Gene test interpretation: CFTR

INTRODUCTION — This monograph discusses the interpretation of genetic testing for pathologic variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene and possible actions based on results. It does not discuss the indications for testing and is not intended to replace clinical judgment in the decision to test or in the clinical care of the individual who was tested. These subjects are discussed separately in UpToDate [1]. (See 'UpToDate topics' below.)

HOW TO READ THE REPORT

General principles

●Assess report for accuracy and interpretation (table 1).

●If the report is from direct-to-consumer testing or a research study and the results would impact clinical care, the patient should be retested using a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory with established procedures assuring proper specimen collection and labeling.

●The diagnosis of CF should rarely, if ever, be based on the results of genetic testing alone in the absence of clinical manifestations or physiologic corroboration of CFTR dysfunction (ie, abnormal sweat chloride test, transepithelial nasal potential difference measurement, or intestinal current measurement).

Though >2100 deoxyribonucleic acid (DNA) sequence variants have been identified in the CFTR gene, certainty regarding the potential to cause disease exists for only a subset. When genetic testing consists of genotyping a specific panel of CFTR variants known to cause CF, all variants tested are, by definition, known to be pathogenic; the report typically describes positive results as either mutations or pathogenic variants (table 2). In contrast, when CFTR gene sequencing is performed, the report will typically include all identified variants with classification of each variant as pathogenic, likely pathogenic, likely benign, or benign by the testing laboratory based on accumulated data (table 3) [2]. Variants for which pathogenicity cannot be characterized are called variants of uncertain significance (VUSs) and may be reclassified as more data emerge. (See "Secondary findings from genetic testing", section on 'Definitions and classification of variants'.)

CF-specific caveats

●Positive results – As CF is an autosomal recessive disease, genetic testing is considered definitively positive if the individual either [3]:

•Is homozygous for a variant known to be pathogenic; or

•Is compound heterozygous for two pathogenic variants that are in trans (one in each CFTR gene copy), established by confirming one variant was inherited from each parent.

Genotypes meeting the above criteria but including likely pathogenic variants can be considered positive if the clinical diagnosis is certain (eg, typical features of CF with a positive sweat chloride test).

Genotypes meeting the above criteria that include one or more VUS should be interpreted cautiously, particularly in cases where both variants are VUSs.

●Negative results – The clinical implication of a negative test depends on the method used [3]:

•Genotyping or targeted mutation analysis – A CF genotyping panel typically tests for 23 to >100 of the more commonly observed pathogenic variants that cause CF (generally those with a population frequency of at least 1 percent) (table 4). A negative test result only means that the individual does not carry any of the CFTR mutations tested. A negative result does not completely eliminate the possibility of being a CF carrier or, in the case of fetal testing, of having an affected child. This is because patients can have one or more rare variants that are not represented on such panels. The uncertainty is greatest in individuals of non-European ancestry, for whom data regarding the identity and frequency of pathogenic CF variants are lacking, or in individuals with a history of parental consanguinity. (See 'Negative screen' below.)

If the patient has a family history of CF or relatives who are CFTR variant carriers, the patient should be tested explicitly for the variants known to exist in the kindred. If such information is not available, sequencing is recommended rather than genotyping.

•Sequencing – There are several methods for gene sequencing, each with strengths and limitations. A negative sequencing test means only that no variants were identified in the parts of the gene that were adequately sequenced or that the only identified variants are ones known to be benign (ie, ones that do not cause disease). False negative results can arise if a pathogenic variant is missed in a gene region poorly covered by the sequencing assay, including regions that are difficult to sequence due to technical limitations or noncoding gene regions that are not included in assay. If a diagnosis of CF is suspected clinically, practitioners should verify the adequacy of sequence coverage (comments are often included in the report) and consult with a specialist if suspicions remain.

●False positives – Additional testing may be necessary to exclude false positive results. As an example, variants I506V, I507V, and F508C may lead to false positive reports of F508del or F507del. The laboratory usually performs this testing automatically (ie, reflex testing).

●5T/7T/9T testing – Additional testing, usually performed automatically (ie, reflex testing), helps predict the phenotype for certain variants. For example, when R117H is identified, testing for 5T/7T/9T variants helps predict the likelihood of congenital bilateral absence of the vas deferens.

●Compound heterozygotes – If genetic testing reveals two or more pathogenic variants at different positions within the gene, further evaluation is needed to evaluate whether the variants reside on the same chromosome (ie, they are in "cis," both inherited from one parent) or if each are on separate chromosomes (ie, in "trans," inherited from each parent separately) (figure 1). Because CF is autosomal recessive, only variants in trans cause disease. Cis versus trans positioning can be resolved by genotyping both variants in informative relatives (parents, siblings, children). The observation of only one of two variants in at least one of these individuals is typically sufficient to establish trans positioning.

ASYMPTOMATIC ADULTS

Carrier screening — Carrier screening is routinely offered to all females planning pregnancy or in early pregnancy. At a minimum, screening examines a panel of the 23 most common mutations (pathogenic variants) in the United States (table 4). (See "Cystic fibrosis: Carrier screening".)

For the purpose of prenatal screening and fetal diagnosis, predicting the potential fetal phenotype can be difficult. If the fetus shares at least one CFTR variant with an affected relative, the phenotype may be similar to that of the affected relative. However, there can be significant inter-individual phenotype variation among relatives with the same genotype, and the phenotype may be modified by the variant inherited from the other parent and other factors. These uncertainties must be shared with the patient. (See "Cystic fibrosis: Carrier screening", section on 'Prediction of phenotype after prenatal diagnosis'.)

Information on predicted phenotype is presented in separate searchable databases. (See 'Genetics resources' below.)

The clinical implication of variants identified depends on the specific variants and background risk (eg, family history). Partner testing informs the risk of having a child affected with CF with each pregnancy. Patient values and preferences will impact decisions about pregnancy. Moreover, disease-modifying therapies have dramatically improved the prognosis for CF, with a life expectancy for children born with CF today of at least 44 to 53 years (figure 2). Thus, shared decision-making must acknowledge uncertainties regarding the ability to predict the future of patients born with CF today.

Some pregnant individuals will use the screening results to avoid the birth of an affected child, while others will use it to plan for the birth of an affected child. Couples at risk who are not pregnant may elect to conceive using noncarrier donor gametes or in vitro fertilization (IVF) with preimplantation genetic testing and selection of unaffected embryos. (See "Preimplantation genetic testing".)

Some couples who would not terminate an affected pregnancy may decide not to be screened prenatally but to await the results of newborn screening.

Positive screen — Individuals with a positive screening test should receive genetic counseling, including the information that partner testing is indicated (algorithm 1).

If the father is unavailable for testing, the risk of an affected child is calculated based on the mother's test results and the background carrier rate for her partner's race and ethnicity (table 5).

If both partners carry a known pathogenic variant, there is a one in four chance with every pregnancy that their child will be affected. Affected offspring can be identified prenatally using chorionic villus sampling or amniocentesis. Because obstetric and neonatal management is not changed by the prenatal diagnosis of CF, couples who would not terminate an affected pregnancy may elect to delay testing until after birth to avoid the small risk of miscarriage from invasive diagnostic procedures. (See 'Newborn screening' below.)

Negative screen — A negative screening test only means that the individual does not carry any of the CF mutations included in the screening panel; a negative result does not eliminate the possibility of being a CF carrier. The degree to which the risk of being a carrier is reduced after a negative test is determined by the number of pathogenic variants screened and the patient's race and ethnicity. There is considerable variability across ethnicity and race with regard to the most common CF-causing mutations (table 5).

If there is a family history of CF, carrier screening should include the variants present in the affected relative in addition to those in the standard panel. If the affected relative had no or incomplete CF testing and is not available for testing, and standard screening of relative is negative, CFTR sequencing should be offered.

When a CF carrier's partner has a negative screen, further testing of the screen-negative partner may be indicated. A genetic counselor can help determine the next step (eg, CFTR sequencing versus expanded screening panels followed, if negative, by CFTR sequencing), taking into account background risk and insurance coverage.

Incidental finding — An individual may unexpectedly discover that they carry a pathogenic or likely pathogenic CFTR variant through testing done for research purposes, exome or genome testing performed for other clinical indications, or direct-to-consumer testing. If confirmed by repeat testing, genetic counseling is indicated (algorithm 1). Those with two copies of a pathogenic or likely pathogenic variant should be evaluated at a certified CF center.

Although some laboratories now offer cell-free DNA screening for CF, this test is not recommended because its diagnostic accuracy is unconfirmed. If a patient chooses this method of screening and the result is positive for CF, testing of the parents and the fetus by the standard methods outlined above is recommended. (See 'Carrier screening' above.)

NEWBORN SCREENING — Newborn screening typically measures blood levels of immunoreactive trypsinogen (IRT), with or without concurrent CFTR gene testing. If IRT is abnormal, genetic testing is indicated. (See "Cystic fibrosis: Clinical manifestations and diagnosis", section on 'Diagnosis'.)

Infants with one or more pathogenic CFTR variants are referred for sweat chloride testing, which distinguishes affected children from carriers in most cases. Infants with indeterminate results may be given a provisional diagnosis of CFTR-related metabolic syndrome (CRMS), also referred to as CF screen positive, inconclusive diagnosis (CFSPID).

Some cases of CF will be missed by newborn screening. CF should be suspected in individuals with suggestive symptoms, even when results of the newborn screen are negative or equivocal. CFTR gene sequencing may identify pathogenic variants in this setting.

SYMPTOMATIC INDIVIDUALS — Because universal newborn screening for CF is used in the United States, it is much less common for adults to have undiagnosed CF. However, individuals with milder CF or only a single feature of CF are more likely to present later in childhood or adulthood and to have uncommon CFTR variants not included in the standard screening panels. (See "Cystic fibrosis: Clinical manifestations and diagnosis", section on 'Regional variation in screening'.)

Thus, CFTR gene testing should be considered in individuals with a chronic condition that can be associated with CF such as pulmonary disease, recurrent pancreatitis, nasal polyps, chronic sinusitis, or male infertility due to absence of the vas deferens. (See 'Spectrum of disease' below.)

Such testing is usually conducted by a subspecialist. Genetic testing and counseling of such patients is complicated and often benefits from CF center expertise.

Due to the limitations of sequencing (particularly false negative results and the common findings of variants of unknown significance [VUSs]), genetic testing for CF should be performed in conjunction with sweat chloride testing to establish a diagnosis.

PATIENTS WITH ESTABLISHED DIAGNOSIS OF CF

Spectrum of disease — CF is a life-limiting, systemic disease with an autosomal recessive transmission pattern.

●Classic CF – Features of classic CF include bronchiectasis, sinusitis, pancreatitis, pancreatic exocrine insufficiency, malabsorption, CF-related diabetes mellitus, hepatobiliary disease with cirrhosis and/or portal hypertension, and absence of the vas deferens in males. Disease manifestations and severity vary widely and are impacted by inherited variants, environmental factors, and treatment. (See "Cystic fibrosis: Clinical manifestations and diagnosis".)

●CFTR-related disorders – Related but less severe conditions called CFTR-related disorders generally involve only one affected organ system, such as recurrent pancreatitis, recurrent bronchitis, or congenital absence of the vase deferens.

The most common pathogenic CFTR variants, F508del and W1282X, are highly penetrant and result in a reasonably consistent phenotype; all patients with F508del or W1282X who have another pathogenic variant will develop classic CF. In contrast, it is difficult to predict the phenotype if there is one pathogenic variant and one variant of uncertain significance (VUS). This may result in classic CF, a CFTR-related disorder, or a carrier state. As data accumulate, a VUS may be reclassified as pathogenic or benign, so clinicians must seek an updated interpretation before providing counseling.

Prognosis — CFTR genotype provides limited prognostic value with respect to overall survival or rate of lung function decline. From data collected prior to the widespread introduction of disease-modifier drugs, individuals with two known severe CFTR variants (Class I, II, and III CFTR mutations) have high annual rates of lung function decline and overall mortality compared with those with only one or no severe CFTR variants [4,5].

Treatment response — Results of CFTR gene testing helps guide treatment of CF because mutation class impacts treatment options. CF modifier medications result in dramatic improvements in lung function, reduced exacerbation rates, and better quality of life for individuals with responsive genotypes; this includes the majority of patients with CF [6,7]. However, up to 10 percent of individuals have CFTR genotypes that are unresponsive to therapy, including those with Class I (nonsense) mutations (table 6). (See "Cystic fibrosis: Genetics and pathogenesis" and "Cystic fibrosis: Treatment with CFTR modulators".)

CONSIDERATIONS FOR RELATIVES — CF is an autosomal recessive disorder. Relatives of individuals with pathogenic CFTR variants are at increased risk of being carriers and should be offered genetic counseling.

●Parents of patients with CF – Barring nonpaternity, unaffected parents of individuals with CF are usually obligate carriers.

●Siblings of patients with CF – Each full sibling of an individual with CF has a 25 percent chance of being affected, a 50 percent chance of being an asymptomatic carrier, and a 25 percent chance of being a noncarrier. This means unaffected siblings of a child with classic CF have a two in three chance (66 percent) of being a carrier.

●Offspring of patients with CF – An affected individual will transmit one pathogenic variant to offspring; therefore, offspring will either be asymptomatic carriers or will have CF depending on the genotype of the other parent.

●Siblings of a CF carrier – Each full sibling of a known carrier has a 50 percent chance of being a carrier.

RESOURCES

UpToDate topics

●(See "Cystic fibrosis: Carrier screening".)

●(See "Cystic fibrosis: Genetics and pathogenesis".)

●(See "Cystic fibrosis: Clinical manifestations and diagnosis".)

Genetics resources

Resources for locating a genetic counselor or specialized CF center:

●National Society of Genetic Counselors (NSGC)

●Cystic Fibrosis Foundation (www.cff.org)

Resources for determining pathogenicity of variants:

●Clinical and Functional Translation of CFTR International Consortium (cftr2.org)

●Cystic Fibrosis Mutation Database (www.genet.sickkids.on.ca/cftr/)

●CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr/)