Functional Genetic Diversity in the High-Affinity Carnitine Transporter OCTN2 (SLC22A5)

Abstract

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n ϭ 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, Ϫ207GϾC. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (L-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p Ͻ 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V max for L-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the Ϫ207G allele showed increased L-carnitine transport compared with the Ϫ207C/C homozygotes (p Ͻ 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.

Cardiomyopathy and carnitine deficiency

“…2B). These results agree with previous expression studies in HEK 293 cells [23] and in CHO cells [19]. Statistical analysis indicated that heterozygosity for primary carnitine deficiency was not a risk factor for cardiomyopathy (odds ratio=0.55 with a 95% confidence interval of 0.09 to 3.33, i.e.…”

“…With this assumption, only 2/324 patients with cardiomyopathy were heterozygous for mutations in the carnitine transporter gene. This compares with a rate of 0.5-1% in the general population estimated from the 1:40,000-1:120,000 prevalence of primary carnitine deficiency [3][4][5] and with a frequency of 1.11% obtained from the analysis of 270 normal controls [23] and our expression studies of the changes identified (Fig. 2B).…”

“…1. DNA sequencing identified polymorphisms not affecting the amino acid residues and described in the normal population [23] in the DNA of 31 patients and variants of unknown significance in 7 patients (Table I). The L144F variant has been reported in normal controls, while T264M, E317K, and I312V have not yet been reported.…”

“…2B). Both patients carrying these mutations had dilated cardiomyopathy.Variations in the SLC22A5 gene in the general population were recently reported [23]. It is unclear how many of these variations are normal polymorphisms or might represent mutations.…”

Genetics of P450 oxidoreductase: Sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations

“…DNA was collected from 842 unrelated individuals in the San Francisco Bay Area as part of the Studies of Pharmacogenetics in Ethnically Diverse Populations (SOPHIE) project (32). Subjects identified themselves as African American, Caucasian American, Chinese American, or Mexican American by having all four grandparents of the stated ethnicity.…”