Miyuki Shimane

Molecular and Functional Identification of Sodium Ion-dependent, High Affinity Human Carnitine Transporter OCTN2*

Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake ofl-[3H]carnitine was strongly enhanced in a sodium-dependent manner with K m value of 4.34 μm, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediatedl-[3H]carnitine transport was inhibited by the d-isomer, acetyl-d,l-carnitine, and γ-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, β-hydroxybutyric acid, γ-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis (jvs; ref. 6). Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 (ref. 9). Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Loss of LKB1 Kinase Activity in Peutz-Jeghers Syndrome, and Evidence for Allelic and Locus Heterogeneity

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. There is an increased risk of benign and malignant tumors in the gastrointestinal tract and in extraintestinal tissues. One PJS locus has been mapped to chromosome 19p13.3; a second locus is suspected on chromosome 19q13.4 in a minority of families. The PJS gene on 19p13.3 has recently been cloned, and it encodes the serine/threonine kinase LKB1. The gene, which is ubiquitously expressed, is composed of 10 exons spanning 23 kb. Several LKB1 mutations have been reported in heterozygosity in PJS patients. In this study, we screened for LKB1 mutations in nine PJS families of American, Spanish, Portuguese, French, Turkish, and Indian origin and detected seven novel mutations. These included two frameshift mutations, one four-amino-acid deletion, two amino-acid substitutions, and two splicing errors. Expression of mutant LKB1 proteins (K78I, D176N, W308C, and L67P) and assessment of their autophosphorylation activity revealed a loss of the kinase activity in all of these mutants. These results provide direct evidence that the elimination of the kinase activity of LKB1 is probably responsible for the development of the PJS phenotypes. In two Indian families, we failed to detect any LKB1 mutation; in one of these families, we previously had detected linkage to markers on 19q13.3-4, which suggests locus heterogeneity of PJS. The elucidation of the molecular etiology of PJS and the positional cloning of the second potential PJS gene will further elucidate the involvement of kinases/phosphatases in the development of cancer-predisposing syndromes.

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 Reyes 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.