The cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) gene encodes an epithelial ion channel. Although one mutation remains the most common cause of CF (F508del), there have been more than 2000 reported variations in CFTR . For the most part, individuals who carry only one mutation (heterozygotes) have no symptoms; individuals who inherit deleterious mutations from both parents have CF. However, growing awareness of CFTR mutations that do not ever or do not always cause CF, and individuals with mild or single-organ system manifestations of CFTR-related disease have made this Mendelian relationship more complex.
Key points
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There are more than 2000 mutations in the cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) gene described, though not all result in CF.
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Genotyping CFTR can help establish a CF diagnosis (if mutations are characterized) and can be helpful to identify patients eligible for mutation-specific therapies.
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There is a wide range of phenotypes and severity among patients with CF.
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The traditional legacy manner of naming mutations used by CF clinicians and scientists is not in line with other genetic nomenclatures.
The history of the discovery of cystic fibrosis transmembrane conductance regulator as the gene responsible for cystic fibrosis
Very soon after cystic fibrosis (CF) was recognized and characterized, clinicians caring for patients with CF observed that the disease occurred in families with a Mendelian autosomal recessive inheritance pattern. The study of CF was an important test case for the molecular tools used to study how DNA and genes are responsible for inherited traits. Beginning with restriction fragment length polymorphism analysis that allowed DNA segments differing by restriction enzyme sites to be sorted with inheritance of a phenotype, the gene responsible for CF could be localized to the long arm of chromosome 7. Coincident with these genetic advances, researchers examining the biochemical defect in the disease by studying the epithelial tissues affected by CF discovered that these tissues did not allow normal chloride transport. The genetic and biochemical investigational pathways converged with the identification of the CF transmembrane conductance regulator ( CFTR ) gene and its most common mutation (F508del) in 1989. Immediately after the gene was identified, it became apparent that multiple mutations in this gene could be responsible for CF ( Box 1 ).
The terms mutation and variant can both be used to describe a genetic difference from that which is commonly seen in a population (sometimes referred to as wild-type). In some writing, mutation implies that the genetic change is deleterious and variant is used when the change does not result in disease; however, both terms are used in the literature, somewhat interchangeably. The Human Genome Variation Society, American College of Medical Genetics and Genomics, and the Association for Molecular Pathology recommend use of the term variant , recognizing the potential negative connotations of the term mutation . Similarly, the terms polymorphism or single nucleotide polymorphism have been used to describe sequence changes occurring at greater than 1% frequency in the population but also to describe sequence changes that do not lead to disease (regardless of frequency). Therefore, consensus has been to use the term variant or sequence alteration to describe any DNA change, with further explanation given if the variant has been deemed to have an effect on the gene, protein, or person. However, for the purpose of this article and this issue, mutation is used to describe DNA changes because it is more familiar to most clinicians. When known, terms such as CF-causing mutation or mutation of unknown significance may be used to appropriately define disease liability.
The history of the discovery of cystic fibrosis transmembrane conductance regulator as the gene responsible for cystic fibrosis
Very soon after cystic fibrosis (CF) was recognized and characterized, clinicians caring for patients with CF observed that the disease occurred in families with a Mendelian autosomal recessive inheritance pattern. The study of CF was an important test case for the molecular tools used to study how DNA and genes are responsible for inherited traits. Beginning with restriction fragment length polymorphism analysis that allowed DNA segments differing by restriction enzyme sites to be sorted with inheritance of a phenotype, the gene responsible for CF could be localized to the long arm of chromosome 7. Coincident with these genetic advances, researchers examining the biochemical defect in the disease by studying the epithelial tissues affected by CF discovered that these tissues did not allow normal chloride transport. The genetic and biochemical investigational pathways converged with the identification of the CF transmembrane conductance regulator ( CFTR ) gene and its most common mutation (F508del) in 1989. Immediately after the gene was identified, it became apparent that multiple mutations in this gene could be responsible for CF ( Box 1 ).
The terms mutation and variant can both be used to describe a genetic difference from that which is commonly seen in a population (sometimes referred to as wild-type). In some writing, mutation implies that the genetic change is deleterious and variant is used when the change does not result in disease; however, both terms are used in the literature, somewhat interchangeably. The Human Genome Variation Society, American College of Medical Genetics and Genomics, and the Association for Molecular Pathology recommend use of the term variant , recognizing the potential negative connotations of the term mutation . Similarly, the terms polymorphism or single nucleotide polymorphism have been used to describe sequence changes occurring at greater than 1% frequency in the population but also to describe sequence changes that do not lead to disease (regardless of frequency). Therefore, consensus has been to use the term variant or sequence alteration to describe any DNA change, with further explanation given if the variant has been deemed to have an effect on the gene, protein, or person. However, for the purpose of this article and this issue, mutation is used to describe DNA changes because it is more familiar to most clinicians. When known, terms such as CF-causing mutation or mutation of unknown significance may be used to appropriately define disease liability.
Resources to describe and characterize variation in cystic fibrosis transmembrane conductance regulator
As understanding of CF has increased and more patients have been identified, advancing DNA technology has become a greater part of regular medical care and the number of CFTR mutations recognized has grown to more than 2000. Researchers at the University of Toronto and the Hospital for Sick Children began cataloging CFTR mutations in 1989 and continue to curate a publicly available online database of all genetic variations described in CFTR (the CF Mutation Database; http://www.genet.sickkids.on.ca/cftr/app ). This resource has limited clinical information and/or laboratory-based functional testing in some cases but was not established as a resource to determine whether or not a mutation causes CF. There are examples of benign CFTR variants being mistaken for causative mutations if functional investigations are not carried out or if other variants present are not recognized. The ramifications of an incorrect mutation annotation are now more severe as the CFTR genotype is increasingly being used as part of the CF diagnosis and to inform treatment.
To meet the growing need for a resource that characterizes mutations, the US CF Foundation amassed a team of international CF researchers to create the Clinical and Functional Translation of CFTR (CFTR2) database. CFTR2 was designed with the goal to identify and annotate the mutations that cause CF among all the mutations described in CFTR . The research group, with tremendous assistance of registries and national CF foundations, has collected genotype and phenotype data from patients with CF around the world (though most hail from North America and Europe). From the list of CFTR mutations observed in patients with CF, 3 separate analyses were used to evaluate and annotate these mutations: clinical characteristics, functional testing, and population/penetrance analysis. Sweat chloride concentration was used as a clinical filter because it is standardized, widely performed, and reflects CF severity. Functional evaluation was performed to quantify the effect of the mutation on how much protein is made, how it is processed, and whether the protein functions normally. Finally, population and penetrance analysis compared how much the mutation is seen in CF cases versus in the general population. CFTR mutations seen more commonly in the general population are suspicious that they are nonpenetrant (do not always result in CF) (see Box 3 on penetrance/expressivity later). This evaluation yields 3 possible outcomes ( Box 2 ): a mutation could be characterized as CF causing, as a mutation of varying clinical consequence (MVCC), or as non-CF causing. The initial phase of the project studied the most common 159 CFTR mutations that had an allele frequency of 0.01% or greater in a group of nearly 40,000 patients, with subsequent updates resulting in 88,664 patients with CF studied and a total of 276 mutations annotated. Table 1 summarizes available online resources to help interpret CFTR mutations.
The CFTR2 group is performing ongoing analysis of CFTR mutations to determine their likelihood to result in CF (disease liability). The following annotations are made based on the criteria detailed here:
CF-causing mutations : Mutations satisfying the 3 criteria listed next are characterized as CF-causing mutations.
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Clinical: Patients with one copy of the mutation and another copy of a known CF-causing mutation (such as F508del) have clinical features that satisfy the CF diagnostic criteria ; specifically, the mean sweat chloride of these patients is 60 mEq/L or greater.
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Functional: Mutations expected to result in a premature termination codon (class 1) are presumed dysfunctional and undergo no further testing. Mutations expected to result in an amino acid substitution or alter splicing efficiency are tested experimentally and deemed CF causing if they result in less than 10% of wild-type (nonmutated) CFTR protein levels or chloride conductance.
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Population/penetrance: Mutations with no evidence of reduced or nonpenetrance (that is, evidence supports that they will always cause CF when in trans with another CF-causing mutation) were used. Because there is no way to confirm full penetrance (without knowing everyone in the world’s genotype and whether or not they have CF), the double negative of the following is intentional: CF-causing mutations have no evidence of non penetrance.
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Mutation of varying clinical consequence (MVCC): Mutations not meeting clinical or functional criteria listed earlier but that have no evidence of nonpenetrance are characterized as MVCCs. These mutations may not always result in CF.
Non–CF-causing mutations : Mutations not meeting clinical and/or functional criteria listed earlier and that do have evidence of nonpenetrance are characterized as non-CF causing. Mutations are deemed to be nonpenetrant based on a study of fathers of CF offspring and evaluations of population frequencies of mutations. Rare individuals who have one non–CF-causing mutation in trans with a CF-causing mutation have presented with CFTR-related disorders or mild CF symptoms. For this reason, non–CF-causing mutations are not labeled as neutral or benign. However, these individuals are not expected to have life-limiting CF and represent only a small portion of those carrying non–CF-causing mutation in trans with a CF-causing mutation.
| Resource | Web Address | Information |
|---|---|---|
| CF Mutation Database | http://www.genet.sickkids.on.ca/app | Original site that described all variation in CFTR gene; useful to determine if a mutation has ever been seen before, describing a mutation using legacy and new nomenclature, and searching by location; contains some clinical and functional information, submitted by the clinic or laboratory that described the mutation |
| CFTR2 | http://cftr2.org/index.php | Database of CFTR mutations seen in patients with CF; mutations annotated to describe their disease liability; useful to determine if a mutation is CF causing, of varying clinical consequence, or non-CF causing |
| The following Web sites are not CF specific but may be helpful as they describe variation across the genome. | ||
| 1000 genomes | http://browser.1000genomes.org/index.html | Data from the HapMAP project; variants in exon as well as intron |
| UCSC | https://genome.ucsc.edu/ | Source that allows examination of variation in the entire genome |
| ExAC | http://exac.broadinstitute.org/ | Summary information from several bioinformatics resources; mostly exonic data |
Nomenclature and terminology
Nomenclature recommendations for mutations within CFTR have changed over time, creating a challenge for clinicians, patients, and researchers. The same mutation within CFTR may have different names that are, or were at one time, accurate.
The CFTR DNA sequence (like nearly all genes) contains both exons (sections of DNA that code for the protein) and introns (sections between exons that do not code for protein). As DNA is transcribed into mRNA, the introns are removed in a process called splicing. The CFTR gene was originally thought to have 24 exons, which were numbered as such. Subsequent findings indicated that there were actually 27, resulting in the addition of exons 6b, 14b, and 17b (exons 6, 14, and 17 renamed to 6a, 14a, and 17a, respectively). Codon (protein) numbering has always begun with the first ATG (methionine) being codon 1, but the original nucleotide numbering of the CFTR gene began 132 bases 5′ from the A of the ATG initiation codon, such that this A existed at nucleotide position 133.
Following the development of the Human Genome Variation Society (HGVS), an attempt to standardize genetic nomenclature across all genes was undertaken. This attempt resulted in the renumbering of CFTR nucleotides such that coding DNA position +1 now begins at the A of the initiation codon and differs from the colloquial numbering system by 132 bases. The amino acid numbering remains unchanged. Thus, the common mutation F508del ([delta]F508) was previously numbered to nucleotide position 1653 but now corresponds to c.1521. The 27 exons were also renumbered 1 to 27, eliminating the a and b versions. The CFTR mutation names before these changes are referred to as the legacy system.
The HGVS recommendations state that mutations should be described first at the DNA level and may also be described at the protein level. But because the CF community initially characterized most mutations using legacy nomenclature, most CFTR mutations have at least 3 names that may be recognizable and used in medical records. The legacy mutation G542X is now referred to as c.1624G>T (HGVS cDNA nomenclature) or p.Gly542Ter (HGVS protein nomenclature). Similarly, legacy mutation 2184insA is now known as c.2052delA or p.Lys684AsnfsX38. The difference in nucleotide numbering is due to the change in starting point described earlier.
When referring to a specific mutation in a patient’s medical record, it is recommended to list HGVS and legacy names, if known. This practice decreases the chance for misinterpretation by those reviewing the medical records and gives greater assurance that a patient’s mutations will be correctly recorded. It is also recommended to either include a copy of the genetic testing report in a patient’s record or, if original records cannot be included, copy the nomenclature from the report verbatim, as incorrect transcription of a mutation name can result in confusion. Nomenclature for several common CFTR mutations is described in Table 2 . Online resources, such as the CF Mutation Database and CFTR2, can be used to translate nomenclature.
| Legacy Name | HGVS cDNA | HGVS Protein | Other Known or Accepted Names |
|---|---|---|---|
| [delta]F508 | c.1521_1523delCTT | p.Phe508del | F508del, 1653delCTT, ΔF508 |
| G551D | c.1652G>A | p.Gly551Asp | — |
| 3849 + 10kbC>T | c.3717 + 12191C>T | No protein name | — |
| N1303K | c.3909C>G | c.Asn1303Lys | — |
| 621 + 1G>T | c.489 + 1G>T | No protein name | — |
| 3659delC | c.3528delC | p.Lys1177SerfsX15 | — |
| 2183AA>G | c.2051_2052delAAinsG | p.Lys684SerfsX38 | 2183delAA>G |
| 1716G/A | c.1584G>A | p.(Glu528 = ) | E528E |
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