ClinVar Genomic variation as it relates to human health

ACMG classification criteria: PS3, PM3, PP3

This sequence change replaces methionine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 467 of the SLC3A1 protein (p.Met467Thr). This variant is present in population databases (rs121912691, gnomAD 0.6%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individual(s) with cystinuria (PMID: 8054986, 8792820, 21677404). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 18115). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on SLC3A1 protein function. Experimental studies have shown that this missense change affects SLC3A1 function (PMID: 8054986, 9083097, 18332091). For these reasons, this variant has been classified as Pathogenic.

The SLC3A1 c.1400T>C; p.Met467Thr variant (rs121912691), is the most common variant in the SLC3A1 gene, and is reported in the literature in the homozygous or compound heterozygous state in multiple individuals and families affected with cystinuria (Bisceglia 1996, Calonge 1994, Gucev 2011, Halbritter 2015, Popovska-Jankovic 2013, Tanzer 2006). This variant is reported as pathogenic by multiple laboratories in ClinVar (Variation ID: 18115), and is found in the general population with an overall allele frequency of 0.24% (682/282,552 alleles, including 4 homozygotes) in the Genome Aggregation Database. The methionine at codon 467 is highly conserved, but computational analyses (SIFT, PolyPhen-2) predict that this variant is tolerated. However, functional analyses of the p.Met467Thr variant protein shows mislocalization leading to decreased transport activity (Bartoccioni 2008, Calonge 1994, Chillaron 1997). Additionally, other variants at this codon (c.1399A>G, p.Met467Val; c.1400T>A, p.Met467Lys) have been reported in individuals with cystinuria and are considered pathogenic (Bisceglia 1996, Calonge 1994, Popovska-Jankovic 2013, Shen 2017). Based on available information, the p.Met467Thr variant is considered to be pathogenic. Pathogenic variants in SLC3A1 are associated with cystinuria (MIM: 220100); both autosomal dominant and autosomal recessive transmission has been reported. References: Bartoccioni P et al. Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype. Hum Mol Genet. 2008 Jun 15;17(12):1845-54. Bisceglia L et al. Molecular analysis of the cystinuria disease gene: identification of four new mutations, one large deletion, and one polymorphism. Hum Genet. 1996 Oct;98(4):447-51. Calonge MJ et al. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat Genet. 1994 Apr;6(4):420-5. Chillaron J et al. An intracellular trafficking defect in type I cystinuria rBAT mutants M467T and M467K. J Biol Chem. 1997 Apr 4;272(14):9543-9. Gucev Z et al. Cystinuria AA (B): digenic inheritance with three mutations in two cystinuria genes. J Genet. 2011 Apr;90(1):157-9. Halbritter J et al. Fourteen monogenic genes account for 15% of nephrolithiasis/nephrocalcinosis. J Am Soc Nephrol. 2015 Mar;26(3):543-51. Popovska-Jankovic K et al. Molecular characterization of cystinuria in south-eastern European countries. Urolithiasis. 2013 Feb;41(1):21-30. Shen L et al. Clinical and genetic characterization of Chinese pediatric cystine stone patients. J Pediatr Urol. 2017 Dec;13(6):629.e1-629.e5. Tanzer F et al. Analysis of a 1-year-old cystinuric patient with recurrent renal stones. Int J Urol. 2006 Oct;13(10):1347-9.

The SLC3A1 c.1400T>C (p.Met467Thr) variant is well documented as the most common pathogenic variant associated with type 1 cystinuria. The p.Met467Thr variant has been found in 25 affected homozygotes, 43 affected compound heterozygotes, 22 affected heterozygotes, and 22 unaffected individuals (Calonge et al. 1994; Gasparini et al. 1995; Bisceglia et al. 1996; Harnevik et al. 2001; Botzenhart et al. 2002; Schmidt et al. 2004; Font-Llitjos et al. 2005; Guillen et al. 2005; Skopková et al. 2005; Tanzer et al. 2006; Tanzer et al. 2007; Barbosa et al. 2012; Popovska-Jankovic et al. 2013; Rhodes et al. 2015). The p.Met467Thr variant was found in a heterozygous state in four of 811 controls and is reported at a frequency of 0.00454 in the European (non-Finnish) population of the Exome Aggregation Consortium. The Met467 amino acid residue is very highly conserved in all known SLC3A1 sequences. Calonge et al. (1994) performed functional studies on the p.Met467Thr variant using expression analysis in a Xenopus oocyte system, demonstrating that the variant significantly altered amino acid transport and abolished 80% of normal activity. Chillaron et al. (1997) and Bartoccioni et al. (2008) subsequently demonstrated that the p.Met467Thr variant displays a trafficking defect that maintains an intracellular location rather than being located on the cell surface. Based on the collective evidence, the p.Met467Thr variant is classified as pathogenic for cystinuria. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The p.Met467Thr (NM_000341.3 c.1400T>C) variant in SLC3A1 has been reported in a t least 4 homozygous and 11 compound heterozygous individuals with cystinuria ( Halbritter 2015, Tostivint 2017, Gucev 2011, and Calonge 1994), and segregated i n 3 affected siblings in 2 families (Calonge 1994). This variant has also been r eported in ClinVar (Variation ID# 18115) as pathogenic by multiple laboratories. Functional studies provide some support that this variant impacts the protein ( Chillaron 1997, Bartoccioni 2008, and Calonge 1994). This variant has been ident ified in 0.57% (58/10144) of Ashkenazi Jewish chromosomes by the Genome Aggregat ion Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs121912691, rs121 912692), though its frequency is low enough to be consistent with a recessive ca rrier frequency. In summary, the p.Met467Thr variant is pathogenic for cystinuri a in an autosomal recessive manner based upon frequent biallelic observations in affected individuals, segregation and functional studies.

Reported as the most common pathogenic variant in SLC3A1, accounting for 29.3% of disease-causing alleles across multiple ethnic groups (Eggermann et al., 2012); Published functional studies demonstrate the absence of transport activity in mammalian cells transfected with the M467T variant (Bartoccioni et al., 2008); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect; This variant is associated with the following publications: (PMID: 14991253, 12234283, 10798361, 8054986, 18332091, 25296721, 9083097, 21677404, 8792820, 12820697, 10805464, 17010017, 30609409, 28812535, 15635077, 22480232, 16138908, 11748844, 28646536, 31589614, 10562926, 30069816, 33226606)

Variant summary: SLC3A1 c.1400T>C (p.Met467Thr) results in a non-conservative amino acid change located in the Glycosyl hydrolase, family 13, catalytic domain (IPR006047) of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.0025 in 251156 control chromosomes in the gnomAD database, including 4 homozygotes. c.1400T>C has been reported in the literature in multiple individuals affected with Cystinuria in homozygous (examples: Calonge_1994, Tanzer_2007, Harnevik_2001), compound heterozygous (Harnevik_2001) and heterozygous state (examples: Tanzer_2007, Harnevik_2001). The variant also segregated with the disease (Calonge_1994). These data indicate that the variant is very likely to be associated with disease. Multiple studies have shown that this variant impairs normal transport activity of the protein (examples: Calonge_1994, Bartoccioni_2008). A different variant affecting this residue (c.1400T>A, p.Met467Lys) has been classified pathogenic in ClinVar (CV ID 18116). The following publications have been ascertained in the context of this evaluation (PMID: 18332091, 8054986, 17880288, 11748844). ClinVar contains an entry for this variant (Variation ID: 18115). Based on the evidence outlined above, the variant was classified as pathogenic.

The c.1400T>C (p.M467T) alteration is located in exon 8 (coding exon 8) of the SLC3A1 gene. This alteration results from a T to C substitution at nucleotide position 1400, causing the methionine (M) at amino acid position 467 to be replaced by a threonine (T). Based on data from gnomAD, the C allele has an overall frequency of 0.241% (682/282552) total alleles studied. The highest observed frequency was 0.56% (58/10362) of Ashkenazi Jewish alleles. This is the most common alteration seen in individuals with cystinuria, accounting for approximately 30% of the known disease-causing alleles and detectable in nearly all ethnic groups (Eggermann, 2012). Numerous individuals affected with cystinuria and harboring this variant, in either the homozygous or compound heterozygous state, have been reported in the literature, and this alteration has been found to segregate with disease in multiple families (Calonge, 1994; Harnevik, 2001; Font-Llitjos, 2005; Halbritter, 2015; Rhodes, 2015). This amino acid position is well conserved in available vertebrate species. Functional studies of this variant, as well as a different variant at the same amino acid (p.M467K), have demonstrated that these alterations lead to delayed transport of the protein and failure to form functional heterotetramers (Calonge, 1994; Chillaron, 1997; Bartoccioni, 2008). This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

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 cystinuria (MIM#220100). (I) 0108 - This gene is associated with both recessive and dominant disease. Although predominantly associated with recessive disease, there are reports of affected carriers (PMID: 15635077, 25964309). (I) 0200 - Variant is predicted to result in a missense amino acid change from methionine to threonine. (I) 0251 - Variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (674 heterozygotes, 4 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (21 heterozygotes, 1 homozygote). (I) 0502 - Missense variant with conflicting in silico predictions and uninformative conservation. (I) 0600 - Variant is located in the annotated Alpha amylase catalytic domain or motif (NCBI). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant is reported as a common pathogenic variant in patients with cystinuria (ClinVar, PMID: 8054986, 25964309). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

PP1, PP3, PM3_very_strong, PS3_supporting, PS4_moderate

NM_000341.3:c.1400T>C in the SLC3A1 gene has an allele frequency of 0.006 in Ashkenazi Jewish subpopulation in the gnomAD database. Tostivint I et al. identified this variant in at least 12 patients with cystinuria, including 2 homozygotes and 10 compound heterozygotes: c.266T>C/c.1400T>C; c.1400T>C/c.592delG; c.1400T>C/c.1354C>T; c.1400T>C/c.1500+1G>T; etc.(PMID: 28646536 ). The p.Met467Thr (NM_000341.3 c.1400T>C) variant in SLC3A1 has also been found segregating in 3 affected siblings in one family (PMID: 8054986). Functional studies have shown that this missense change affects protein stability, impairs oligomerization and reduces amino acid transport in vitro (PMID: 18332091; 9083097; 8054986). Taken together, we interprete this variant as Pathogenic/Likely pathogenic variant. ACMG/AMP Criteria applied: PS3, PM3_Strong, PP1, PP4.

The SLC3A1 p.Met467Thr variant was identified in 19 of 782 proband chromosomes (frequency: 0.0448) from individuals or families with Cystinuria and kidney stones (Rhodes_2015_PMID:25964309; Halbritter_2015_PMID:25296721; Popovska-Jankovic_2013_PMID:23532419). The variant was identified in dbSNP (ID: rs121912691) and in ClinVar (classified as pathogenic by 7 submitters including GeneDx and Invitae; associated condition is Cystinuria). The variant was identified in control databases in 682 of 282552 chromosomes (4 homozygous) at a frequency of 0.002414 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: Ashkenazi Jewish in 58 of 10362 chromosomes (freq: 0.005597), European (non-Finnish) in 517 of 128916 chromosomes (freq: 0.00401), Other in 19 of 7206 chromosomes (freq: 0.002637), Latino in 35 of 35416 chromosomes (freq: 0.000988), European (Finnish) in 24 of 25124 chromosomes (freq: 0.000955), African in 20 of 24972 chromosomes (freq: 0.000801) and South Asian in 9 of 30610 chromosomes (freq: 0.000294); it was not observed in the East Asian populations. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. The p.Met467 residue is conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. However, functional studies using Xenopus oocytes showed M467T mutants to have reduced transport activity (Bartoccioni_2008_PMID:18332091, Chillarón_1997_PMID:9083097, Calonge_1994_PMID:8054986). In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.

The SLC3A1 c.1400T>C variant is predicted to result in the amino acid substitution p.Met467Thr. This variant has been reported to be causative for cystinuria in the homozygous and compound heterozygous state in multiple unrelated individuals (Calonge et al. 1994. PubMed: 8054986; Halbritter et al. 2015. PubMed: 25296721). Functional studies demonstrate the p.Met467Thr variant results in destabilized heterodimers (Bartoccioni et al. 2008. PubMed: 18332091). Autosomal recessive and dominant inheritance of pathogenic variants in the SLC3A1 gene has been reported previously (OMIM #220100); however, heterozygous carriers of the p.Met467Arg variant have been reported as unaffected (Calonge et al. 1994. PubMed: 8054986; Gucev et al. 2011. PubMed: 21677404). This variant is reported in 0.56% of alleles in individuals of Ashkenazi Jewish descent in gnomAD. This variant is interpreted as pathogenic for autosomal recessive SLC3A1-related disorders.