ClinVar Genomic variation as it relates to human health

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.

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 1070 of the CFTR protein (p.Arg1070Gln). This variant is present in population databases (rs78769542, gnomAD 0.5%), including at least one homozygous and/or hemizygous individual. This missense change has been observed in individual(s) with cystic fibrosis or congenital bilateral absence of the vas deferens. While this variant is commonly found in cis with p.Ser466*, it has also been observed without p.Ser466* in affected individuals and is expected to be causative for CFTR-related conditions whether p.Ser466* is present or not (PMID: 12955726, 18467194, 18951463, Invitae). Some individuals with this variant may present with milder symptoms. ClinVar contains an entry for this variant (Variation ID: 35866). 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 not expected to disrupt CFTR protein function with a negative predictive value of 80%. Experimental studies have shown that this missense change affects CFTR function (PMID: 8662892, 8702904, 11242048, 23891399). This variant disrupts the p.Arg1070 amino acid residue in CFTR. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 7539342, 16189704, 21520337, 23974870). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic.

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: PS3, PM3, PM5, PP3, PP4, BS2

The CFTR c.3209G>A (p.Arg1070Gln) variant has been reported in the published literature in individuals with cystic fibrosis (PMID: 7683628 (1993), 22658665 (2012), 30561903 (2019), 18951463 (2008), 29504914 (2018), 36272381 (2023)) and congenital bilateral absence of the vas deferens (CBAVD) (PMID: 12955726 (2003)). It was also found to cause a mild phenotype in the compound heterozygous state with a second CF-causing variant and was often associated with pancreatic insufficient CF in the presence of S466* variant in cis (PMID: 18951463 (2008), 22658665 (2012)). Published functional studies indicate that this variant affects chloride channel gating causing lower open probability of the channel and lower bicarbonate transport (PMID: 8662892 (1996), 11242048 (2001)). The frequency of this variant in the general population, 0.0047 (143/30596 chromosomes (Genome Aggregation Database, http://gnomad.broadinstitute.org)), is uninformative in the assessment of its pathogenicity. Based on the available information, this variant is classified as pathogenic.

CFTR: PS3, PS4, PP4:Moderate, PP3

The c.3209G>A; p.Arg1070Gln variant (rs78769542) has been observed in the compound heterozygous state in patients diagnosed with cystic fibrosis with pancreatic insufficiency (see link to CFTR2 database), or mild and atypical CFTR-related disorders, such as chronic pancreatitis and congenital absence of vas deferens (Feldmann 2003, Krasnov 2008), but its effects in the homozygous state are unknown. It is reported as pathogenic or likely pathogenic by several laboratories in Clinvar (Variation ID: 35866) and is observed in the South Asian population at an overall frequency of 0.46% (141/ 30766 alleles, 3 homozygotes) in the Genome Aggregation Database. The arginine at codon 1070 is highly conserved and computational algorithms (PolyPhen-2, SIFT) predict that the variant has an impact on the protein. Functional characterization of the variant protein is inconclusive on the expression level of the mature protein (Cotten 1996, Seibert 1996, Sosnay 2013, Van Goor 2014), but indicates an observable decrease in anion transport activity (Choi 2001, Seibert 1996, Sosnay 2013, Van Goor 2014). Based on available information, the p.Arg1070Gln variant is classified as pathogenic, with a variable presentation of clinical phenotypes. References: Link to CFTR2 database: https://www.cftr2.org/ Choi J et al. Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis. Nature. 2001;410(6824):94-7. Cotten J et al. Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1996;271(35):21279-84. Feldmann D et al. CFTR genotypes in patients with normal or borderline sweat chloride levels. Hum Mutat. 2003;22(4):340. Krasnov K et al. Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships. Hum Mutat. 2008;29(11):1364-72. Seibert F et al. Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity. J Biol Chem. 1996;271(25):15139-45. Sosnay PR et al. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet. 2013;45(10):1160-7. Van Goor F et al. Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function. J Cyst Fibros. 2014;13(1):29-36.

when the variant is in trans with another CF-causing variation, can either result in CF or in a CFTR-RD

The p.R1070Q pathogenic mutation (also known as c.3209G>A), located in coding exon 20 of the CFTR gene, results from a G to A substitution at nucleotide position 3209. The arginine at codon 1070 is replaced by glutamine, an amino acid with highly similar properties. Functional studies have shown that this mutation does not affect protein processing (Seibert FS et al. J. Biol. Chem., 1996 Jun;271:15139-45; Sosnay PR et al. Nat. Genet., 2013 Oct;45:1160-7), but it was observed to decrease the open probability of the channel (Seibert FS et al. J. Biol. Chem., 1996 Jun;271:15139-45). This mutation has been reported as a variant of varying clinical consequence (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 March 30, 2018); when in trans with a second disease causing mutation it may result in cystic fibrosis, CFTR-related disorders, or no phenotype (Bombieri C et al. Semin Respir Crit Care Med, 2015 Apr;36:180-93; Girardet A et al. Eur. J. Hum. Genet., 2016 Apr;24:469-78). Of note, this mutation has also been observed as part of a complex allele with p.S466* (Krasnov KV et al. Hum. Mutat., 2008 Nov;29:1364-72; Lucarelli M et al. Mol. Med., 2015 Apr;21:257-75). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

The observed missense c.3209G>A(p.Arg1070Gln) variant in CFTR gene has been observed in the compound heterozygous state in multiple individuals affected with cystic fibrosis (Feldmann et al., 2003, Krasnov et al., 2008). Experimental studies have shown that this missense change affects CFTR function (Van Goor et al., 2014). The p.Arg1070Gln variant has been reported with allele frequency of 0.06% in gnomAD Exomes. This variant has been submitted to the ClinVar database as Uncertain Significance / Likely Pathogenic / Pathogenic (multiple submissions). The amino acid change p.Arg1070Gln in CFTR is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Pathogenic. In absence of another reportable variant in CFTR gene, the molecular diagnosis is not confirmed.

Variant summary: CFTR c.3209G>A (p.Arg1070Gln) results in a conservative amino acid change located in the ABC transporter type 1, transmembrane domain (IPR011527) of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00061 in 251348 control chromosomes in the gnomAD database, including 3 homozygotes. This frequency is not significantly higher than estimated for a pathogenic variant in CFTR causing Non-Classic Cystic Fibrosis (0.00061 vs 0.013), allowing no conclusion about variant significance. This variant has been reported in several patients with classic CF, non-classic CF, or CBAVD and in several patients it was observed in cis with p.S466X forming a complex allele (example, Krasnov_2008, Mercier_1994, Kanavakis_1005, Savov_1994, Tzetis_2007, Feldmann_2003, Sosnay_2013, Furlan_2016, Lucarelli_2015, Palermo_2016, Behar_2017, Ivanov_2018, Salvatore_2019, Frentescu_2008, Noni_2023). Available patient data suggests that in compound heterozygotes, this variant typically results in mild disease such as CF/NC and/or CBAVD, while in cis with p.S466X variant, it causes classic CF (Krasnov_2008). However, the variant has also been reported in trans with F508del in a CF patient with sweat chloride levels >60 mmol/L (without p.S466X in cis; Noni_2023). Although this variant is able to mature and reach the cell surface and was found to have no significant effect on chloride transport, it was found to cause relatively subtle channel defects with functional consequences of a lower open probability of the channel, lower cyclic AMP-stimulated iodide efflux, and lower bicarbonate transport (Seibert_1996, Krasnov_2008, Sosnay_2013, Choi_2001). In these studies, maturation assays were performed on HEK-293, MDCK and COS cells, and channel function and channel transport assays were performed on HEK293 and CHO cells. In FRT cells, however, chloride transport as well as maturation was 20.5% of normal but increased to 32.9% of normal after Ivacaftor treatment (Van Goor_2014) and 42.64% of normal chloride channel conductance relative to wild type (Bihler_2024). These functional differences might be due to the use of different cell lines in different studies and technical backgrounds; however, all of these studies are suggestive of a mild functional impairment for the variant in isolation. A different variant affecting the same codon has been classified as pathogenic by our lab (c.3208C>T, p.Arg1070Trp), supporting the critical relevance of codon 1070 to CFTR protein function. However, due to the varying and typically mild disease phenotypes in individuals with this variant, as well as the allele frequency and presence of homozygotes in the gnomAD database, this variant likely represents a hypomorphic allele. The following publications have been ascertained in the context of this evaluation (PMID: 25910067, 1284534, 25087612, 23974870, 8662892, 16049310, 7512860, 30561903, 27171515, 36272381, 19707853, 7529319, 17489851, 23891399, 15880796, 7544320, 29504914, 30930780, 26014425, 27209008, 18467194, 12955726, 11242048, 12007216, 28546993, 18951463, 38388235). ClinVar contains an entry for this variant (Variation ID: 35866). Based on the evidence outlined above, the variant was classified as likely pathogenic.

The missense c.3209G>A (p.Arg1070Gln) variant in CFTR gene has been observed in multiple individuals affected with CFTR-related disorders (Feldmann et al., 2003, Frenţescu et al., 2008; Krasnov et al., 2008). Experimental studies showed that this variant cause relatively subtle channel defects with functional consequences of a lower open probability of the channel, lower cyclic AMP-stimulated iodide efflux, lower bicarbonate and anion transport (Seibert et al., 1996; Cotten et al., 1996; Krasnov et al., 2008; Sosnay et al., 2013). The p.Arg1070Gln variant is present with allele frequency of 0.06% in gnomAD Exomes. This variant has been submitted to the ClinVar database as Uncertain Significance / Likely Pathogenic / Pathogenic (multiple submissions). Computational evidence (Polyphen - Probably damaging, SIFT - Tolerated and MutationTaster - Disease causing) predicts conflicting evidence on protein structure and function for this variant. The reference amino acid of p.Arg1070Gln in CFTR is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. The amino acid Arg at position 1070 is changed to a Gln changing protein sequence and it might alter its composition and physico-chemical properties. For these reasons, this variant has been classified as Pathogenic.

The CFTR c.3209G>A variant is predicted to result in the amino acid substitution p.Arg1070Gln. This variant has been reported to be causative for cystic fibrosis (Mercier et al. 1993. PubMed ID: 7683628; Krasnov et al. 2008. PubMed ID: 18951463; Mickle et al. 2000. PubMed: 10762539; www.CFTR2.org). This variant is reported in 0.47% of alleles in individuals of South Asian descent in gnomAD. This variant is interpreted as pathogenic.