American Journal of Respiratory and Critical Care Medicine

To the Editor:

With the advent of whole-genome sequencing, the interpretation of DNA variants in noncoding genomic regions has increased in relevance. This study highlights the importance of intronic variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which is responsible for cystic fibrosis (CF) (1). We report the high prevalence of a recently discovered intronic variant, c.3874-4522A>G, in patients with significant disease and propose its inclusion in future targeted diagnostic gene panels.

Genetic testing of CFTR is one of the most frequently performed genetic analyses worldwide and is used to establish a diagnosis of CF in symptomatic individuals, to determine carrier status in the general population, as part of newborn screening, and for CFTR modulator therapy (2). CF is primarily a clinical diagnosis, based on consensus clinical and laboratory criteria (sweat chloride ≥60 mEq/L and/or two CF-causing mutations [in trans]), and/or abnormal values of electrophysiological measurements (i.e., nasal potential difference or intestinal current measurements). More than 2,000 sequence variations have been described in the CFTR gene, often with geographic or ethnic variations in frequency (www.genet.sickkids.on.ca/cftr/). To date, only 312 of these have been curated as CF-causing in the CFTR2 database (3) (https://www.cftr2.org/).

There are principally two methods that are used for CFTR testing in symptomatic individuals: targeted screening for specific known variants and full CFTR sequencing. Targeted mutation analysis (TMA) is usually performed using one of many commercially available kits, focusing on 30–50 pathogenic variants. The most common pathogenic CFTR variant is F508del, which is present on ∼66% of CF alleles worldwide (4); however, there is extensive heterogeneity in the remaining CF alleles. European Society of Human Genetics guidelines (5) recommend testing for all CF-causing mutations with a frequency of >1% in the local population. If TMA detects only a single heterozygous pathogenic variant (or none) in a patient with a high index of suspicion, it is usually followed by sequencing of the entire CFTR coding region and possibly flanking intronic sequences, and multiplex ligation-dependent probe amplification for copy number variants. Current methods detect CF alleles in ∼85–95% of white European individuals with CF (3).

Undetected CFTR mutations may lie within introns or regulatory regions, which are not routinely analyzed. Three pathogenic intronic variants are known: c.3718-2477C>T (3849+10kbC>T; intron 22), c.1680-886A>G (1811+1.6kbA>G; intron 12), and c.870-1113-870-1110delGAAT (intron 7). They all activate cryptic splice sites, resulting in the inclusion of cryptic exons in the CFTR transcript. Using next-generation sequencing (NGS) of the whole CFTR gene, other investigators and we have identified a common intronic variant that may account for many incomplete CF genetic diagnoses (68).

Using NGS of the whole CFTR gene, we performed a molecular genetic diagnosis of CF in 74 patients in whom a previous TMA had not revealed two pathogenic variants; 21 patients were heterozygous for a single pathogenic CFTR variant. In 57 patients (40 adults and 17 children) the suspicion for CF was high (owing to a clinical phenotype and/or a sweat chloride of ≥30 mmol/L) and in 17 patients (8 adults) the suspicion was low (atypical phenotype and/or sweat chloride of <30 mmol/L), but the cause of bronchiectasis had not yet been elucidated. Single-nucleotide and copy number variants were called, filtered, and classified according to American College of Medical Genetics and Genomics guidelines (9), using an in-house bioinformatics pipeline and interrogation of the gnomAD genome browser (http://gnomad.broadinstitute.org), Human Gene Mutation (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/), and CFTR2 (https://www.cftr2.org/) databases.

Twenty (27%) of the 74 patients (all with a high clinical suspicion of CF) were found to have two pathogenic CFTR variants, confirming the diagnosis of CF. Twelve of these patients were compound heterozygous for the c.3874-4522A>G variant in intron 23. All had extensive bronchiectasis and impaired lung function (Table 1), two were pancreatic insufficient, and all males were infertile. All had evidence of abnormal ion transport—with varying levels of residual CFTR function—with a median (range) sweat chloride of 46.0 (23.5–69.0) mmol/L and/or limited chloride secretory capacity on nasal potential difference. The c.3874-4522A>G variant was confirmed to be inherited in trans with the second pathogenic variant in patient 4, whose parents were available for study.

Table 1. Clinical Findings in Patients with the CFTR c.3784-4522A>G Variant

ID  GenotypeSexDoBSymptom Onset (yr)Sweat Cl* (mmol/L)Nasal PD (Total Cl Secretion, mV)Respiratory CharacteristicsPancreatic InsufficiencyChronic Rhinosinus SymptomsAzoospermia/CBAVDOther Comorbidities
12  Recent FEV1 [L (%)]Recent FVC [L (%)]RadiologySputum Microbiology
P1  p.(Phe508del); c.3784-4522A>GF19854444492.84.2Widespread BESANoYesN/ADepression, osteoporosis, previous nasal polypectomy, history of pancreatitis
P2p.(Phe508del); c.3784-4522A>GM19790.53235Not done1.75 (43)2.50 (49)Widespread BEPA, intermittent AF growthNoYesYesABPA, osteoporosis, adrenal insufficiency, previous bronchial arterial embolization  
P3 p(lle507del); c.3784-4522A>GF1978164751Not done1.33 (50)2.26 (74)Widespread BESA and PANoYesN/ASmall right-sided pneumothorax, recurrent significant hemoptysis, referred for transplant 
P4 p.(Phe508del); c.3784-4522A>GF198433383311.92.9Bilateral UL and RLL BESA and PANoNoN/ANone 
P5 p.(Ile444ArgfsTer3); c.3784-4522A>GM19971853Insufficient sample8N/AN/AWidespread BEPAYesYesUnknown§Low BMI 
P6 p.(Phe508del); c.3784-4522A>GF197820232461.21.7Bilateral UL BESAYesNoN/AGORD, vitamin D deficiency, previous episodes of recurrent pancreatitis 
P7 p.(Phe508del); c.3784-4522A>GM1976194848101.16 (31)1.50 (32)Widespread BESA and PA, MabNoYesYesT2RF on NIV and LTOT, microcytic anemia, recurrent rib fractures, previous embolization and pancreatitis 
P8 p.(Phe508del); c.3784-4522A>GM1975205255Not done2.85 (77)3.74 (83)Bilateral UL BESA and PANoYesYesMild, presumed CF-related liver disease 
P9 p.(Phe508del); c.3784-4522A>GF1982116870Not done0.85 (26)1.33 (34)Widespread BEPA, MabNoNoUnknownNone 
P10 p.(Phe508del); c.3784-4522A>GM196217644841.51 (41)2.63 (55)Widespread BEPANoNoYesCF bone disease, LTOT, hypothyroidism 
P11 p.(Phe508 del); c.3784-4522A>GM1994544Insufficient sample164.43 (71)5.26 (81)Widespread BEPANoYesYesNo 
P12 p.(Phe508del); c.3784-4522A>GM1986173640Not done3.51 (86)5.02 (104)Widespread BESANoYesYesNo 

Definition of abbreviations: ABPA = allergic bronchopulmonary aspergillosis; AF = Aspergillus fumigatus; BE = bronchiectasis; BMI = body mass index; CBAVD = congenital bilateral absence of the vas deferens; CF =  cystic fibrosis; Cl = chloride; DoB = date of birth; GORD = gastroesophageal reflux disease; LTOT = long-term oxygen therapy; Mab = Mycobacterium abscessus; N/A = not applicable; NIV = noninvasive ventilation; PA = Pseudomonas aeruginosa; PD = potential difference; RLL = right lower lobe; SA = Staphylococcus aureus; T2RF = type 2 respiratory failure; UL = upper lobe.

None of the patients had CF diabetes.

*Sweat Cl 1 and 2 refer to duplicate measurements.

Total chloride secretion = net change after perfusion with zero Cl solution followed by zero Cl with isoprenaline.

Chronic infection unless stated otherwise.

§Presumed infertile (he has no children but has not yet been formally tested).

The variant was first described by Roth and colleagues in a patient with residual chloride secretion (10). It was observed in a single patient during NGS screening of the whole CFTR gene in 18 patients with CF (6). In another patient it was shown to introduce a cryptic splice site, resulting in a cryptic exon (8), which if transcribed and translated would result in premature CFTR protein truncation. The variant was recently reported in 10 of 19 patients (eight families) who had been found to harbor pathogenic intronic variants (7). These patients were older at disease onset, more likely to be pancreatic-sufficient, and have an equivocal sweat test (7). The variant is extremely rare in control cohorts, with an allele frequency of 0.0035% in the gnomAD database (5/143,000 alleles, gnomAD v3).

We estimated the frequency of the c.3874-4522A>G variant in the UK CF population from our genotype-positive adult CF cohort (547 patients, 138 different pathogenic variants). The c.3874-4522A>G variant is the seventh most common, in 2.2% of patients. If one excludes F508del (67% of pathogenic alleles in the cohort), c.3874-4522A>G accounts for 3.3% of other pathogenic CFTR alleles. It has been found in patients from different ethnic backgrounds and occurs on different haplotypes (7). This suggests that the c.3874-4522A>G variant should be included in newborn screening tests and in commercial kits for first-line TMA.

Our experience with CFTR indicates that the surveillance of noncoding regions of other genes will go some way toward identifying missing variants and increasing diagnostic yield. This is particularly relevant for patients with a suspected autosomal-recessive disease who are heterozygous for a single pathogenic variant but have not received a formal diagnosis because of an incomplete genotype. Moreover, most of the intronic CFTR variants cause aberrant splicing, which may be amenable to treatment with current CFTR modulators (11) or investigational therapeutic techniques such as CRISP/Cas9 targeted excision and antisense oligonucleotide therapeutics, or standard gene therapy. This study is the first comprehensive analysis of CFTR in a large cohort of patients. Although whole-genome sequencing may not be necessary for disorders caused by pathogenic variants in a few major genes, we propose that the entire noncoding and coding regions of such genes should be included when “first-line” investigations remain equivocal.

The authors thank Sam Wilkinson, Lizzie Briggs, and Laura Brett for excellent technical assistance, and Juliana Burgess, Registry Research Nurse, for her assistance with data retrieval and processing.

1. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:10661073.
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3. Sosnay PR, Siklosi KR, Van Goor F, Kaniecki K, Yu H, Sharma N, et al. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet 2013;45:11601167.
4. Bobadilla JL, Macek M Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations—correlation with incidence data and application to screening. Hum Mutat 2002;19:575606.
5. Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, et al. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders—updated European recommendations. Eur J Hum Genet 2009;17:5165.
6. Bonini J, Varilh J, Raynal C, Thèze C, Beyne E, Audrezet MP, et al. Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis. Genet Med 2015;17:796806.
7. Bergougnoux A, Délétang K, Pommier A, Varilh J, Houriez F, Altieri JP, et al. Functional characterization and phenotypic spectrum of three recurrent disease-causing deep intronic variants of the CFTR gene. J Cyst Fibros 2019;18:468475.
8. Ellingford JM, Beaman G, Webb K, O’Callaghan C, Hirst RA, Black GCM, et al.; On behalf of the 100,000 Genomes Project. Whole genome sequencing enables definitive diagnosis of cystic fibrosis and primary ciliary dyskinesia [preprint]. bioRxiv 2018 [accessed 2018 Oct]. Available from: https://www.biorxiv.org/content/10.1101/438838v1.
9. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al.; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405424.
10. Roth EK, Hirtz S, Duerr J, Wenning D, Eichler I, Seydewitz HH, et al. The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients. PLoS One 2011;6:e24445.
11. Rowe SM, Daines C, Ringshausen FC, Kerem E, Wilson J, Tullis E, et al. Tezacaftor-ivacaftor in residual-function heterozygotes with cystic fibrosis. N Engl J Med 2017;377:20242035.
*Corresponding author (e-mail: ).

Originally Published in Press as DOI: 10.1164/rccm.201908-1541LE on February 4, 2020

Author disclosures are available with the text of this letter at www.atsjournals.org.

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