Genetic Epidemiology of the Carnitine Transporter OCTN2 Gene in a Japanese Population and Phenotypic Characterization in Japanese Pedigrees with Primary Systemic Carnitine Deficiency

Akio Koizumi 3 Jun-ichi Nozaki 3 Toshihiro Ohura 2 3 Tsuyoshi Kayo 3 Yasuhiko Wada 3 Jun-ichi Nezu 1 3 Rikiya Ohashi 0 3 Ikumi Tamai 0 3 Yutaka Shoji 3 6 Goro Takada 3 6 Satoshi Kibira 3 5 Toyojiro Matsuishi 3 4 Akira Tsuji 0 3 0 Department of Pharmacobio-Dynamics, Faculty of Pharmaceutical Sciences, Kanazawa University , Kanazawa 920-0934, Japan 1 Chugai Research Institute for Molecular Medicine , 153-2 Nagai Niihari, Ibaraki 300-4101, Japan 2 Department of Pediatrics, Tohoku University School of Medicine , Sendai 980-8575, Japan 3 Department of Hygiene, Akita University School of Medicine , Akita 010-8543, Japan 4 Department of Pediatrics and Child Health, Kurume University School of Medicine , Kurume 830-0011, Japan 5 Second Department of Internal Medicine, Akita University School of Medicine , Akita 010-8543, Japan 6 Department of Pediatrics, Akita University School of Medicine , Akita 010-8543, Japan 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 m M for females and 38 m M 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 - 1.39164) compared with the wild-types. Sequencing of DNA isolated from three deceased siblings (1.58 years) in two families retrospectively confirmed that all three deceased subjects were homozygous for the OCTN2 mutations. Primary systemic carnitine deficiency (SCD; MIM 212140) is a rare hereditary disease (1). This disease is treatable but is often overlooked (2,3). Although there is no direct evidence, as yet, several lines of circumstantial evidence imply that SCD may be one of the causes of sudden infant death syndrome (36). The OCTN2 gene (7) encodes for a Na-dependent carnitine transporter that was mapped at 5q31.1 where a genetic locus for SCD has been assigned by linkage analysis (8). It has been proven that mutations of OCTN2 impair carnitine transport in SCD patients (912). Due to the lack of a proper marker for diagnosis at the molecular level no genetic epidemiological studies have been done in the general population. The primary goal of the present study was, therefore, aimed at obtaining the prevalence of heterozygotes of OCTN2 mutations in the general population. Cardiac complications have been reported (13) in an SCD sibling who was judged to be heterozygous by serum freecarnitine level. Furthermore, in several families very young siblings of affected patients have been reported to die unexpectedly of unknown causes (3,14). There was, however, no direct retrospective molecular diagnosis of these deceased children to determine whether these children also suffered from SCD. These observations raise two questions: (i) whether the deceased children were homozygous for the OCTN2 mutations; No. of subtype 64 1 4 48 Description of mutation Letters in bold indicate mutated nucleotides. Italic letters indicate missense or nonsense mutations. aInsertion of A in the 3'-UTR, resulting in an in-frame shift in this region. bUnclassified, undetermined as to either A or B haplotype. and (ii) whether heterozygotes for the OCTN2 mutations develop cardiac complications. Population-based study A total of 557 males (mean age SD 32.4 9.31 years, range 1868, median 31) and 416 females (mean age SD 25.7 6.13 years, range 1862, median 24) participated in the first stage of screening. The serum free-carnitine concentrations were distributed normally with the mean serum freecarnitine SD for males being 50.4 7.62 m M and for females 40.2 7.19 m M. The mean was significantly higher in males than in females (P < 0.01). The screening cut-off levels of serum free-carnitine concentrations were set at 38 m M for males and 28 m M for females, which corresponded to the lowest 5% limit values (i.e. mean 1.64 SD). Any individual with a serum free-carnitine level below the cut-off value was tentatively identified as a carrier candidate and those whose serum free-carnitine levels were above the cut-off values were assigned to the normal group. All members of the carrier candidate group and randomly selected persons from the normal group (50 males and 30 females) were asked to return for further examination. Nineteen males (73% response rate) and 17 females (85% response rate) from the carrier candidate group, and 45 males (90% response rate) and 24 females (80% response rate) from the normal group, returned for the second phase. Fourteen (seven males, seven females) of the carrier candidate group consistently had serum free-carnitine levels below the 5% lowest limit: mean SD was 35.2 1.4 m M for males with a range of 33.837.2, and 25.2 2.5 m M for females with a range of 21.027.1. In contrast, the remaining members of the carrier candidate group and the normal group had serum free-carnitine levels above the 5% lowest limit: mean SD was 52.4 8.2 m M for males (n = 57) with a range of 38.4 71.3, and 39.0 5.3 m M for females (n = 34) with a range of 30.648.2. All 10 exons of the organic cation transporter (OCTN2) gene were sequenced in 105 subjects from members of the carrier candidates and normal groups. The structure pattern of the sequence polymorphisms allowed us to resolve the linkage and infer haplotypes. Haplotype A was found in 69 out of 210 chromosomes (32.9%), haplotype B was found in 71 out of 210 (33.8%) and haplotype C was found in 68 out of 210 (32.3%) (Table 1). A truncated mutation, Trp132X (Fig. 1A), was found in three participants who had serum free-carnitine levels lower than the 5% lowest limit at two separate determinations. This mutation was also found in the AK family and was derived from maternal ancestors (10). The genotypes of these three carriers enabled us to infer multiple site linkage (...truncated)