High-affinity Carnitine Transporter OCTN2 Research Articles

Identification of SLC22A5 Gene Mutation in a Family with Carnitine Uptake Defect.

Primary systemic carnitine deficiency is caused by homozygous or compound heterozygous mutation in the SLC22A5 gene on chromosome 5q31. The most common presentations are in infancy and early childhood with either metabolic decompensation or cardiac and myopathic manifestations. We report a case of 9-year-old boy with dysmorphic appearance and hypertrophic cardiomyopathy. Tandem MS spectrometry analysis was compatible with carnitine uptake defect (CUD). His sister had died due to sudden infant death at 19 months. His second 4-year-old sister's echocardiographic examination revealed hypertrophic cardiomyopathy, also suffering from easy fatigability. Her tandem MS spectrometry analyses resulted in CUD. We sequenced all the exons of the SLC22A5 gene encoding the high affinity carnitine transporter OCTN2 in the DNA. And one new mutation (c.1427T>G → p.Leu476Arg) was found in the boy and his sister in homozygous form, leading to the synthesis of an altered protein which causes CUD. The parent's molecular diagnosis supported the carrier status. In order to explore the genetic background of the patient's dysmorphic appearance, an array-CGH analysis was performed that revealed nine copy number variations only. Here we report a novel SLC22A5 mutation with the novel hallmark of its association with dysmorphologic feature.

Diagnoses of newborns and mothers with carnitine uptake defects through newborn screening

Carnitine uptake defect (CUD) is an autosomal recessive fatty acid oxidation defect caused by a deficiency of the high-affinity carnitine transporter OCTN2. CUD patients may present with hypoketotic hypoglycemia, hepatic encephalopathy or dilated cardiomyopathy. Tandem mass spectrometry screening of newborns can detect CUD, although transplacental transport of free carnitine from the mother may cause a higher free carnitine level and cause false negatives during newborn screening. From Jan 2001 to July 2009, newborns were screened for low free carnitine levels at the National Taiwan University Hospital screening center. Confirmation tests included dried blood spot free acylcarnitine levels and mutation analyses for both babies and their mothers. Sixteen newborns had confirmation tests for persistent low free carnitine levels; four had CUD, six had mothers with CUD, and six cases were false positives. All babies born to mothers with CUD had transient carnitine deficiency. The six mothers with CUD were put on carnitine supplementation (50–100mg/kg/day). One mother had dilated cardiomyopathy at diagnosis and her cardiac function improved after treatment. Analysis of the SLC22A5 gene revealed that p.S467C was the most common mutation in mothers with CUD, while p.R254X was the most common mutation in newborns and children with CUD. Newborn screening allows for the detection of CUD both in newborns and mothers, with an incidence in newborns of one in 67,000 (95% CI: one in 31,600–512,000) and a prevalence in mothers of one in 33,000 (95% CI: one in 18,700–169,000). Detection of CUD in mothers may prevent them from developing dilated cardiomyopathy.

Functional Genetic Variation in the Basal Promoter of the Organic Cation/Carnitine Transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5)

The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.

Cardiomyopathy and carnitine deficiency

Carnitine is essential for the transfer of long-chain fatty acids across the mitochondrial membrane for subsequent β-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 two 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.

Carnitine Membrane Transporter Deficiency: A Rare Treatable Cause of Cardiomyopathy and Anemia

Carnitine transporter defect is an autosomal recessive disorder caused by mutations in the SLC22A5 gene that encodes the high-affinity carnitine transporter OCTN2. Affected patients can present with predominant metabolic or cardiac manifestations. Early recognition of this disorder in a context of life-threatening cardiac failure and treatment with carnitine can be lifesaving in this inborn error of fatty acid oxidation. Here we describe a boy with a severe cardiomyopathy and severe anemia who improved with carnitine therapy. Physiopathology of anemia, a probably less recognized symptom of carnitine deficiency, is also discussed.

Carnitine/organic cation transporter OCTN2-mediated transport of carnitine in primary-cultured epididymal epithelial cells

Carnitine is essential for the acquisition of motility and maturation of spermatozoa in the epididymis, and is accumulated in epididymal fluid. In this study, carnitine transport into primary-cultured rat epididymal epithelial cells was characterized to clarify the nature of the transporter molecules involved. Uptake of carnitine by primary-cultured epididymal epithelial cells was time, Na(+) and concentration dependent. Kinetic analysis of carnitine uptake by the cells revealed the involvement of high- and low-affinity transport systems with Km values of 21 microM and 2.2 mM respectively. The uptake of carnitine by the cells was significantly reduced by inhibitors of carnitine/organic cation transporter (OCTN2), such as carnitine analogues and cationic compounds. In RT-PCR analysis, OCTN2 expression was detected. These results demonstrated that the high-affinity carnitine transporter OCTN2, which is localized at the basolateral membrane of epididymal epithelial cells, mediates carnitine supply into those cells from the systemic circulation as the first step of permeation from blood to spermatozoa.

OCTN2-mediated transport of carnitine in isolated Sertoli cells

Carnitine is extensively accumulated in epididymis. Carnitine is also accumulated in testis at higher concentration than in the plasma and is used in spite of the presence of the blood-testis barrier. In this study, we examined the characteristics of carnitine transport in primary-cultured rat Sertoli cells, which constitute a part of the blood-testis barrier. Uptake of [3H]carnitine (11.4 nM) from the basal side of Sertoli cells was Na+-dependent and was significantly decreased in the presence of 10 microM (48.0 +/- 7.4% of control) or 100 microM unlabeled carnitine (14.6 +/- 5.7% of control). Furthermore, the uptake was significantly inhibited in the presence of 100 microM acetyl-L-carnitine, 100 microM gamma-butyrobetaine or 500 microM quinidine. In RT-PCR analysis, the high-affinity carnitine transporter OCTN2 was detected in rat whole testis tissue and primary-cultured Sertoli cells. In contrast, the low-affinity carnitine transporter ATB(0,+) was detected in rat whole testis tissue, but not in primary cultured Sertoli cells. These results demonstrate that OCTN2 mediates carnitine supply to Sertoli cells from the circulation.

Carnitine transporter defect due to a novel mutation in the SLC22A5 gene presenting with peripheral neuropathy

The carnitine transporter defect (McKusick 212140) is an autosomal recessive disorder caused by mutations in the SLC22A5 gene, which encodes the high-affinity carnitine transporter OCTN2 (Wang et al 2001). Diagnosis is suspected when plasma carnitine levels are extremely low and secondary causes of carnitine loss are excluded. The disease can present with recurrent Reye-like episodes of hypoketotic hypoglycaemia or with cardiomyopathy associated with myopathy (Stanley et al 1991). Here we report novel clinical findings in a 3-year-old with primary carnitine deficiency.

Carnitine transport: pathophysiology and metabolism of known molecular defects.

Early-onset dilatative and/or hypertrophic cardiomyopathy with episodic hypoglycaemic coma and very low serum and tissue concentrations of carnitine should alert the clinician to the probability of the plasmalemmal high-affinity carnitine transporter defect. The diagnosis can be established by demonstration of impaired carnitine uptake in cultured skin fibroblasts or lymphoblasts and confirmed by mutation analysis of the human OCTN2 gene in the affected child and obligate heterozygote parents. The institution of high-dose oral carnitine supplementation reverses the pathology in this otherwise lethal autosomal recessive disease of childhood, and carnitine therapy from birth in prospectively screened siblings may altogether prevent the development of the clinical phenotype. Heterozygotes may be at risk for cardiomyopathy in later adult life, particularly in the presence of additional risk factors such as hypertension and competitive pharmacological agents. OCTN2 belongs to a family of organic cation/carnitine transporters that function primarily in the elimination of cationic drugs and other xenobiotics in kidney, intestine, liver and placenta. The high- and low-affinity human carnitine transporters, OCTN2 and OCTN1, are multifunctional polyspecific organic cation transporters; therefore, defects in these transporters may have widespread implications for the absorption and/or elimination of a number of key pharmacological agents such as cephalosporins, verapamil, quinidine and valproic acid. A third organic/cation carnitine transporter with high specificity for carnitine, Octn3, has been cloned in mice. The juvenile visceral steatosis (jvs) mouse serves as an excellent clinical, biochemical and molecular model for the high-affinity carnitine transporter OCTN2 defect and is due to a spontaneous point mutation in the murine Octn2 gene on mouse chromosome 11, which is syntenic to the human locus at 5q31 that harbours the human OCTN2 gene.

Na +-coupled transport of l-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney

The mechanism of Na +-dependent transport of l-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of l-carnitine via human OCTN2 was characterized. Uptake of l-[ 3H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na + gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of l-[ 3H]carnitine was specifically dependent on Na + and the osmolarity effect showed that Na + significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of l-carnitine by OCTN2. In addition, the fluxes of l-carnitine and Na + were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na + is coupled with flux of l-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of l-carnitine after glomerular filtration in the kidney.

GFP–Human High-Affinity Carnitine Transporter OCTN2 Protein: Subcellular Localization and Functional Restoration of Carnitine Uptake in Mutant Cell Lines with the Carnitine Transporter Defect

Individuals with the plasmalemmal high-affinity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts. The cDNA encoding the high-affinity sodium-dependent human carnitine transporter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lines with the CUD. Transfection of cell lines from 12 unrelated patients (nine fibroblast and three lymphoblastoid) with a GFP construct harboring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Transient and stable expression of the recombinant GFP–human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable transfection of the lymphoblastoid cell lines were achieved. There was functional restoration of carnitine uptake in the transfected mutant cell lines, thereby confirming the identity of the transfected cDNA. In addition, we report the first demonstration of the subcellular localization of an in-frame fusion GFP–human high-affinity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser-scanning fluorescence microscopy.