ClinVar Genomic variation as it relates to human health

The CFTR c.1054C>T; p.Arg352Trp variant (rs193922497) is reported in individuals with cystic fibrosis (CF) (Schrijver 2005), mildly elevated sweat chloride (McGinniss 2005), and congenital absence of the vas deferens (CBAVD) (Picci 2010). In an individual with CBAVD, this variant was reported in trans to the pathogenic p.Phe508del variant (Picci 2010). Further, in testing performed at ARUP Laboratories, the p.Arg352Trp variant has been observed in multiple individuals with CFTR-related disorders that carry a second pathogenic variant, though affected individuals primarily exhibit mild forms of disease instead of classic CF. This variant is reported in ClinVar (Variation ID: 35816) and it is found in the Latino population with an allele frequency of 0.3% (105/35414 alleles) in the Genome Aggregation Database. The arginine at residue 352 is highly conserved, computational algorithms (PolyPhen-2, SIFT) predict that the variant is deleterious, and functional analyses report chloride channel activity of 11% of wildtype (CFTR2 database). Additionally, another variant in the same codon, p.Arg352Gln, is considered pathogenic (Sosnay 2013). Based on available information, the variant is classified as likely pathogenic. References: CFTR2 database: https://cftr2.org McGinniss M et al. Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples. Hum Genet. 2005 Dec; 118(3-4):331-8. Picci L et al. Identification of a D579G homozygote cystic fibrosis patient with pancreatic sufficiency and minor lung involvement. Hum Mutat. 1999; 13(2):173. Schrijver I et al. Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum. J Mol Diagn. 2005; 7(2):289-99. Sosnay P et al. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet. 2013; 45(10):1160-7.

Observed in multiple unrelated individuals with clinical features of cystic fibrosis or congenital absence of the vas deferens, some who also harbored second CFTR variants, but familial segregation information and additional clinical information was not included (Schrijver et al., 2005; Kanavakis et al., 2003; Picci et al., 2010); Observed in infants ascertained through newborn screening with negative or borderline sweat chloride test results, who also harbored additional CFTR variants, but segregation data was absent for some of these individuals (Soultan et al., 2008; Lilley et al., 2010); Classified as a variant of varying clinical consequence in a well-curated database (CFTR2); In silico analysis, which includes protein predictors and evolutionary conservation, supports that this variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 19897426, 21228398, 18421494, 10094564, 12752573, 16126774, 22043142, 29805046, 30888834, 36409994, 36670555, 17272608, 16189704, 19014821, 15858154)

The CFTR c.1054C>T variant is predicted to result in the amino acid substitution p.Arg352Trp. This variant has been reported in a patient with cystic fibrosis and a compound heterozygous individual with CBAVD (Schrijver et al 2005. PubMed ID: 15858154; Picci et al. 2010. PubMed ID: 19897426). This variant was also reported in a heterozygote in large-scale childhood disease carrier screening study (HGMD #CM962462 in Supplemental Table S9, Bell et al. 2011. PubMed ID: 21228398). However, no family or functional studies were conducted to confirm the pathogenicity of the p.Arg352Trp change in any of these studies. Two other missense changes at the same position, p.Arg352Gln and p.Arg352Gly, have also been reported in patients with cystic fibrosis (see Supplementary table 1 in Sosnay et al. 2013. PubMed ID: 23974870; Cremonesi et al. 1992. PubMed ID: 1284538). The amino acid residue p.Arg352 of the CFTR protein has been highly conserved during evolution. In ClinVar this variant is interpreted as uncertain and likely pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/35816/). At this time, its clinical significance is uncertain due to the absence of conclusive functional and genetic evidence.

The frequency of this variant in the general population, 0.003 (105/35414 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in individuals with cystic fibrosis (CF) (PMID: 12752573 (2003), 16784904 (2007), 17272608 (2007)). It was also reported in an individual with congenital bilateral absence of the vas deferens (CVABD) (PMID: 19897426 (2010)), an individual atypical CF (PMID: 16189704 (2005)), and multiple individuals with borderline/elevated sweat chloride results (PMID: 19014821 (2008), 22043142 (2010)). It was also identified in healthy, unaffected individuals (PMID: 16126774 (2005), 26755536 (2019)). An in vitro study found that this variant in combination with the CFTR p.Phe508del variant resulted in 12.1% of normal CFTR activity, however, the effect of this variant alone on CFTR protein function/activity was not established (PMID: 30888834 (2019)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, we are unable to determine the clinical significance of this variant.

The p.R352W variant (also known as c.1054C>T), located in coding exon 8 of the CFTR gene, results from a C to T substitution at nucleotide position 1054. The arginine at codon 352 is replaced by tryptophan, an amino acid with dissimilar properties. The p.R352W variant has been reported as a variant of varying clinical consequences (VVCC) (Sosnay PR et al. Pediatr. Clin. North Am., 2016 08;63:585-98; The Clinical and Functional TRanslation of CFTR (CFTR2); available at http://cftr2.org. Accessed September 17, 2020). In individuals with abnormal newborn screening results, this variant was identified in conjunction with p.F508del (phase unknown) in two individuals (Lilley M et al. Paediatr Child Health, 2010 Nov;15:590-4; Castellani C et al. Arch. Dis. Child., 2017 07;102:644-646) and was identified as part of a complex allele p.[R352W;P750L] and in trans with p.F508del in two individuals (McGinniss MJ et al. Hum. Genet., 2005 Dec;118:331-8; Soultan ZN et al. J. Pediatr., 2008 Dec;153:857-9). This variant was also identified in an adult male with congenital bilateral absence of the vas deferens (CBAVD) in conjunction with p.F508del; phase info was not provided (Picci L et al. J. Cyst. Fibros., 2010 Jan;9:29-35). Using a Xenopus laevis oocyte model, variants affecting the positive charge at codon 352 (p.R352A, p.R352E, p.R352Q) were observed to alter pore structure by disrupting the interaction between R352 and D993; however, p.R352W was not studied (Cui G et al. J. Membr. Biol., 2008 Mar;222:91-106). Based on internal structural analysis, R352W is deleterious and disrupts a characteristic residue critical for function (Aubin CN et al. J. Gen. Physiol., 2006 Nov;128:535-45; Cui G et al. J. Membr. Biol., 2008 Mar;222:91-106; Mornon JP et al. Cell. Mol. Life Sci., 2015 Apr;72:1377-403; (Liu F et al. Cell, 2017 03;169:85-95.e8). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on available evidence to date, the clinical significance of this alteration remains unclear.

Variant summary: CFTR c.1054C>T (p.Arg352Trp) results in a non-conservative amino acid change in the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00041 in 251136 control chromosomes, predominantly at a frequency of 0.003 within the Latino subpopulation in the gnomAD database. This frequency is not significantly higher than estimated for a pathogenic variant in CFTR causing CFTR-Related Diseases (0.00041 vs 0.013), allowing no conclusion about variant significance. c.1054C>T has been reported in the literature in both asymptomatic individuals and subjects diagnosed with multiple CFTR-related phenotypes, including congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis (CF). The variant has been reported in compound heterozygosity with the severe pathogenic variant p.Phe508del in individuals diagnosed with CBAVD (e.g. Schwarz_2009, Picci_2010) and in patients followed after a positive newborn screening test, but whom did not meet diagnostic criteria for a diagnosis of CF (e.g. Lilley_2010, Catellani_2016). These data suggest that the variant could be a "mild" mutation, which when combined with a more severe mutation can contribute to less severe CFTR-related phenotypes. c.1054C>T has also been reported in multiple CF patients, however without a second mutation and/or other clinical details available (e.g. Kavanakis_2003, Schrijver_2005, Ciminelli_2007, Giusti_2007, Kharazzi_2015, Salinas_2023). These data indicate that the variant may be associated with disease. At least one publication reports experimental evidence indicating that the variant, when expressed in-vitro in combination with the p.Phe508del mutation, results in approximately 12% of normal CFTR activity (e.g. McCague_2019). In addition, other functional studies investigating the Arg352 residue have reported that this region is important for stabilizing the channel's tertiary structure and predict that mutations in this amino acid may have a functional impact on the protein (e.g. Guinamard_1999, Cui_2008). Finally, when expressed alone in vitro, channel Cl- conductance and overall protein expression were reduced to <10% of wild type controls (e.g. Bihler_2024). A different amino acid substitution at this residue, p.Arg352Gln, has been classified by our laboratory as pathogenic, supporting the critical relevance of codon 352 to CFTR function. The CFTR2 database reports that this variant has varying consequences and that when combined with another CF-causing variant, some patients have CF, while others do not. The following publications have been ascertained in the context of this evaluation (PMID: 21228398, 38388235, 26755536, 11504857, 16784904, 18421494, 17272608, 10220340, 12752573, 26574590, 22043142, 25880441, 30888834, 16189704, 19897426, 29805046, 36409994, 15858154, NO_PMID). ClinVar contains an entry for this variant (Variation ID: 35816). Based on the evidence outlined above, the variant was classified as likely pathogenic.

The CFTR c.1054C>T (p.Arg352Trp) variant is a missense variant that has been reported in at least four studies, in which it is found in a compound heterozygous state with a second variant in four individuals, including in one individual with congenital bilateral absence of the vas deferens and in three newborns who underwent additional screening for cystic fibrosis (CF) following abnormal newborn screening for this condition (McGinniss et al. 2005; Soultan et al. 2008; Lilley et al. 2010; Picci et al. 2010). Two of the newborns were found to carry the p.Arg352Trp variant in cis with another missense variant on one allele and a classic CF variant on the other allele. The CF status of these newborns is not known. Control data are unavailable for this variant, which is reported at a frequency of 0.004764 in the Latino population of the Exome Aggregation Consortium. The evidence for this variant is limited. The p.Arg352Trp variant is therefore classified as a variant of unknown significance but suspicious for pathogenicity for CFTR-related disorders. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 352 of the CFTR protein (p.Arg352Trp). This variant is present in population databases (rs193922497, gnomAD 0.3%). This missense change has been observed in individuals with CFTR-related conditions (PMID: 19897426, 26574590). ClinVar contains an entry for this variant (Variation ID: 35816). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt CFTR protein function. This variant disrupts the p.Arg352 amino acid residue in CFTR. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 7544319, 20522854, 27086061, 28646244). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.