Expression of the organic cation/carnitine transporter family (Octn1,-2 and-3) in mdx muscle and heart: Implications for early carnitine therapy in Duchenne muscular dystrophy to improve cellular carnitine homeostasis.

INTRODUCTION: Carnitine is essential for long-chain fatty acid oxidation in muscle and heart. Tissue stores are regulated by organic cation/Cn transporter plasmalemmal Octn2. We previously demonstrated low carnitine in quadriceps/gluteus and heart of adult mdx mice. METHODS: We studied protein and mRNA expression of Octn2, mitochondrial Octn1 and peroxisomal Octn3 in adult male C57BL/10ScSn-DMD mdx/J quadriceps, heart, and diaphragm compared to C57BL/10SnJ mice. RESULTS: We demonstrated reduction in mOctn2 expression on Western blot and similar expression of mOctn1 and mOctn3 in mdx quadriceps, heart and diaphragm. There was a significant upregulation of mOctn1 and mOctn2 mRNA by qRT-PCR in mdx quadriceps and of mOctn2 and mOctn3 mRNA in mdx heart. We showed upregulation of mdx mOctn1 and mOctn3 mRNA but no increase in protein expression. DISCUSSION: Dystrophin deficiency likely disrupts Octn2 expression decreasing muscle carnitine uptake thus contributing to membranotoxic long-chain acyl-CoAs with sarcolemmal and organellar membrane oxidative injury providing a treatment rationale for early L-carnitine in DMD.

Carnitine uptake defect due to a 5'UTR mutation in a pedigree with false positives and false negatives on Newborn screening.

Carnitine Uptake Defect (CUD) is an autosomal recessive disorder due to mutations in the SLC22A5 gene. Classically patients present in infancy with profound muscle weakness and cardiomyopathy with characteristic EKG findings. Later presentations include recurrent hypoketotic hypoglycemia, proximal limb girdle myopathy,and/or recurrent muscle pain. Newborn screening detects most of these clinical variants but in addition has identified maternal CUD often in asymptomatic women. We describe a family ascertained through 3 newborn screening (NBS) positive infants found to be unaffected themselves but in whom the mothers (sisters) were affected. There were also two affected children born to an affected male and his heterozygous wife who were false negatives on NBS but had increased fractional excretion of free carnitine in the urine. Analysis on a Next Generation Sequencing panel specifically designed to fully cover newborn screening disease targets showed a homozygous change in the five probands (SLC22A5; NM_003060:c.-149G > A; p.?). The mutation segregates with the CUD within the family. It is in the 5' UTR and has a frequency within the gnomAd database of 0.001198. Plasma carnitine was decreased and fractional excretion of free carnitine was increased in all affected individuals. Functional carnitine uptake studies in cultured skin fibroblasts of one proband showed carnitine uptake at the 5 µM concentration to be 6% of controls. Relative expression of OCTN2 mRNA to beta-actin mRNA by qRT-PCR was increased in a proband relative to controls by a factor of 465-fold. Western blotting revealed a 120 kDa protein band, as well as a weaker 240 kDa band in the proband, the significance of which is unknown at this time.

Attention deficit/hyperactivity disorder as an associated feature in OCTN2 deficiency with novel deletion (p.T440-Y449).

This boy presented with ADHD at 3 years and at 8 years was hyperactive with no documented hypoglycemia and had myopathy, cardiomyopathy, and very low serum carnitine. L-carnitine improved his exercise intolerance, cardiomyopathy, and behavior. Analysis of SLC22A5 revealed a premature stop codon (p.R282*) and a novel in-frame deletion (p.T440-Y449).

Novel myophosphorylase mutation (p.Arg94Pro) with progressive exercise intolerance.

We present a 16-year-old girl with a unique clinical phenotype characterized by rapidly progressive exercise intolerance, transient exertional weakness, and progressive muscle cramps involving all limbs and bulbar muscles, following a first myoglobinuric episode at age 15 years, arising from homozygosity for a novel missense mutation (c.281G>C) in PYGM.

NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders.

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.

The mdx mouse as a model for carnitine deficiency in the pathogenesis of Duchenne muscular dystrophy.

INTRODUCTION: Muscle and cardiac metabolism are dependent on the oxidation of fats and glucose for adenosine triphosphate production, for which L-carnitine is an essential cofactor. METHODS: We measured muscle carnitine concentrations in skeletal muscles, diaphragm, and ventricles of C57BL/10ScSn-DMDmdx/J mice (n = 10) and compared them with wild-type C57BL/6J (n = 3), C57BL/10 (n = 10), and C3H (n = 12) mice. Citrate synthase (CS) activity was measured in quadriceps/gluteals and ventricles of mdx and wild-type mice. RESULTS: We found significantly lower tissue carnitine in quadriceps/gluteus (P < 0.05) and ventricle (P < 0.05), but not diaphragm of mdx mice, when compared with controls. CS activity was increased in mdx quadriceps/gluteus (P < 0.03) and ventricle (P < 0.02). This suggests compensatory mitochondrial biogenesis. CONCLUSIONS: Decreased tissue carnitine has implications for reduced fatty acid and glucose oxidation in mdx quadriceps/gluteus and ventricle. The mdx mouse may be a useful model for studying the role of muscle carnitine deficiency in DMD bioenergetic insufficiency and providing a targeted and timed rationale for L-carnitine therapy.

Vulnerability to oxidative stress in vitro in pathophysiology of mitochondrial short-chain acyl-CoA dehydrogenase deficiency: response to antioxidants.

OBJECTIVE: To elucidate the pathophysiology of SCAD deficient patients who have a unique neurological phenotype, among fatty acid oxidation disorders, with early developmental delay, CNS malformations, intractable seizures, myopathy and clinical signs suggesting oxidative stress. METHODS: We studied skin fibroblast cultures from patients homozygous for ACADS common variant c.625G>A (n = 10), compound heterozygous for c.625G>A/c.319C>T (n = 3) or homozygous for pathogenic c.319C>T (n = 2) and c.1138C>T (n = 2) mutations compared to fibroblasts from patients with carnitine palmitoyltransferase 2 (CPT2) (n = 5), mitochondrial trifunctional protein (MTP)/long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) (n = 7), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiencies (n = 4) and normal controls (n = 9). All were exposed to 50 µM menadione at 37°C. Additional conditions included exposure to 39°C and/or hypoglycemia. Time to 100% cell death was confirmed with trypan blue dye exclusion. Experiments were repeated with antioxidants (Vitamins C and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue. RESULTS: The most significant risk factor for vulnerability to menadione-induced oxidative stress was the presence of a FAO defect. SCADD fibroblasts were the most vulnerable compared to other FAO disorders and controls, and were similarly affected, independent of genotype. Cell death was exacerbated by hyperthermia and/or hypoglycemia. Hyperthermia was a more significant independent risk factor than hypoglycemia. Rescue significantly prolonged survival. Incubation with antioxidants and Bezafibrate significantly increased viability of SCADD fibroblasts. INTERPRETATION: Vulnerability to oxidative stress likely contributes to neurotoxicity of SCADD regardless of ACADS genotype and is significantly exacerbated by hyperthermia. We recommend rigorous temperature control in SCADD patients during acute illness. Antioxidants and Bezafibrate may also prove instrumental in their management.

Upregulation of mammary gland OCTNs maintains carnitine homeostasis in suckling infants.

BACKGROUND: Transport of L-carnitine, essential cofactor of fatty acid metabolism, into breast milk is critical for the normal growth and development of the suckling infant. OBJECTIVE: To increase understanding of developmental expression of carnitine/organic cation (Octn) transporter family at different stages of murine breast development for carnitine delivery. METHODS: We applied our transporter-specific antibodies to mOctn1, mOctn2 and mOctn3 to sections of mammary glands of virginal non-lactating, pregnant, late lactating and post-lactating C3H females. RESULTS: We demonstrated differential expression of mOctn1, -2 and -3 in epithelial ducts, specialized myoepithelial cells and fatty stroma. There was notable upregulation of all three Octns and mRNA by RT-PCR concurrent with an increase in epithelial ducts in breasts of pregnant (15days gestation) and lactating mice (15-days post-partum) compared to virginal 6 week old females, and notable downregulation in expression of Octns 15 days after cessation of lactation. In lactating murine mammary gland at 15 days post-partum, there was a marked increase of fat globules in epithelial ducts. Octn1 and Octn2 had similar expression patterns in lactating gland cells which formed fat globules that were exocytosed into the lumen of alveoli along with transporters Octn1 and Octn2. Octn3 was primarily localized to myoepithelial cells surrounding the ducts at all stages of breast development. CONCLUSIONS: There is a dynamic upregulation of the Octn family in pregnant and lactating breasts which likely provides the suckling infant with adequate carnitine for the rapid postnatal upregulation of fatty acid oxidation and ketogenesis critical for cerebral energy metabolism during fasting hypoglycemia.

Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise.

OBJECTIVE: Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using (31)P-magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high-intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end-exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle. METHODS AND RESULTS: We studied CFTR expression in human skeletal muscle by Western blot with anti-CFTR antibody (Ab) L12B4 and demonstrated a single band with expected molecular weight of 168kDa. We isolated the cDNA by reverse transcription polymerase chain reaction and directly sequenced a 975bp segment (c. 3,600-4,575) that was identical to the human CFTR sequence. We showed punctate staining of CFTR in sarcoplasm and sarcolemma by immunofluorescence microscopy with L12B4 Ab and secondary Alexa 488-labeled Ab. We confirmed CFTR expression in the sarcotubular network and sarcolemma by electron microscopy, using immunogold-labeled anti-CFTR Ab. We observed activation of CFTR Cl(-) channels with iodide efflux, on addition of forskolin, 3-isobutyl-1-methyl-xanthine, and 8-chlorphenylthio-cyclic adenosine monophosphate, in wild-type C57BL/6J isolated muscle fibers in contrast to no efflux from mutant F508del-CFTR muscle. INTERPRETATION: We speculate that a defect in sarcoplasmic reticulum CFTR Cl(-) channels could alter the electrochemical gradient, causing dysregulation of Ca(2+) homeostasis, for example, ryanodine receptor or sarco(endo)plasmic reticulum Ca(2+) adenosine triphosphatases essential to excitation-contraction coupling leading to exercise intolerance and muscle weakness in CF.

Organic cation/carnitine transporter family expression patterns in adult murine heart.

Organic cation/carnitine transporters transport carnitine, drugs, and xenobiotics (e.g. choline, acetylcarnitine, quinidine, and verapamil), and are expressed in muscle, heart, blood vessels, etc. To characterize expression patterns of mOctn1, -2, and -3 in adult murine heart, we applied our transporter-specific antibodies to mOctn1, -2, and -3, followed by secondary antibody and DAB peroxidase detection to adult murine heart sections counterstained with hematoxylin. All three transporters showed strong expression in cardiomyocytes, lamina fibrosa of cardiac valves, great arteries and intermuscular arterioles, and a striking differential expression in the vagal innervated sinoatrial and atrioventricular nodes. The hOCTN2 deficiency presents with carnitine-responsive cardiomyopathy. Octn1, -2, and -3 are expressed in many regions of murine heart with a pattern suggestive of potential roles in modulating myocardial bioenergetics, valvular function, and acetylcholine generation for parasympathetic vagal innervation of the cardiac conduction system. This distribution may play a role in the hypertrophic cardiomyopathy seen in hOCTN2 deficiency, and may also affect the absorption/elimination of organic cationic cardiac drugs.

Short-chain acyl-CoA dehydrogenase gene mutation (c.319C>T) presents with clinical heterogeneity and is candidate founder mutation in individuals of Ashkenazi Jewish origin.

We report 10 children (7 male, 3 female), 3 homozygous for c.319C>T mutation and 7 heterozygous for c.319C>T on one allele and c.625G>A variant on the other in the short-chain acyl-CoA dehydrogenase (SCAD) gene (ACADS). All were of Ashkenazi Jewish origin in which group we found a c.319C>T heterozygote frequency of 1:15 suggesting the presence of a founder mutation or selective advantage. Phenotype was variable with onset from birth to early childhood. Features included hypotonia (8/10), developmental delay (8/10), myopathy (4/10) with multicore changes in two and lipid storage in one, facial weakness (3/10), lethargy (5/10), feeding difficulties (4/10) and congenital abnormalities (3/7). One female with multiminicore myopathy had progressive external ophthalmoplegia, ptosis and cardiomyopathy with pneumonia and respiratory failure. Two brothers presented with psychosis, pyramidal signs, and multifocal white matter abnormalities on MRI brain suggesting additional genetic factors. Two other infants also had white matter changes. Elevated butyrylcarnitine (4/8), ethylmalonic aciduria (9/9), methylsuccinic aciduria (6/7), decreased butyrate oxidation in lymphoblasts (2/4) and decreased SCAD activity in fibroblasts or muscle (3/3) were shown. Expression studies of c.319C>T in mouse liver mitochondria showed it to be inactivating. c.625G>A is a common variant in ACADS that may confer disease susceptibility. Five healthy parents were heterozygous for c.319C>T and c.625G>A, suggesting reduced penetrance or broad clinical spectrum. We conclude that the c.319C>T mutation can lead to wide clinical and biochemical phenotypic variability, suggesting a complex multifactorial/polygenic condition. This should be screened for in individuals with multicore myopathy, particularly among the Ashkenazim.

Expression patterns of the organic cation/carnitine transporter family in adult murine brain.

UNLABELLED: Organic cation/carnitine transporters transport carnitine, drugs, and xenobiotics (e.g. choline, acetylcarnitine, betaine, valproic acid), and are expressed in muscle, heart, blood vessels, kidney, gut, etc. OBJECTIVE: To characterize expression patterns of mOctn1, -2 and -3 in murine brain. METHODS: We applied our transporter-specific antibodies to mOctn1, -2 and -3, followed by 2 0 antibody and DAB peroxidase detection to serial adult murine brain sections counterstained with hematoxylin. RESULTS: All three transporters showed strong expression in the external plexiform layer of the olfactory bulb and in olfactory nerve, the molecular layer and neuronal processes of input fibres extending vertically in motor cortex, in the dendritic arborization of the cornu ammonis and dendate gyrus (hippocampus), neuronal processes in the arcuate nucleus (hypothalamus), choroid plexus cells, and neuronal cell bodies and dendrites of cranial nerve nuclei V and VII. In the cerebellum, all three transporters were strongly expressed in dendritic processes of Purkinje cells, but Octn1 and -2 were expressed more strongly than Octn3 in Purkinje cell bodies. In spinal cord, Octn1, -2 and -3 were prominent in axons and dendritic end-arborizations of spinal cord neurons in both ascending and descending white matter tracts, whereas Octn3 was also strongly expressed in grey matter, specifically in anterior horn cell bodies. Octn3 was weakly expressed in glomerular layer neuronal cell bodies of olfactory bulb. CONCLUSIONS: hOCTN2 deficiency presents with carnitine-responsive cardiomyopathy, myopathy and hypoglycemic, hypoketotic coma with strokes, seizures and delays. In mouse, Octn1, -2 and -3 are expressed in many regions throughout the central nervous system with a pattern suggestive of roles in modulating cerebral bioenergetics and in acetylcholine generation for neurotransmission in olfactory, satiety, limbic, memory, motor and sensory functions. This distribution may play a role in the pattern of neurological injury that occurs in hOCTN2 deficiency during catabolic episodes of hypoglycemic, hypoketotic encephalopathy and which may manifest with cognitive impairment, hypotonia and seizures.

Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria.

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.

OCTN3 is a mammalian peroxisomal membrane carnitine transporter.

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.

Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn's disease at 5q31.

Campylobacter jejuni and Mycobacterium paratuberculosis have been implicated in the pathogenesis of Crohn's disease. The presence of bacterial metabolites in the colonic lumen causing a specific breakdown of fatty acid oxidation in colonic epithelial cells has been suggested as an initiating event in inflammatory bowel disease (IBD). l-Carnitine is a small highly polar zwitterion that plays an essential role in fatty acid oxidation and ATP generation in intestinal bioenergetic metabolism. The organic cation/carnitine transporters, OCTN1 and OCTN2, function primarily in the transport of l-carnitine and elimination of cationic drugs in the intestine. High-resolution linkage disequilibrium mapping has identified a region of about 250kb in size at 5q31 (IBD5) encompassing the OCTN1 and -2 genes, to confer susceptibility to Crohn's disease. Recently, two variants in the OCTN1 and OCTN2 genes have been shown to form a haplotype which is associated with susceptibility to Crohn's. We show that OCTN1 and OCTN2 are strongly expressed in target areas for IBD such as ileum and colon. Further, we have now identified a nine amino acid epitope shared by this functional variant of OCTN1 (Leu503Phe) (which decreases the efficiency of carnitine transport), and by C. jejuni (9 aa) and M. paratuberculosis (6 aa). The prevalence of this variant of OCTN1 (Phe503:Leu503) is 3-fold lower in unaffected individuals of Jewish origin (1:3.44) compared to unaffected individuals of non-Jewish origin (1:1). We hypothesize that a specific antibody raised to this epitope during C. jejuni or M. paratuberculosis enterocolitis would cross-react with the intestinal epithelial cell functional variant of OCTN1, an already less efficient carnitine transporter, leading to an impairment of mitochondrial beta-oxidation which may then serve as an initiating event in IBD. This impairment of l-carnitine transport by OCTN1 may respond to high-dose l-carnitine therapy.

Characterization of organic cation/carnitine transporter family in human sperm.

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K(m) of 3.39+/-1.16 microM; V(max) of 0.23+/-0.14 pmol/min/mg sperm protein; and mean+/-SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K(m) of 25.9+/-14.7 microM; V(max) of 1.49+/-1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K(m) of 412.6+/-191 microM; V(max) of 32.7+/-20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to L-carnitine supplementation.

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5).

Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No. NW_000039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31, which has been shown to confer susceptibility to Crohn's disease. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohn's disease.

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.