PharmGKB Level of Evidence 1A: Level 1A clinical annotations describe variant-drug combinations that have variant-specific prescribing guidance available in a current clinical guideline annotation or an FDA-approved drug label annotation. Annotations of drug labels or clinical guidelines must give prescribing guidance for specific variants (e.g. CYP2C9*3, HLA-B*57:01) or provide mapping from defined allele functions to diplotypes and phenotypes to be used as supporting evidence for a level 1A clinical annotation. Level 1A clinical annotations must also be supported by at least one publication in addition to a clinical guideline or drug label with variant-specific prescribing guidance.

Variant summary: The CFTR c.1647T>G (p.Ser549Arg) variant involves the alteration of a non-conserved nucleotide. 5/5 in silico tools predict a damaging outcome for this variant. This variant is absent in 120694 control chromosomes and was reported in numerous affected individuals in the literature. Functional studies show the variant to result in defective channel gating as the predominant defect, resulting in less than 1% chloride conductance compared to WT (Yu_2012, Sosnay_2013). In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic, including CFTR2. Ivacaftor addition caused a >10-fold increase in CFTR-mediated chloride transport in FRT cells expressing S549R-CFTR (Yu_2012) and this drug has been approved by the U.S. Food and Drug Administration to treat patients with CF who are 2 years and older with at least one copy of this mutation (CFTR2 database). Taken together, this variant is classified as pathogenic.

The p.S549R pathogenic mutation (also known as c.1647T>G and 1779T>G), located in coding exon 12 of the CFTR gene, results from a T to G substitution at nucleotide position 1647. The serine at codon 549 is replaced by arginine, an amino acid with dissimilar properties. This mutation has been described in the homozygous state in 15 individuals of Middle Eastern descent with pancreatic insufficiency, severe pulmonary disease, and elevated sweat chloride levels (Frossard PM et al. Eur. Respir. J., 1999 Jan;13:100-2). This pathogenic mutation is associated with elevated sweat chloride levels and pancreatic insufficiency; in vitro functional studies showed this mutation results very little to no to chloride conductance compared to wild type (Sosnay PR et al. Nat. Genet., 2013 Oct;45:1160-7). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Disease-causing CFTR variant. See www.CFTR2.org for phenotype information.

This variant is classified as Pathogenic. Evidence in support of pathogenic classification: Variant is present in gnomAD <0.01 for a recessive condition (v4: 11 heterozygote(s), 0 homozygote(s)). An alternative nucleotide substitution resulting in the same amino acid change is also present in gnomAD (v4: 2 heterozygote(s), 0 homozygote(s)); This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been classified as pathogenic by the CFTR2 expert panel for classic cystic fibrosis (ClinVar). Additional information: Variant is predicted to result in a missense amino acid change from serine to arginine; This variant is heterozygous; This gene is associated with autosomal recessive disease; An alternative amino acid change at the same position has been observed in gnomAD (v4: 64 heterozygote(s), 0 homozygote(s)); Loss of function is a known mechanism of disease in this gene and is associated with cystic fibrosis (MIM#219700); This variant has been shown to be paternally inherited by trio analysis.

CFTR: PM3:Very Strong, PM2, PP3

The c.1647T>G variant in CFTR is a missense variant predicted to cause substitution of serine to arginine at amino acid 549. This variant is rare in the general population with a frequency below the threshold expected for the associated phenotype(s). This variant has been observed in one or more individuals affected with the associated recessive disease, as either homozygous or compound heterozygous with a second variant (PMID: 30996306). Given the available evidence, this variant is classified as Pathogenic.

NM_000492.3(CFTR):c.1647T>G(S549R) is classified as pathogenic in the context of cystic fibrosis and is associated with the classic form of disease. Sources cited for classification include the following: PMID 23974870, 10401194 and 18456578. Classification of NM_000492.3(CFTR):c.1647T>G(S549R) is based on the following criteria: This is a well-established pathogenic variant in the literature that has been observed more frequently in patients with clinical diagnoses than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

This variant was identified in 1 patient with a clinically confirmed diagnosis of cystic fibrosis. The variant was classified in the context of a project re-classifying variants in the German Cystic Fibrosis Registry (Muko.e.V.). Link: https://www.muko.info/angebote/qualitaetsmanagement/register/cf-einrichtungen/mukoweb. Criteria applied: PS1, PS3_MOD, PM2, PM3_STR, PM5_STR, PP3, PP4

PS1,PS3,PM3(strong),PM2,PM5

This sequence change replaces serine, which is neutral and polar, with arginine, which is basic and polar, at codon 549 of the CFTR protein (p.Ser549Arg). This variant is present in population databases (rs121909005, gnomAD 0.006%). This missense change has been observed in individual(s) with cystic fibrosis or congenital absence of the vas deferens (PMID: 10923036, 11005149, 16840743, 23082198). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. ClinVar contains an entry for this variant (Variation ID: 40190). Invitae Evidence Modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) indicates that this missense variant is expected to disrupt CFTR protein function with a positive predictive value of 80%. Experimental studies have shown that this missense change affects CFTR function (PMID: 1712898, 10764788, 12080183, 23974870). For these reasons, this variant has been classified as Pathogenic.

The CFTR c.1647T>G variant is predicted to result in the amino acid substitution p.Ser549Arg. This variant has been documented as causative for cystic fibrosis (Kerem et al. 1990. PubMed ID: 2236053; Table S2 - Sosnay et al. 2013. PubMed ID: 23974870; cftr2.org). This variant is reported in 0.0058% of alleles in individuals of Latino descent in gnomAD. In summary, this variant is interpreted as pathogenic.