disease has substantial non-CFTR (CF transmembrane conductance regulator) genetic influence. Identification of genetic modifiers has prognostic and therapeutic importance. Objectives: Identify genetic modifier loci and genes/pathways associated with pulmonary disease severity. Methods: Whole-genome sequencing data on 4,248 unique pwCF with pancreatic insufficiency and lung function measures were combined with imputed genotypes from an additional 3,592 patients with pancreatic insufficiency from the United States, Canada, and France. This report describes association of approximately 15.9 million SNPs using the quantitative Kulich normal residual mortality-adjusted (KNoRMA) lung disease phenotype in 7,840 pwCF using premodulator lung function data. Measurements and Main Results: Testing included common and rare SNPs, transcriptome-wide association, gene-level, and pathway analyses. Pathway analyses identified novel associations with genes that have key roles in organ development, and we hypothesize that these genes may relate to dysanapsis and/or variability in lung repair. Results confirmed and extended previous genome-wide association study findings. These whole-genome sequencing data provide finely mapped genetic information to support mechanistic studies. No novel primary associations with common single variants or rare variants were found. Multilocus effects at chr5p13 (SLC9A3/CEP72) and chr11p13 (EHF/APIP) were identified. Variant effect size estimates at associated loci were consistently ordered across the cohorts, indicating possible age or birth cohort effects. Conclusions: This premodulator genomic, transcriptomic, and pathway association study of 7,840 pwCF will facilitate mechanistic and postmodulator genetic studies and the development of novel therapeutics for CF lung disease.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Genome-Wide Association Study/methods , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Patient Acuity , Lung , Microtubule-Associated Proteins/geneticsABSTRACT
Cystic fibrosis (CF) is a multiorgan disease caused by a wide variety of mutations in the cystic fibrosis transmembrane conductance regulator gene. As treatment has progressed from symptom mitigation to targeting of specific molecular defects, genetics has played an important role in identifying the proper precision therapies for each individual. Novel therapeutic approaches are focused on expanding treatment to a greater number of individuals as well as working toward a cure. This review discusses the role of genetics in our understanding of CF with a particular emphasis on how genetics informs the exciting landscape of current and novel CF therapies.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Cystic Fibrosis/therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/therapeutic use , MutationABSTRACT
Individuals with cystic fibrosis (CF) develop complications of the gastrointestinal tract influenced by genetic variants outside of CFTR. Cystic fibrosis-related diabetes (CFRD) is a distinct form of diabetes with a variable age of onset that occurs frequently in individuals with CF, while meconium ileus (MI) is a severe neonatal intestinal obstruction affecting â¼20% of newborns with CF. CFRD and MI are slightly correlated traits with previous evidence of overlap in their genetic architectures. To better understand the genetic commonality between CFRD and MI, we used whole-genome-sequencing data from the CF Genome Project to perform genome-wide association. These analyses revealed variants at 11 loci (6 not previously identified) that associated with MI and at 12 loci (5 not previously identified) that associated with CFRD. Of these, variants at SLC26A9, CEBPB, and PRSS1 associated with both traits; variants at SLC26A9 and CEBPB increased risk for both traits, while variants at PRSS1, the higher-risk alleles for CFRD, conferred lower risk for MI. Furthermore, common and rare variants within the SLC26A9 locus associated with MI only or CFRD only. As expected, different loci modify risk of CFRD and MI; however, a subset exhibit pleiotropic effects indicating etiologic and mechanistic overlap between these two otherwise distinct complications of CF.
Subject(s)
Cystic Fibrosis , Diabetes Mellitus , Infant, Newborn, Diseases , Intestinal Obstruction , Cystic Fibrosis/complications , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Diabetes Mellitus/genetics , Genome-Wide Association Study , Humans , Infant, Newborn , Intestinal Obstruction/complications , Intestinal Obstruction/geneticsABSTRACT
Whole-genome sequencing (WGS) is the gold standard for fully characterizing genetic variation but is still prohibitively expensive for large samples. To reduce costs, many studies sequence only a subset of individuals or genomic regions, and genotype imputation is used to infer genotypes for the remaining individuals or regions without sequencing data. However, not all variants can be well imputed, and the current state-of-the-art imputation quality metric, denoted as standard Rsq, is poorly calibrated for lower-frequency variants. Here, we propose MagicalRsq, a machine-learning-based method that integrates variant-level imputation and population genetics statistics, to provide a better calibrated imputation quality metric. Leveraging WGS data from the Cystic Fibrosis Genome Project (CFGP), and whole-exome sequence data from UK BioBank (UKB), we performed comprehensive experiments to evaluate the performance of MagicalRsq compared to standard Rsq for partially sequenced studies. We found that MagicalRsq aligns better with true R2 than standard Rsq in almost every situation evaluated, for both European and African ancestry samples. For example, when applying models trained from 1,992 CFGP sequenced samples to an independent 3,103 samples with no sequencing but TOPMed imputation from array genotypes, MagicalRsq, compared to standard Rsq, achieved net gains of 1.4 million rare, 117k low-frequency, and 18k common variants, where net gains were gained numbers of correctly distinguished variants by MagicalRsq over standard Rsq. MagicalRsq can serve as an improved post-imputation quality metric and will benefit downstream analysis by better distinguishing well-imputed variants from those poorly imputed. MagicalRsq is freely available on GitHub.
Subject(s)
Genome-Wide Association Study , Polymorphism, Single Nucleotide , Humans , Genome-Wide Association Study/methods , Polymorphism, Single Nucleotide/genetics , Calibration , Genotype , Machine LearningABSTRACT
In December 2020, the U.S. Food and Drug Administration (FDA) expanded the list of CFTR variants approved for treatment with CFTR modulators drugs from 39 to 183. Clinicians should be aware that individuals harboring certain variants approved for treatment may not respond to or benefit from this therapy. After review, the expert panel leading the CFTR2 project identified four categories of variants that may not result in a clinical response to modulator treatment: 15 variants assigned as non CF-causing; 45 variants of unknown significance; six variants known or suspected to cause mis-splicing as their primary defect rather than an amino acid substitution; and eight variants known to occur together in cis with another deleterious variant not expected to lead to CFTR protein (nonsense or frameshift). The potential risks and benefits of CFTR modulator therapy should be considered carefully for individuals harboring these variants.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Mutation , RNA SplicingABSTRACT
The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl-]; 87-105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl-] (75-123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o-) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Aminophenols/pharmacology , Aminophenols/therapeutic use , Chlorides/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Phenotype , QuinolonesABSTRACT
Cystic fibrosis (CF) is a severe genetic disorder that can cause multiple comorbidities affecting the lungs, the pancreas, the luminal digestive system and beyond. In our previous genome-wide association studies (GWAS), we genotyped approximately 8,000 CF samples using a mixture of different genotyping platforms. More recently, the Cystic Fibrosis Genome Project (CFGP) performed deep (approximately 30×) whole genome sequencing (WGS) of 5,095 samples to better understand the genetic mechanisms underlying clinical heterogeneity among patients with CF. For mixtures of GWAS array and WGS data, genotype imputation has proven effective in increasing effective sample size. Therefore, we first performed imputation for the approximately 8,000 CF samples with GWAS array genotype using the Trans-Omics for Precision Medicine (TOPMed) freeze 8 reference panel. Our results demonstrate that TOPMed can provide high-quality imputation for patients with CF, boosting genomic coverage from approximately 0.3-4.2 million genotyped markers to approximately 11-43 million well-imputed markers, and significantly improving polygenic risk score (PRS) prediction accuracy. Furthermore, we built a CF-specific CFGP reference panel based on WGS data of patients with CF. We demonstrate that despite having approximately 3% the sample size of TOPMed, our CFGP reference panel can still outperform TOPMed when imputing some CF disease-causing variants, likely owing to allele and haplotype differences between patients with CF and general populations. We anticipate our imputed data for 4,656 samples without WGS data will benefit our subsequent genetic association studies, and the CFGP reference panel built from CF WGS samples will benefit other investigators studying CF.
ABSTRACT
INTRODUCTION: Loss-of-function variants in both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF); however, there is evidence that reduction in CFTR function due to the presence of one deleterious variant can have clinical consequences. Here, we hypothesise that CFTR variants in individuals with a history of smoking are associated with chronic obstructive pulmonary disease (COPD) and related phenotypes. METHODS: Whole-genome sequencing was performed through the National Heart, Lung, and Blood Institute TOPMed (TransOmics in Precision Medicine) programme in 8597 subjects from the COPDGene (Genetic Epidemiology of COPD) study, an observational study of current and former smokers. We extracted clinically annotated CFTR variants and performed single-variant and variant-set testing for COPD and related phenotypes. Replication was performed in 2118 subjects from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study. RESULTS: We identified 301 coding variants within the CFTR gene boundary: 147 of these have been reported in individuals with CF, including 36 CF-causing variants. We found that CF-causing variants were associated with chronic bronchitis in variant-set testing in COPDGene (one-sided p=0.0025; OR 1.53) and in meta-analysis of COPDGene and ECLIPSE (one-sided p=0.0060; OR 1.52). Single-variant testing revealed that the F508del variant was associated with chronic bronchitis in COPDGene (one-sided p=0.015; OR 1.47). In addition, we identified 32 subjects with two or more CFTR variants on separate alleles and these subjects were enriched for COPD cases (p=0.010). CONCLUSIONS: Cigarette smokers who carry one deleterious CFTR variant have higher rates of chronic bronchitis, while presence of two CFTR variants may be associated with COPD. These results indicate that genetically mediated reduction in CFTR function contributes to COPD related phenotypes, in particular chronic bronchitis.
Subject(s)
Bronchitis, Chronic , Cystic Fibrosis , Pulmonary Disease, Chronic Obstructive , Bronchitis, Chronic/complications , Cystic Fibrosis/complications , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Observational Studies as Topic , Pulmonary Disease, Chronic Obstructive/epidemiology , SmokersABSTRACT
PURPOSE: The growing size of public variant repositories prompted us to test the accuracy of pathogenicity prediction of DNA variants using population data alone. METHODS: Under the a priori assumption that the ratio of the prevalence of variants in healthy population vs that in affected populations form 2 distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability to a variant belonging to either distribution. RESULTS: The approach, termed Bayesian prevalence ratio (BayPR), accurately parsed 300 of 313 expertly curated CFTR variants: 284 of 296 pathogenic/likely pathogenic variants in 1 distribution and 16 of 17 benign/likely benign variants in another. BayPR produced an area under the receiver operating characteristic curve of 0.99 for 103 functionally confirmed missense CFTR variants, which is equal to or exceeds 10 commonly used algorithms (area under the receiver operating characteristic curve range = 0.54-0.99). Application of BayPR to expertly curated variants in 8 genes associated with 7 Mendelian conditions led to the assignment of a disease-causing probability of ≥80% to 1350 of 1374 (98.3%) pathogenic/likely pathogenic variants and of ≤20% to 22 of 23 (95.7%) benign/likely benign variants. CONCLUSION: Irrespective of the variant type or functional effect, the BayPR approach provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only the variant counts in affected and unaffected population samples.
Subject(s)
Algorithms , Mutation, Missense , Bayes Theorem , Humans , ROC CurveABSTRACT
BACKGROUND: Cystic fibrosis (CF) is a recessive condition caused by variants in each CF transmembrane conductance regulator (CFTR) allele. Clinically affected individuals without two identified causal variants typically have no further interrogation of CFTR beyond examination of coding regions, but the development of variant-specific CFTR-targeted treatments necessitates complete understanding of CFTR genotype. METHODS: Whole genome sequences were analyzed on 5,058 individuals with CF. We focused on the full CFTR gene sequence and identified disease-causing variants in three phases: screening for known and structural variants; discovery of novel loss-of-function variants; and investigation of remaining variants. RESULTS: All variants identified in the first two phases and coding region variants found in the third phase were interpreted according to CFTR2 or ACMG criteria (n = 371; 16 [4.3%] previously unreported). Full gene sequencing enabled delineation of 18 structural variants (large insertions or deletions), of which two were novel. Additional CFTR variants of uncertain effect were found in 76 F508del homozygotes and in 21 individuals with other combinations of CF-causing variants. Both causative variants were identified in 98.1% (n = 4,960) of subjects, an increase of 2.3 percentage points from the 95.8% (n = 4,847) who had a registry- or chart-reported disease-causing CFTR genotype. Of the remaining 98 individuals, 78 carried one variant that has been associated with CF (CF-causing [n = 70] or resulting in varying clinical consequences n = 8]). CONCLUSIONS: Complete CFTR gene sequencing in 5,058 individuals with CF identified at least one DNA variant in 99.6% of the cohort that is targetable by current molecular or emerging gene-based therapeutic technologies.
Subject(s)
Cystic Fibrosis , Alleles , Cohort Studies , Cystic Fibrosis/diagnosis , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Genotype , Humans , MutationABSTRACT
Chronic Pseudomonas aeruginosa (Pa) infection is associated with increased morbidity and mortality in people with cystic fibrosis (CF). There is no gold standard definition of chronic Pa infection in CF. We compared chronic Pa definitions using encounter-based versus annualized data in the Early Pseudomonas Infection Control (EPIC) Observational study cohort, and subsequently compared annualized chronic Pa definitions across a range of U.S. cohorts spanning decades of CF care. We found that an annualized chronic Pa definition requiring at least 1 Pa+ culture in 3 of 4 consecutive years ("Green 3/4") resulted in chronic Pa metrics similar to established encounter-based modified Leeds criteria definitions, including a similar age at and proportion who fulfilled chronic Pa criteria, and a similar proportion with sustained Pa infection after meeting the chronic Pa definition. The Green 3/4 chronic Pa definition will be valuable for longitudinal analyses in cohorts with limited culture frequency.