Tyrosine Residues Affecting Sodium Stimulation of Carnitine Transport in the OCTN2 Carnitine/Organic Cation Transporter

Filippo, Cristina Amat Di San; Longo, Nicola

doi:10.1074/jbc.m309171200

Cited by 20 publications

(27 citation statements)

References 21 publications

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“…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.…”

Section: Discussionsupporting

confidence: 82%

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Cardiomyopathy and carnitine deficiency

Filippo

Taylor

Mestroni

et al. 2008

Molecular Genetics and Metabolism

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Carnitine is essential for the transfer of long-chain fatty acids across the mitochondrial membrane for subsequent beta-oxidation. A defect in the high affinity carnitine transporter OCTN2 causes autosomal recessive primary carnitine deficiency that can present with hypoketotic hypoglycemia, mainly in infancy, or cardiomyopathy. Heterozygotes for primary carnitine deficiency can have mildly reduced plasma carnitine levels and can develop benign cardiac hypertrophy. In animal models, heterozygotes for this disease have a higher incidence of cardiomyopathy with aging. This study tested whether heterozygosity for primary carnitine deficiency was associated with cardiomyopathy. The frequency of mutations in the SLC22A5 gene encoding the OCTN2 carnitine transporter was determined in 324 patients with cardiomyopathy and compared to that described in the normal population. Missense variations identified in normal controls and patients with cardiomyopathy were expressed in Chinese Hamster Ovary cells to confirm a functional effect. Exons 2-10 of the SLC22A5 gene were amplified by PCR in the presence of LCGreen I™ and analyzed by dye-binding/high-resolution thermal denaturation. Exon 1 of the gene was sequenced in all patients. Heterozygosity for a few variants (L144F, T264M, I312V, E317K and R488H) was found in 6/324 patients with cardiomyopathy. Expression of these variants in CHO cells indicated that T264M decreased, E317K increased, while L144F, I312V, and R488H did not significantly affect carnitine transport. Expression in CHO cells of all the variants identified in a normal population indicated that only 2 had a functional effect (L17F and Y449D), while L144F, V481I, V481F, M530V, and P549S did not change significantly carnitine transport. The frequency of variants affecting carnitine transport was 2/324 patients with cardiomyopathy (0.61%) not significantly different from frequency of 3/270 (1.11%) in the general population. These results indicate that heterozygosity for primary carnitine deficiency is not more frequent in patients with unselected types of cardiomyopathy and is unlikely to be an important cause of cardiomyopathy in humans. KeywordsSLC22A5; OCTN2; carnitine transporter; cardiomyopathy; carnitine Primary carnitine deficiency (OMIM# 212140) is an autosomal recessive disorder of the carnitine cycle that results in defective fatty acid oxidation [1,2]. It has a frequency of ranging *Correspondence to: Nicola Longo, M.D., Ph.D., Division of Medical Genetics, Department of Pediatrics, University of Utah, 2C412 SOM, 50 North Medical Drive, Salt Lake City UT 84132, USA. Phone 801 585 2457; Fax 801 587 7690, E-mail: Nicola.Longo@hsc.utah.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the pr...

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Section: Discussionsupporting

confidence: 82%

“…The final clones were sequenced to confirm the presence of the mutation and the absence of PCR artifacts. The clones were transfected into CHO cells using lipofectamine [18,19]. Cells were selected for 2 weeks in 0.8 mg/ml of G418 and then used for the transport assay.…”

Section: Methodsmentioning

confidence: 99%

Cardiomyopathy and carnitine deficiency

Filippo

Taylor

Mestroni

et al. 2008

Molecular Genetics and Metabolism

Self Cite

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“…It is noteworthy that one OCTN2 variant, Tyr449Asp, had been identified previously in the heterozygous state in a patient who died of sudden cardiac arrest at 3 months of age (Vockley et al, 2000;Amat di San Filippo and Longo, 2004). This patient was found on autopsy to have moderately reduced carnitine transport activity (to 57% of control) and reduced very long-chain acyl-CoA dehydrogenase (VLCAD) activity (to 46% of control) in cultured fibroblasts.…”

Section: Resultsmentioning

confidence: 99%

“…Five of these had previously been reported on the NCBI single nucleotide polymorphism database (dbSNP): two high- frequency synonymous SNPs (c.285CϾT and c.807GϾA), one amino acid substitution (L144F), one intronic SNP (IVS4 ϩ 13CϾT), and one SNP in the 3Ј-untranslated region. In addition, one rare variant (Tyr449Asp) had been identified previously in a patient with a suspected carnitine transport defect (Amat di San Filippo and Longo, 2004).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2006

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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.

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“…Patients with carnitine deficiency exhibit encephalopathy, progressive cardiomyopathy, hypoglycemia, and skeletal myopathy. More than 15 mutations in SLC22A5 have been associated with primary carnitine deficiency Seth et al, 1999;Wang et al, 1999Wang et al, , 2000aMayatepek et al, 2000;Cederbaum et al, 2002;Spiekerkoetter et al, 2003;Amat di San Filippo and Longo, 2004;Makhseed et al, 2004;Melegh et al, 2004). One of these mutations (P478L) causes complete loss of carnitine uptake but still retains transport activity of organic cations .…”

Section: G Heartmentioning

confidence: 99%