ClinVar Genomic variation as it relates to human health

The SLC22A5 c.1345T>G (p.Tyr449Asp) variant is a missense variant that has been reported in a total of three individuals with systemic primary carnitine deficiency, including in one who carried the variant in a compound heterozygous state and in one who carried the variant in a heterozygous state with an unidentified second variant. The third individual was heterozygous but also carried a variant in the VLCAD gene, which is associated with very-long-chain acyl-CoA dehydrogenase deficiency, a disease with phenotypic overlap to systemic primary carnitine deficiency (Amat di San Filippo and Longo 2004; Li et al. 2010). The p.Tyr449Asp variant was reported in a heterozygous state in one of 270 ethnically diverse healthy volunteers (Urban et al. 2006) and is reported at a frequency of 0.00605 in the African population of the 1000 Genomes Project. Functional studies in CHO cells (Amat di San Filippo and Longo 2004; Amat di San Filippo et al. 2008) and HEK 293 cells (Urban et al. 2006) indicate the variant impairs carnitine transport, reduces sodium stimulation of the channel, and affects substrate specificity but does not affect membrane localization or the kinetic constant of the channel. Based on the evidence, the p.Tyr449Asp variant is classified as a variant of unknown significance but suspicious for pathogenicity for systemic primary carnitine deficiency. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The SLC22A5 c.1345T>G, p.Tyr449Asp variant (rs11568514) has been reported in individuals with primary carnitine deficiency (Amat di San Filippo 2004, Li 2010). Functional characterization of the variant protein indicates a decrease in carnitine transport in response to sodium, and an altered preference for tetraethylammonium cation (Amat di San Filippo 2004, Urban 2006). The p.Tyr449Asp variant is located in the intracellular loop between transmembrane domains 10 and 11, and another alteration in this region (p.Glu452Lys) have also been shown to affect sodium-dependent carnitine transport (Wang 2000), suggesting that this region has functional significance. The variant is listed as pathogenic in ClinVar (Variation ID: 25420), and observed in the general population databases at a frequency of 0.2 percent in the 1000 Genomes Project (8/5008 alleles), 0.1 percent in the Exome Variant Server (16/13006 alleles), and 0.03 percent in the Genome Aggregation Database (90/277216 alleles). The tyrosine at residue 449 is moderately conserved, and computational algorithms (Align GVGD, Mutation Taster, PolyPhen-2, SIFT) predict that the variant has an impact on the protein. Based on the above information, the variant is classified as pathogenic. References: Amat di San Filippo C et al. Tyrosine residues affecting sodium stimulation of carnitine transport in the OCTN2 carnitine/organic cation transporter. J Biol Chem. 2004; 279(8):7247-53. Li F et al. Molecular spectrum of SLC22A5 (OCTN2) gene mutations detected in 143 subjects evaluated for systemic carnitine deficiency. Hum Mutat. 2010; 31(8):E1632-51. Urban T et al. Functional genetic diversity in the high-affinity carnitine transporter OCTN2 (SLC22A5). Mol Pharmacol. 2006; 70(5):1602-11. Wang Y et al. Abnormal sodium stimulation of carnitine transport in primary carnitine deficiency. J Biol Chem. 2000; 275(27):20782-6.

This sequence change replaces tyrosine, which is neutral and polar, with aspartic acid, which is acidic and polar, at codon 449 of the SLC22A5 protein (p.Tyr449Asp). This variant is present in population databases (rs11568514, gnomAD 0.4%). This missense change has been observed in individual(s) with primary carnitine deficiency (PMID: 28841266; Invitae). 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: 25420). 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 SLC22A5 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects SLC22A5 function (PMID: 14665638, 16652335, 16931768, 18337137). For these reasons, this variant has been classified as Pathogenic.

• The p.Tyr449Asp variant in the SLC22A5 gene has been previously reported in at least 6 individuals with systemic primary carnitine deficiency (Li et al., 2010; Vockley et al., 2000; Amat di San Filippo et al., 2004; Frigeni et al., 2017; Guevara-Campos et al., 2019). In one individual, this variant was observed in trans with another likely pathogenic/pathogenic variant (p.Leu269Argfs*26); and another individual was homozygous for this variant, consistent with autosomal recessive inheritance (Frigeni et al., 2017; ARUP Primary Carnitine Deficiency and SLC22A5 Database). • The p.Tyr449Asp variant has been identified in 92/24,958 African/African-American chromosomes by the Genome Aggregation Database (http://gnomad.broadinstitute.org/). • Functional studies of this variant are supportive of a deleterious effect to the protein; however, it is unclear if this would be sufficient to be disease-causing (Amat di San Filippo et al., 2004; Urban et al., 2006; Frigeni et al., 2017). • The p.Tyr449Asp variant is located in the intracellular loop between transmembrane domains 10 and 11 of SLC22A5 (Amat di San Filippo et al., 2004). Other nearby disease-associated variants have been described in this domain, including at least one that has been shown to reduce carnitine transport (Wang et al., 2000). • Computational tools predict that this variant is deleterious; however, the accuracy of in silico algorithms is limited. • These data were assessed using the ACMG/AMP variant interpretation guidelines. In summary, there is sufficient evidence to classify the p.Tyr449Asp variant as likely pathogenic for systemic primary carnitine deficiency in an autosomal recessive manner based on the information above. [ACMG evidence codes used: PS3_moderate; PM3; PM1; PP3]

ACMG classification criteria: PM2, PP3, BP1

Reported in an individual with 12.4% carnitine transport activity in fibroblasts who also harbored a frameshift variant in SLC22A5, however it is not known whether the two variants occurred on the same (in cis) or opposite (in trans) allele (PMID: 28841266); Reported in two individuals who were heterozygous for this single variant in SLC22A5 (PMID: 15714519, 14665638); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Published expression studies are conflicting as to whether the Y449D variant reduces carnitine transport to a level expected to be pathogenic (PMID: 16931768, 14665638); This variant is associated with the following publications: (PMID: 26828774, 18673259, 16602102, 23379544, 14665638, 25087612, 23757202, 20574985, 19940846, 16652335, 18337137, 31200524, 31980526, 32778825, 34637945, 35281663, 16931768, 38166572, 36343260, 15714519, 28841266)

Variant summary: SLC22A5 c.1345T>G (p.Tyr449Asp) results in a non-conservative amino acid change located in the Major facilitator superfamily domain (IPR020846) of 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 0.00025 in 251478 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in SLC22A5 causing Systemic Primary Carnitine Deficiency (0.00025 vs 0.0046), allowing no conclusion about variant significance. c.1345T>G has been reported in the literature as a non-informative genotype (second allele not specified) and at-least one compound heterozygous genotype in individuals affected with features of Systemic Primary Carnitine Deficiency (e.g, Amat di San Fillippo_2004, Dobrowlski_2005, Frigeni_2017, Li_2010). These data indicate that the variant may be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (example, Amat di San Fillippo_2004, Urban_2006). The most pronounced variant effect results in 18% of normal carnitine transporter activity in-vitro. The following publications have been ascertained in the context of this evaluation (PMID: 15714519, 28841266, 14665638, 31200524, 20574985, 16931768). ClinVar contains an entry for this variant (Variation ID: 25420). Based on the evidence outlined above, the variant was classified as likely pathogenic.

PP3, PM3_supporting, PS3_moderate