Variant summary: SLC22A5 c.1319C>T (p.Thr440Met) 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 1.2e-05 in 251494 control chromosomes. c.1319C>T has been widely reported in the literature in multiple individuals affected with Systemic Primary Carnitine Deficiency and has been subsequently cited by others (example, Lamhonwah_2002, Wattanasirichaigoon_2006, Amat_2006, Rose_2012, Li_2010, Angelini_2015). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (example, Lamhonwah_2002). The most pronounced variant effect results in <10% of normal carnitine uptake in skin fibroblasts from a homozygous affected individual. Six clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change replaces threonine, which is neutral and polar, with methionine, which is neutral and non-polar, at codon 440 of the SLC22A5 protein (p.Thr440Met). This variant is present in population databases (rs72552732, gnomAD 0.003%). This missense change has been observed in individuals with primary carnitine deficiency (PMID: 12210323, 16652335, 16830263, 20574985, 21922592, 24997454). This variant is also known as c.1540C>T. ClinVar contains an entry for this variant (Variation ID: 25416). Invitae Evidence Modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) 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: 12210323, 21922592). For these reasons, this variant has been classified as Pathogenic.

PM1, PM2, PP2, PP3, PP5

Across a selection of the literature, the SLC22A5 c.1319C>T (p.Thr440Met) missense variant has been reported in ten individuals with systemic primary carnitine deficiency, including in one in a homozygous state, in eight in a compound heterozygous state, and in one in a heterozygous state (Lamhonwah et al. 2002; Amat di San Filippo et al. 2006; Li et al. 2010). The variant is also found in three unaffected heterozygotes. The p.Thr440Met variant is reported at a frequency of 0.00003 in the European (non-Finnish) population of the Exome Aggregation Consortium based on two alleles in an area of good coverage, so the variant is presumed to be rare. Functional studies performed on cultured patient skin fibroblasts and in CHO cells were performed to evaluate carnitine uptake and transport levels compared to wildtype. The p.Thr440Met variant was found to have carnitine transport level that was less than 10% of wildtype (Amat di San Filippo et al. 2006). Lamhonwah et al. (2002) reported that the carnitine uptake values ranged from 2.8% in a patient homozygous for the variant to 20% in a compound heterozygote, in comparison to controls. Based on the collective evidence, the p.Thr440Met variant is classified as pathogenic 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.1319C>T; p.Thr440Met variant (rs72552732), is reported in the literature in a homozygous or compound heterozygous state in multiple individuals affected with primary carnitine deficiency (Angelini 2015, Frigeni 2017, Lamhonwah 2002, Li 2010, Thompson 2018, Wattanasirichaigoon 2006) and cardiomyopathy (Papadopoulou-Legbelou 2017). This variant is reported as pathogenic/likely pathogenic by multiple laboratories in ClinVar (Variation ID: 25416), and is found in the non-Finnish European population with an overall allele frequency of 0.0027% (3/111718 alleles) in the Genome Aggregation Database. Functional analyses of the variant protein shows impaired carntitine transport (Amat di San Filippo 2006). The threonine at codon 440 is highly conserved, and computational analyses (SIFT, PolyPhen-2) predict that this variant is deleterious. Based on available information, this variant is considered to be pathogenic. References: Amat di San Filippo C et al. Pharmacological rescue of carnitine transport in primary carnitine deficiency. Hum Mutat. 2006 Jun;27(6):513-23. Angelini C et al. Spectrum of metabolic myopathies. Biochim Biophys Acta. 2015 Apr;1852(4):615-21. Frigeni M et al. Functional and molecular studies in primary carnitine deficiency. Hum Mutat. 2017 Dec;38(12):1684-1699. Lamhonwah AM et al. Novel OCTN2 mutations: no genotype-phenotype correlations: early carnitine therapy prevents cardiomyopathy. Am J Med Genet. 2002 Aug 15;111(3):271-84. Li FY et al. Molecular spectrum of SLC22A5 (OCTN2) gene mutations detected in 143 subjects evaluated for systemic carnitine deficiency. Hum Mutat. 2010 Aug;31(8):E1632-51. Papadopoulou-Legbelou K et al. Dilated Cardiomyopathy as the Only Clinical Manifestation of Carnitine Transporter Deficiency. Indian J Pediatr. 2017 Mar;84(3):231-233. Thompson ML et al. Genomic sequencing identifies secondary findings in a cohort of parent study participants. Genet Med. 2018 Apr 12. doi: 10.1038/gim.2018.53. (Epub ahead of print) Wattanasirichaigoon D et al. Pericardial effusion in primary systemic carnitine deficiency. J Inherit Metab Dis. 2006 Aug;29(4):589.

Published functional studies in CHO cells demonstrate a damaging effect on carnitine transport activity (Frigeni et al., 2017); Not observed at a significant frequency in large population cohorts (Lek et al., 2016); 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: 27807682, 29790872, 16865412, 12210323, 16830263, 20574985, 16652335, 24997454, 28841266, 23379544, 21922592, 16602102, 32276632)

PP1, PP3, PP4, PM2, PM3, PS3, PS4

The SLC22A5 c.1319C>T variant is predicted to result in the amino acid substitution p.Thr440Met. This variant has been reported, in the homozygous or compound heterozygous state, in multiple patients with systemic primary carnitine deficiency (e.g., Lamhonwah et al. 2002. PubMed ID: 12210323; Amat di San Filippo et al. 2006. PubMed ID: 16652335; Li et al. 2010. PubMed ID: 20574985; Angelini. 2015. PubMed ID: 24997454). In in vitro assays, the p.Thr440Met substitution has been shown to nearly abolish carnitine transport (Amat di San Filippo et al. 2006. PubMed ID: 16652335; Frigeni et al. 2017. PubMed ID: 28841266). This variant is reported in 0.0026% of alleles in individuals of European (Non-Finnish) descent in gnomAD. Based on the available evidence, we interpret this variant as pathogenic.