Functional and molecular studies in primary carnitine deficiency

Abstract

Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported (n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen-2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency.

Keywords: OCTN2; SLC22A5; carnitine deficiency; carnitine transport; carnitine uptake defect; fatty acid oxidation; mutations; newborn screening.

Figure 1. Carnitine transport by fibroblasts obtained from 358 individuals referred for possible primary carnitine deficiency

³H-Carnitine (0.5 μM) transport was measured for 4 hours at 37°C. Nonsaturable transport, measured in the presence of 2 mM cold carnitine, was subtracted from total transport to obtain saturable carnitine transport. Transport activity was normalized to the one of simultaneous normal controls (100% activity). Each bar represents the average of 6 observations in two separate experiments with the sample numbered from 1 (lowest activity) to 358 (highest activity) indicated on the X-axis. Carnitine transport activity of 20% or less of normal controls was indicative of primary carnitine deficiency (continuous line). Fibroblasts from some of the subjects had carnitine transport higher than their paired normal controls (100% activity, dashed line).

Figure 2. Schematic of the OCTN2 carnitine transporter with location of the 92 variants expressed in CHO cells

The OCTN2 carnitine transporter is composed of 557 amino acids. Hydropathy analysis and conservation with other organic cation transporters indicates that the transporter forms 12 transmembrane spanning domains (rectangles) with both the N- and C-termini facing the cytoplasm. Glycosylation sites are indicated by branching (Filippo, et al., 2011). The 92 OCTN2 variants identified and expressed in CHO cells are indicated by circles. Mutations reducing carnitine transport to 20% or less than wild-type when expressed in CHO cells are indicated by full circles; variants retaining more that 20% residual activity are indicated by empty circles. The A142S and R488H mutations (indicated by black stripes circles) impair carnitine transport when in cis on the same allele, but not alone (Amat di San Filippo, et al., 2006).