Question

Describe the application of molecular testing to newborn screening for cystic fibrosis (CF)

Answer 1

CF is a relatively common, autosomal recessive, and frequently lethal condition caused by mutations in the CF transmembrane conductance regulator gene (CFTR). CFTR consists of 27 exons, spanning approximately 250 kb on 7q31.2.1 CFTR is a member of the ATP-binding cassette transporter family and encodes an anion transporter protein in the epithelium with five domains. Two membrane-spanning domains form a chloride channel pore that plays a role in chlorine and bicarbonate transport and have secondary effects on sodium transport. CFTR protein dysfunction leads to increased salt concentration in sweat and thickened secretions in various organ systems. Numerous genetic mutations have been identified; their characterization and contribution to disease pathogenesis are discussed below. The clinical presentation ranges from multiorgan symptoms, such as chronic respiratory tract infections, failure to thrive, and pancreatic insufficiency starting in infancy, to single-organ manifestations, such as male infertility or chronic sinusitis in adulthood.

The purpose of molecular diagnostic testing (in contrast to molecular testing as part of population screening) is to provide genetic characterization of individuals with clinical or suspected CF. Reasons for pursuing such diagnostic testing include prenatal diagnosis in a carrier couple, newborn screening follow-up, clinical symptoms consistent with CF phenotypes, and a family history of a relative with CF or with a CF-like condition. The benefits of testing include earlier and definitive diagnosis, improved CF-specific care, clarification of atypical cases, and attainment of the information required for providing counseling regarding recurrence risk and fertility options.

CFTR molecular testing is technically straightforward, most commonly using a blood sample and test modalities that include established mutation panels, sequencing, and methods that can detect deletions and duplications usually missed by sequencing. Next-generation sequencing will provide numerous variants and has the potential to combine sequencing with the detection of deletions and duplication. Variants of unknown significance will inevitably present; however, this is seen with Sanger sequencing, as well. Clinicians need to be aware of the limitations of the test being ordered and pay particular attention to test ordering (eg, provide ethnic background, family history of CF, and familial mutations, if available).

For patients with symptoms of CF or a family history, the US guidelines recommend sweat chloride testing with age-appropriate screening thresholds (20 mmol/L, <6 months of age; 40 mmol/L, >6 months of age). If the sweat chloride result is lower than the threshold and two CFTR mutations were not identified with appropriate testing, then the individual is unlikely to have CF.6 If the sweat chloride result is lower than the threshold in the presence of two CF-causing mutations, then the diagnosis of CF can be made. If the sweat chloride test result is higher than the threshold, the diagnosis can be made by confirmatory sweat chloride testing (≥60 mmol/L) or by identification of two CFTR mutations, which is often initially pursued using the ACMG panel, originally designed for carrier screening and, in many ethnic groups, suboptimal for diagnostic testing. With an indeterminate sweat chloride result (higher than the threshold but <60 mmol/L), more extensive CFTR mutation testing is recommended and if two CFTR mutations are then identified, the diagnosis of CF is made. If, however, zero or one CFTR mutation is identified, then CFTR-related disorder or CF both remain possibilities and additional nasal potential difference testing may help distinguish between the two.

For patients with symptoms of CF or a family history, the US guidelines recommend sweat chloride testing with age-appropriate screening thresholds (20 mmol/L, <6 months of age; 40 mmol/L, >6 months of age). If the sweat chloride result is lower than the threshold and two CFTR mutations were not identified with appropriate testing, then the individual is unlikely to have CF.6 If the sweat chloride result is lower than the threshold in the presence of two CF-causing mutations, then the diagnosis of CF can be made. If the sweat chloride test result is higher than the threshold, the diagnosis can be made by confirmatory sweat chloride testing (≥60 mmol/L) or by identification of two CFTR mutations, which is often initially pursued using the ACMG panel, originally designed for carrier screening and, in many ethnic groups, suboptimal for diagnostic testing. With an indeterminate sweat chloride result (higher than the threshold but <60 mmol/L), more extensive CFTR mutation testing is recommended and if two CFTR mutations are then identified, the diagnosis of CF is made. If, however, zero or one CFTR mutation is identified, then CFTR-related disorder or CF both remain possibilities and additional nasal potential difference testing may help distinguish between the two.