Functional analysis of mutations in the OCTN2 transporter causing primary carnitine deficiency: lack of genotype-phenotype correlation

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease is caused by mutations in the novel organic cation transporter OCTN2 (SLC22A5 gene). The disease can present early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. To determine whether the variation in phenotypic severity is due to mutations retaining residual function, we extended mutational analysis of OCTN2 to four additional European families with primary carnitine deficiency. Three patients were homozygous for novel missense mutations (R169W, G242V, A301D). The fourth patient was compound heterozygous for R169W and W351R substitutions. Stable expression of all the mutations in CHO cells confirmed that all mutations abolished carnitine transport, with the exception of the A301D mutation in which residual carnitine transport was 2-3% of the value measured in cells expressing the normal OCTN2 cDNA. Analysis of the patients characterized in molecular detail by our laboratory failed to indicate a correlation between residual carnitine transport and severity of the phenotype or age at presentation.     

Genotype-phenotype correlation in primary carnitine deficiency

Primary carnitine deficiency is caused by defective OCTN2 carnitine transporters encoded by the SLC22A5 gene. Lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy. Recently, asymptomatic mothers with primary carnitine deficiency were identified by low carnitine levels in their infant by newborn screening. Here, we evaluate mutations in the SLC22A5 gene and carnitine transport in fibroblasts from symptomatic patients and asymptomatic women. Carnitine transport was significantly reduced in fibroblasts obtained from all patients with primary carnitine deficiency, but was significantly higher in the asymptomatic women's than in the symptomatic patients' fibroblasts (P < 0.01). By contrast, ergothioneine transport (a selective substrate of the OCTN1 transporter, tested here as a control) was similar in cells from controls and patients with carnitine deficiency. DNA sequencing indicated an increased frequency of nonsense mutations in symptomatic patients (P < 0.001). Expression of the missense mutations in Chinese hamster ovary (CHO) cells indicated that many mutations retained residual carnitine transport activity, with no difference in the average activity of missense mutations identified in symptomatic versus asymptomatic patients. These results indicate that cells from asymptomatic women have on average higher levels of residual carnitine transport activity as compared to that of symptomatic patients due to the presence of at least one missense mutation.     

Carnitine transport by organic cation transporters and systemic carnitine deficiency

The intracellular homeostasis is controlled by different membrane transporters. Organic cation transporters function primarily in the elimination of cationic drugs, endogenous amines, and other xenobiotics in tissues such as the kidney, intestine, and liver. Among these molecules, carnitine is an endogenous amine which is an essential cofactor for mitochondrial beta-oxidation. Recently, a new family of transporters, named OCT (organic cation transporters) has been described. In this minireview, we present the recent knowledge about OCT and focus on carnitine transport, more particularly by the OCTN2. The importance of this sodium-dependent carnitine cotransporter, OCTN2, comes from various recently reported mutations in the gene which give rise to the primary systemic carnitine deficiency (SCD; OMIM 212140). The SCD is an autosomal recessive disorder of fatty acid oxidation characterized by skeletal myopathy, progressive cardiomyopathy, hypoglycemia and hyperammonemia. Most of the OCTN2 mutations identified in humans with SCD result in loss of carnitine transport function. Identifying these mutations will allow an easy targeting of the SCD syndrome. The characteristics of the juvenile visceral steatosis (jvs) mouse, an animal model of SCD showing similar symptoms as humans having this genetic disorder, are also described. These mice have a mutation in the gene encoding the mouse carnitine transporter octn2. Although various OCTN carnitine transporters have been identified and functionally characterized, their membrane localization and regulation are still unknown and must be investigated. This knowledge will also help in designing new drugs that regulate carnitine transport activity.     

Cardiomyopathy and carnitine deficiency

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 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.     

Two novel missense mutations of the OCTN2 gene (W283R and V446F) in a patient with primary systemic carnitine deficiency

Primary systemic carnitine deficiency (SCD) is an autosomal recessive disorder of fatty acid oxidation caused by defective cellular carnitine transport. The disease is characterized by metabolic derangement simulating Reye's syndrome, hypoglcaemia, progressive cardiomyopathy and skeletal myopathy. Recently, it was shown that SCD is caused by mutations in the organic cation/carnitine transporter OCTN2 (SLC22A5). We report two novel mutations, W283R and V446F, which are both missense mutations in an affected infant. In vitro expression studies demonstrated that both are actually function-loss mutations with virtually no uptake activity. This is the first report of compound heterozygosity for two missense mutations in a patient with SCD. Hum Mutat 15:118, 2000.     

Mutations of OCTN2, an organic cation/carnitine transporter, lead to deficient cellular carnitine uptake in primary carnitine deficiency

Systemic primary carnitine deficiency (CDSP, OMIM 212140) is an autosomal recessive disease characterized by low serum and intracellular concentrations of carnitine. CDSP may present with acute metabolic derangement simulating Reye's syndrome within the first 2 years of life. After 3 years of age, patients with CDSP may present with cardiomyopathy and muscle weakness. A linkage with D5S436 in 5q was reported in a family. A recently cloned homologue of the organic cation transporter, OCTN2, which has sodium-dependent carnitine uptake properties, was also mapped to the same locus. We screened for mutation in OCTN2 in a confirmed CDSP family. One truncating mutation (Trp132Stop) and one missense mutation (Pro478Leu) of OCTN2 were identified together with two silent polymorphisms. Expression of the mutant cDNAs revealed virtually no uptake activity for both mutations. Our data indicate that mutations in OCTN2 are responsible for CDSP. Identification of the underlying gene in this disease will allow rapid detection of carriers and postnatal diagnosis of affected patients.     

Novel OCTN2 mutations: no genotype-phenotype correlations: early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile-onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L-carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high-affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 microM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11-bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype.     

A missense mutation in the OCTN2 gene associated with residual carnitine transport activity

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease can present early in life with hypoketotic hypoglycemia and acute metabolic decompensation, or later in life with skeletal or cardiac myopathy. Mutations abolishing the function of OCTN2, an organic cation/carnitine transporter with twelve putative transmembrane spanning domains, were recently demonstrated in patients with early- and late-onset (up to seven years of age) presentation of this syndrome. Most of the reported mutations are null alleles. Here we evaluate the OCTN2 gene in a male patient who presented at seven years of age with severe dilated cardiomyopathy. Plasma carnitine levels were undetectable and carnitine transport by his fibroblasts was reduced to about 3% of normal controls. This patient was homozygous for a single base pair change in exon 8 of the OCTN2 gene (1354G>A) converting the codon for Glu 452 to Lys (E452K) in the predicted intracellular loop between transmembrane domains 10 and 11. Stable expression of the mutant E452K-OCTN2 cDNA in Chinese hamster ovary (CHO) cells caused a partial increase in carnitine transport to 2-4% of the levels measured in the wild type transporter. This reduced transport activity was associated with normal Km toward carnitine (3.1 +/- 1.1 microM), but markedly reduced Vmax. These results indicate that primary carnitine deficiency can be caused by mutations encoding for carnitine transporters with residual activity, and that the E452K affects a domain not involved in carnitine recognition.     

Disorders of carnitine transport and the carnitine cycle

Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta oxidation (carnitine palmitoyl transferase 2, CPT2). Deficiency of the OCTN2 carnitine transporter causes primary carnitine deficiency, characterized by increased losses of carnitine in the urine and decreased carnitine accumulation in tissues. Patients can present with hypoketotic hypoglycemia and hepatic encephalopathy, or with skeletal and cardiac myopathy. This disease responds to carnitine supplementation. Defects in the liver isoform of CPT1 present with recurrent attacks of fasting hypoketotic hypoglycemia. The heart and the muscle, which express a genetically distinct form of CPT1, are usually unaffected. These patients can have elevated levels of plasma carnitine. CACT deficiency presents in most cases in the neonatal period with hypoglycemia, hyperammonemia, and cardiomyopathy with arrhythmia leading to cardiac arrest. Plasma carnitine levels are extremely low. Deficiency of CPT2 present more frequently in adults with rhabdomyolysis triggered by prolonged exercise. More severe variants of CPT2 deficiency present in the neonatal period similarly to CACT deficiency associated or not with multiple congenital anomalies. Treatment for deficiency of CPT1, CPT2, and CACT consists in a low-fat diet supplemented with medium chain triglycerides that can be metabolized by mitochondria independently from carnitine, carnitine supplements, and avoidance of fasting and sustained exercise.     

Analysis of genetic mutations in Chinese patients with systemic primary carnitine deficiency

Systemic primary carnitine deficiency (CDSP) is caused by mutations in SLC22A5 gene, which encodes organic cation transporter 2(OCTN2). CDSP leads to skeletal or cardiac myopathy and hepatic encephalopathy. The present study aimed to identify SLC22A5 gene mutations and analyze the potential relationship between genotype and clinical symptoms in 20 Chinese patients with CDSP. The complete coding region of the SLC22A5 gene including intron-exon boundaries were amplified and sequenced in all patients. Eighteen different mutations were found; of which, nine were novel. The mutations clustering in exons 1 and 4 accounted for 66.7% of all mutant alleles (26/39). The c.760C>T (p. R254X) was the most frequent mutation (25.6%, 10/39), suggesting it as an ethnic founder mutation. The relationship between genotype and phenotype was investigated in patients carrying the R254X mutation. Homozygous patients with R254X were late-onset cases who presented with dilated cardiomyopathy and muscle weakness after 1 year of age. Compound heterozygous patients carrying R254X, combined with other missense mutations occurred in very specific positions, dramatically altered OCTN2 protein function. Based on the analysis of case studies, a clear relationship between free carnitine (C0) level in plasma and OCTN2 genotype was not found in the present work, however, the low plasma C0 level could not indicate disease severity or genotype. Further functional studies with a large sample size are required to understand the relationship between R254X mutation and CDSP.     

Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.     

Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration.     

A founder mutation (R254X) of SLC22A5 (OCTN2) in Chinese primary carnitine deficiency patients

Mutations in the SLC22A5 gene, which encodes for the plasma membrane carnitine transporter OCTN2, cause primary carnitine deficiency (PCD). After our first report of OCTN2 mutations in Chinese, three more Chinese PCD patients were identified. The parents of these families were non-consanguineous and these families were unrelated. Two novel truncating mutations were found: R254X, a single-base mutation at cDNA position 981 (c.981C>T); and Y387X (c.1382T>G). Two probands, one each from Taiwan and Macau, were homozygous for R254X. The other proband from Taiwan carried both R254X and Y387X. Two additional heterozygote carriers of R254X were also identified among 250 control samples, while none was detected for Y387X. The population carrier rate for R254X would be about 1 in 125. Haplotypes of R254X alleles were examined and patients homozygous for R254X were also homozygous for the same haplotype of intragenic and microsatellites markers. Analysis of population frequencies of haplotypes revealed that the chance of 4 chromosomes having arisen as independent events was 0.016. We conclude that R254X is probably a founder mutation in Chinese. Other previously reported mutations found in the Japanese population were also screening in 250 control samples but no carrier was identified, indicating that they were either very rare or not present in Southern Chinese.     

Molecular spectrum of SLC22A5 (OCTN2) gene mutations detected in 143 subjects evaluated for systemic carnitine deficiency

Systemic primary carnitine deficiency (CDSP) is caused by recessive mutations in the SLC22A5 (OCTN2) gene encoding the plasmalemmal carnitine transporter and characterized by hypoketotic hypoglycemia, and skeletal and cardiac myopathy. The entire coding regions of the OCTN2 gene were sequenced in 143 unrelated subjects suspected of having CDSP. In 70 unrelated infants evaluated because of abnormal newborn screening (NBS) results, 48 were found to have at least 1 mutation/unclassified missense variant. Twenty‐eight of 33 mothers whose infants had abnormal NBS results were found to carry at least 1 mutation/unclassified missense variant, including 11 asymptomatic mothers who had 2 mutations. Therefore, sequencing of the OCTN2 gene is recommended for infants with abnormal NBS results and for their mothers. Conversely, 52 unrelated subjects were tested due to clinical indications other than abnormal NBS and only 14 of them were found to have at least one mutation/unclassified variant. Custom designed oligonucleotide array CGH analysis revealed a heterozygous ～1.6 Mb deletion encompassing the entire OCTN2 gene in one subject who was apparently homozygous for the c.680G>A (p.R227H) mutation. Thus, copy number abnormalities at the OCTN2 locus should be considered if by sequencing, an apparently homozygous mutation or only one mutant allele is identified. ©2010 Wiley‐Liss, Inc.     

Disease-associated Glut1 single amino acid substitute mutations S66F, R126C, and T295M constitute Glut1-deficiency states in vitro

Glucose transporter type 1 deficiency syndrome (Glut1DS) is the result of autosomal-dominant loss-of-function mutation of the glucose transporter type 1 gene (GLUT1) leading to brain energy failure and epileptic encephalopathy. In this study, the protein products of the Glut1DS-associated GLUT1 missense mutations, S66F, R126C, and T295M, were characterized using the Glut1-green fluorescent protein (GFP) fusion expressed in CHO cells. Glut1-GFP expression was confirmed by Western blot and confocal microscopy. The applicability of this Glut1-GFP expression model in reporting Glut1 functional deficits was validated by re-confirming the glucose transport defects of the previously reported pathogenic mutations R126H, R126L, and R333W. While S66F, R126C, and T295M mutants were expressed and targeted to the cell membrane, these Glut1 mutants have significantly diminished membrane association and glucose transport activity (p<0.05) relative to the wild-type Glut1 protein. Consistent with the reduced Glut1 membrane association, glucose transport kinetics studies showed that S66F, R126C, and T295M mutants have significantly reduced (p<0.05) Vmax but not Km. Thus, Glut1 single amino acid substitute mutants S66F, R126C, and T295M impair glucose transport function and constitute Glut1-deficiency states in vitro. These results support the pathogenicity of Glut1 S66F, R126C, and T295M in vivo.     

Functional and molecular studies in primary carnitine deficiency

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.     

Fabry disease: 20 novel GLA mutations in 35 families

Thirty two mutations have been found in 35 unrelated patients of European origin with Fabry disease, including 8 females. Twenty of the mutations are novel and comprise of 13 missense: H46Y, W47G, R49P, C94S, F113S, G258R, P259R, Q279H, Q280H, R363H, A377D, P409A, P409T; 1 nonsense: L294X; 5 small deletions: 154delT, 520delT, 909‐918del10, 1152‐1153delCA, 1235‐1236delCT and 1 splice site mutation: IVS5+2t→c. The remaining 12 mutations have all been reported previously. All patients with deletions had the classic form of the disease but it was not possible to predict the phenotype from the missense mutations. © Wiley‐Liss, Inc.     

OCTN2 is associated with carnitine transport capacity of rat skeletal muscles

Aim: Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium-dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2. Methods: Rat hindlimb muscles (soleus, plantaris, and the surface and deep portions of gastrocnemius) were used for Western blotting to detect OCTN2. Tissue carnitine uptake was examined by an integration plot analysis using l -[³H]carnitine as a tracer. Tissue carnitine content was determined by enzymatic cycling methods. The percentage of type I fibres was determined by histochemical analysis. Results: OCTN2 was detected in all skeletal muscles although the amount was lower than that in the kidney. OCTN2 expression was significantly higher in soleus than in the other skeletal muscles. The amount of OCTN2 was positively correlated with the percentage of type I fibres in hindlimb muscles. The integration plot analysis revealed a positive correlation between the uptake clearance of l -[³H]carnitine and the amount of OCTN2 in skeletal muscles. However, the carnitine content in soleus was lower than that in other skeletal muscles. Conclusion: OCTN2 is functionally expressed in skeletal muscles and is involved in the import of carnitine for fatty acid oxidation, especially in highly oxidative muscles.     

Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome

The SLC12A3 gene encodes the thiazide-sensitive Na-Cl co-transporter (NCCT) expressed in the apical membrane of the distal convoluted tubule of the kidney. Inactivating mutations of this gene are responsible for Gitelman syndrome (GS), a disorder inherited as an autosomal recessive trait. We searched for SLC12A3 gene mutations in 21 Italian patients with the clinical and biochemical features of GS (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria, and the absence of nephrocalcinosis). All coding regions with their intron-exon boundaries were analyzed using PCR and SSCP techniques followed by sequencing analysis. We identified 21 different mutations evenly distributed throughout the gene without any mutation hot-spot. Fifteen are novel variants, including 12 missense mutations, one deletion, one deletion-insertion and one splice site mutation: R158Q, T163M, W172R, G316V, G374V, G463E, A464T, S615W, V677M, R852S, R958G, C985Y, 2114-2120delACCAAGT, 2144-2158delGCCTTCTACTCGGATinsTG, and 531-2A>G.     

Genetic epidemiology of the carnitine transporter OCTN2 gene in a Japanese population and phenotypic characterization in Japanese pedigrees with primary systemic carnitine deficiency.

Serum free-carnitine levels were determined in 973 unrelated white collar workers in Akita, Japan. Fourteen of these participants consistently had serum free-carnitine levels below the fifth percentile (28 microM for females and 38 microM for males). The OCTN2 (organic cation transporter) gene was sequenced for these 14 subjects, for 22 subjects whose carnitine levels were below the fifth percentile in the first screening but were normal in the second measurement and in 69 individuals with normal carnitine levels for two separate measurements. Polymorphic sequences defined three major haplotypes with equal frequency. Mutations were identified in nine subjects with low carnitine levels: Trp132X (three individuals), Ser467Cys (four), Trp283Cys (one) and Met179Leu (one). In vitro expression studies in HEK cells indicated that Ser467Cys and Trp283Cys, but not Met179Leu, significantly reduced L-carnitine uptake relative to the normal control. Trp132X and Ser467Cys were associated with specific haplotypes, suggesting a founder effect. A conservative estimate of the overall prevalence of heterozygotes was 1.01% in the Akita prefecture, Japan, giving an estimated incidence of primary systemic carnitine deficiency (MIM 212140) as 1 in 40 000 births. An echocardiographic study of the families of patients with primary carnitine deficiency revealed that the heterozygotes for OCTN2 mutations were predisposed to late onset benign cardiac hypertrophy (odds ratio 15.1, 95% CI 1.39-164) compared with the wild-types. Sequencing of DNA isolated from three deceased siblings (1.5-8 years) in two families retrospectively confirmed that all three deceased subjects were homozygous for the OCTN2 mutations.