A mutation creating an upstream translation initiation codon in SLC22A5 5'UTR is a frequent cause of primary carnitine deficiency

Abstract

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na⁺ -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.

Keywords: 5′-untranslated region; OCTN2 deficiency; carnitine transport; primary or systemic carnitine deficiency.

Figure 1

Molecular characterization of SLC22A5 c.‐149G>A variant. (a) Schematic representation of 5′UTR and exon 1 of SLC22A5. The c.‐149G>A variant introduces a novel translation initiation site. The predicted mutant protein is shown and the partial wild‐type protein. The mutant protein is predicted to result in a premature termination codon in exon 1. (b) Comparison of the sequence context surrounding the wild‐type and mutant AUG (created by c.‐149G>A variant) for the presence of a KOZAK sequence

Figure 2

Functional characterization of SLC22A5 c.‐149G>A variant. HeLa cells were transfected with constructs of the 5′‐UTR and the first 15 (i.e. c.1‐264C_c.15C) (a,c) or 16 (i.e. c.1‐264C_c.16) (b,d) coding nucleotides of exon 1 of SLC22A5 fused to NanoLuc. Left: graphical presentations of the constructs used (a–d). The positions and possible use (arrow) of the wild‐type initiation codon and the novel initiation codon introduced by the c.‐149G>A variant are indicated. Construct a and c have the original ATG in frame with the NanoLuc luciferase open reading frame, whereas construct b has the ATG introduced by c.1‐49G>A in frame with NanoLuc luciferase. Right: relative NanoLuc luciferase activity presented as % of the mean activity of two independently transfected and analyzed wild‐type constructs (a)

Figure 3

Functional consequence of SLC22A5 c.‐149G>A variant. The carnitine‐transport activity was measured in fibroblasts of control subjects and fibroblasts with different mutations in SLC22A5. The genotype of the different cell lines is indicated and the activity is expressed as a percentage of the mean activity of the two control cell lines measured in the same experiment. All measurements were done in duplicate and all cell lines were analyzed in two independent experiments minimally, except for one cell line (*) which was only analyzed in one experiment