ClinVar Genomic variation as it relates to human health

PS3, PM2, PM3_Strong, PP3

The frequency of this variant in the general population is consistent with pathogenicity (http://gnomad.broadinstitute.org). Assessment of experimental evidence suggests this variant results in abnormal protein function. An in vitro study shows the variant protein is retained intracellulary and leads to loss of cotransporter function (PMID: 12039972). In multiple individuals, this variant has been seen with a single recessive pathogenic variant in the same gene, suggesting this variant may also be pathogenic.

This sequence change replaces glycine, which is neutral and non-polar, with arginine, which is basic and polar, at codon 741 of the SLC12A3 protein (p.Gly741Arg). This variant is present in population databases (rs138977195, gnomAD 0.07%). This missense change has been observed in individual(s) with Gitelman syndrome (PMID: 8528245, 17329572, 18391953, 21415153, 22009145, 23328711). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 208612). 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 SLC12A3 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects SLC12A3 function (PMID: 12039972). For these reasons, this variant has been classified as Pathogenic.

Across a selection of the available literature, the SLC12A3 c.2221G>A (p.Gly741Arg) missense variant has been reported in four patients with Gitelman syndrome in a homozygous state and in 79 patients in a compound heterozygous state (Simon et al. 1996; Vargas-Poussou et al. 2011; Glaudemans et al. 2012; Berry et al. 2013; Dongilli et al. 2016). Control data are not available for this variant which is reported at a frequency of 0.00063 in the European (non-Finnish) population of the Exome Aggregation Consortium. Functional studies in Xenopus oocytes demonstrated that the p.Gly741Arg variant resulted in no sodium ion uptake activity, showed intracellular localization rather than plasma membrane localization compared to wild type, and was not glycosylated (De Jong et al. 2002). Based on the collective evidence, the p.Gly741Arg variant is classified as pathogenic for Gitelman syndrome. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

This variant has been previously reported as a compound heterozygous change in patients with Gitelman syndrome (PMID: 8528245, 17329572, 21415153, 22009145, 23328711, 32939031). Functional studies have shown that this change results in reduced sodium uptake (PMID: 12039972). The c.2221G>A (p.Gly741Arg) variant is present in the heterozygous state in the gnomAD population database at a frequency of 0.03685% (104/282254) and is absent in the homozygous state, thus it is presumed to be rare. The c.2221G>A (p.Gly741Arg) variant affects a highly conserved amino acid and is predicted by multiple in silico tools to have a deleterious effect on protein function. Based on the available evidence, the c.2221G>A (p.Gly741Arg) variant is classified as Pathogenic.

The SLC12A3 c.2221G>A variant is classified as Pathogenic (PS3, PM3_strong2, PP3) The SLC12A3 c.2221G>A variant is a single nucleotide change in exon 18/26 of the SLC12A3 gene, which is predicted to change the amino acid glycine at position 741 in the protein to arginine. Functional studies show that the variant results in a non-functional protein (PMID:12039972)(PS3). This variant has been detected in trans with pathogenic variants for this recessive condition (PMID:17329572) (PM3_strong). Computational predictions support a deleterious effect on the gene or gene product (PP3). The variant has been reported in dbSNP (rs138977195), in population databases (gnomAD 62/151816, 0 homs, highest freq 0.079% non-Finnish European) and as disease causing in the HGMD database (CM961300). It has been reported as Conflicting interpretations of pathogenicity by other diagnostic laboratories (ClinVar Variation ID: 208612). The variant has been widely reported in the literature in patients with Gitelman syndrome and has been described as a hotspot mutation (PMID:8528245, 12039972, 17329572, 21415153, 22009145).

The c.2221G>A;p.(Gly741Arg) missense variant has been observed in affected individual(s) and ClinVar contains an entry for this variant (ClinVar ID: 208612; PMID: 18391953; 17329572; 21415153; 22009145; 23328711; 8528245) - PS4. The variant is present at low allele frequencies population databases (rs138977195 – gnomAD 0.0004084%; ABraOM no frequency - http://abraom.ib.usp.br/) - PM2_supporting. The variant co-segregated with disease in multiple affected family members (PMID: 8528245) - PP1. In summary, the currently available evidence indicates that the variant is likely pathogenic.

ACMG criteria used:PS3 PS4 PM1 PM2 PM3 PP3 PP5

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with Gitelman syndrome (MIM#263800). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from glycine to arginine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (104 heterozygotes, 0 homozygotes). (SP) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools and highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated SLC12 family domain (DECIPHER). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been classified as pathogenic by at least fifteen clinical diagnostic laboratories and has been reported as one of the five most frequent pathogenic missense variants associated with Gitelman syndrome in Caucasians (ClinVar; PMID: 35591852). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

proposed classification - variant undergoing re-assessment, contact laboratory

In vitro functional analysis demonstrated significantly diminished metolazone-sensitive sodium uptake levels in cells harboring this variant; immunocytochemical analysis revealed that the altered protein was predominantly localized to the cytoplasm rather than the plasma membrane as observed with the wild-type protein (De Jong et al., 2002); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 8528245, 18391953, 10988270, 9734597, 30201548, 23328711, 17329572, 21415153, 22009145, 26921350, 30596175, 31916727, 31980526, 30136149, 32939031, 31672324, 12039972, 27535533)

This individual is heterozygous for the c.2221G>A variant in the SLC12A3 gene. This variant has been widely reported in numerous patients with Gitelman syndrome in the literature (Vargas-Poussou et al 2011 J Am Soc Nephrol 22:693-703. This variant is considered to be pathogenic according to the ACMG guidelines.

The SLC12A3 c.2221G>A variant is predicted to result in the amino acid substitution p.Gly741Arg. This variant has been reported in many unrelated individuals to be pathogenic for Gitelman syndrome (Simon et al. 1996. PubMed ID: 8528245; De Jong et al. 2002. PubMed ID: 12039972; Supp. Table 1 in Fujimura et al. 2018. PubMed ID: 30596175; Supp. Table S1 in Hureaux et al. 2019. PubMed ID: 31672324; Zacchia et al. 2021. PubMed ID: 33964006). Functional studies indicate that the p.Gly741Arg change impacts protein function (De Jong et al. 2002. PubMed ID: 12039972). This variant is reported in 0.068% of alleles in individuals of European (non-Finnish) descent in gnomAD. This variant is interpreted as pathogenic.