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Background: Pathogenic mitochondrial (mt)DNA mutations, which often cause life‐threatening disorders, are maternally inherited via the cytoplasm of oocytes. Mitochondrial replacement therapy (MRT) is expected to prevent second‐generation transmission of mtDNA mutations. However, MRT may affect the function of respiratory chain complexes comprised of both nuclear and mitochondrial proteins. Methods: Based on the literature and current regulatory guidelines (especially in Japan), we analyzed and reviewed the recent developments in human models of MRT. Main findings: MRT does not compromise pre‐implantation development or stem cell isolation. Mitochondrial function in stem cells after MRT is also normal. Although mtDNA carryover is usually less than 0.5%, even low levels of heteroplasmy can affect the stability of the mtDNA genotype, and directional or stochastic mtDNA drift occurs in a subset of stem cell lines (mtDNA genetic drift). MRT could prevent serious genetic disorders from being passed on to the offspring. However, it should be noted that this technique currently poses significant risks for use in embryos designed for implantation. Conclusion: The maternal genome is fundamentally compatible with different mitochondrial genotypes, and vertical inheritance is not required for normal mitochondrial function. Unresolved questions regarding mtDNA genetic drift can be addressed by basic research using MRT. [ABSTRACT FROM AUTHOR]

The mitochondrial DNA (mtDNA) G13513A mutation in the ND5 subunit gene has been recently reported as a common cause of some phenotypes of mitochondrial myopathy. Until now, the prevalence and characteristics of this mutation in Leigh syndrome (LS) has not been determined. We screened 84 patients with Leigh syndrome (LS) and found the mutation in six (7%) of them. The proportions of mutant mtDNA in muscles were relatively low (42-70%). The onset of symptoms for patients with this mutation was from 9 months to 5 years. It should be noted that five patients had cardiac conduction abnormalities, particularly Wolff-Parkinson-White (WPW) syndrome (three patients). This study suggests that G13513A mutation is a frequent cause of LS and that patients with this mutation may have a characteristic clinical course.

Objectives: To investigate whether the -3826 A-G point mutation of the uncoupling protein 1 (UCP1) gene and the Trp64Arg mutation of the beta3-adrenergic receptor (beta3-AR) gene are associated with increased susceptibility to weight gain and hyperlipidemia in postmenopausal women., Methods: We genotyped 312 Japanese women for UCP1 and beta3-AR gene polymorphisms, and investigated their effects on anthropometrical parameters, serum lipid concentrations, and their changes after 4 years., Results: Although body mass index (BMI), serum triglyceride, total cholesterol, and low-density lipoprotein levels were significantly higher and high-density lipoprotein levels significantly lower in postmenopausal (n=182) than in premenopausal (n=99) women, there was no significant difference in these parameters between carriers and non-carriers of the G allele in the postmenopausal women. In the premenopausal women, BMI and the 4-year change in body weight (BW) of carriers of the G allele were significantly higher than those of non-carriers of the G allele (P=0.022 and 0.048, respectively). In the postmenopausal women, the 4-year change in the level of serum triglyceride of carriers of the G allele was significantly higher (P=0.049), and the change of high-density lipoprotein was significantly lower (P=0.020) than those of non-carriers of the G allele. The beta3-AR polymorphism showed no apparent affect on these parameters., Conclusions: The -3826 A-G polymorphism of the UCP1 gene is associated with an increase in BW of premenopausal women and with the 4-year changes in serum triglyceride and high-density lipoprotein levels in postmenopausal women.

Dubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes. A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members. In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found. In the third patient, a -24C-->T polymorphism and the two mutations c.1901del67 and 2026G-->C were detected. The 2026G-->C mutation was a novel mutation in exon 16 affecting the conversion of Gly(676) to Arg(676) (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers. The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein. It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case.

The effects of regular physical exercise on obesity-associated metabolic abnormalities vary for each individual. In this study, we investigated whether genotypes of genes associated with obesity can predict the effects of exercise on changes in metabolic markers in healthy men. Healthy Japanese men (n=106) performed the exercise program at 50% of their maximal heart rate for 20-60 min a day, 2-3 days each week for 3 months. The levels of fasting plasma glucose (FPG) and serum leptin significantly decreased after the exercise program. Polymorphisms of the beta3-adrenergic receptor (beta3AR) and uncoupling protein-1 (UCP-1) genes were analyzed with RFLP methods. In the Trp/Trp genotype of the beta3AR gene, the levels of serum leptin, FPG and fructosamine (FrAm) decreased significantly after the exercise program, but not in the Arg/Arg genotype. In the AG heterozygote and the GG homozygote of the UCP-1 gene, FPG and FrAm levels were significantly reduced, respectively. In conclusion, gene polymorphism of the beta3AR and UCP-1 was found to be associated with the exercise-mediated improvement in glucose tolerance and leptin resistance in healthy Japanese men.

Leigh syndrome, a severe neurodegenerative disorder, commonly is associated with cytochrome c oxidase deficiency. Recent studies in white patients indicate that SURF-1 gene mutations can cause Leigh syndrome associated with cytochrome c oxidase deficiency. When we measured cytochrome c oxidase activity in cultured lymphoblastoid cells from our Japanese patients with typical Leigh syndrome, three patients demonstrated cytochrome c oxidase deficiency. Three novel mutations of the SURF-1 gene were identified in two of these three patients with cytochrome c oxidase deficiency. All mutations predicted loss of function of the SURF-1 protein; in both patients' cells, cytochrome c oxidase activity was decreased to less than 20% of the control mean. These results indicate that cultured lymphoblastoid cells are useful for elucidating the etiology of Leigh syndrome, and that loss of function of the SURF-1 gene product can be responsible for Leigh syndrome associated with severe cytochrome c oxidase deficiency in Japanese patients.

We have recently identified SLC25A13 on chromosome 7q21.3 as the gene responsible for adult-onset type II citrullinemia (CTLN2) and found seven mutations in the SLC25A13 gene of CTLN2 patients. Most recently, the SLC25A13 mutations have been detected in neonatal/infantile patients with a type of neonatal hepatitis associated with cholestasis (NICCD). In the present study, we identified a novel mutation, E601X, in the SLC25A13 gene and established multiple DNA diagnosis methods for eight mutations by using a genetic analyzer with GeneScan and the single primer extension procedure (SNaPshot). An additional novel missense mutation (variation), E601K, was detected by SNaPshot analysis and was indistinguishable from the mutation E601X detected by the PCR/RFLP method. Multiple DNA diagnoses for the nine mutations revealed that 100 (male/female: 70/30) out of 115 CTLN2 and 38 (14/24) out of 45 NICCD patients tested were homozygotes or compound heterozygotes. The frequency of homozygotes carrying SLC25A13 mutations in both alleles is estimated to be minimally 1 in 21,000 from carrier detection (18 in 1,315 individuals tested) in the Japanese population. The differences in the gender ratio and in mutation types between CTLN2 and NICCD patients are significant. It is, however, unknown whether all homozygotes with mutated SLC25A13 in both alleles suffer from NICCD, CTLN2, both, or neither., (Copyright 2002 Wiley-Liss, Inc.)

Aims/hypothesis: The cumulative effects of several thrifty factors could contribute to the pathogenesis of Type II (non-insulin-dependent) diabetes mellitus. We screened the human UCP1 gene (UCP1) for polymorphisms associated with susceptibility to Type II diabetes., Methods: By using PCR and single-strand conformation polymorphism analysis, UCP1 were screened for mutations in 25 Type II diabetic subjects and 25 healthy control subjects. The allele frequencies of the detected polymorphisms were determined by PCR and restriction fragment length polymorphism analysis in 320 diabetic subjects and 250 control subjects., Results: An A-->C transition in the 5' untranslated region (UTR) of exon 1 (112 bp upstream of the translation initiation codon) and a Met229-->Leu variant were detected. The allele frequencies for the C variant and for the Leu229 variant were higher in the Type II diabetic group than in the control group (p = 0.017 and p = 0.038, respectively). These polymorphisms were in linkage disequilibrium (p < 0.00001). Luciferase assay showed that the former variant in the 5' UTR may affect the promoter activity of UCP1., Conclusion/interpretation: Both the A-->C polymorphism and the Met229-->Leu polymorphism of UCP1 are in linkage disequilibrium and could be one of the diabetes associated single nucleotide polymorphisms (SNPs).

The Trp64Arg mutation of the beta3-adrenergic receptor (beta3AR) gene and A to G polymorphism of the uncoupling protein 1 (UCP1) gene are reported to be associated with weight gain, and both have been shown to have an additive effect on weight gain in Caucasians. Racial differences have also been noted in the beta3AR mutation; however, the effect of UCP1 polymorphism on body weight is not obvious in the Japanese. Thus, we investigated the association of genetic variations in beta3AR and UCP1 genes and the additive effects of these two genes in 214 Japanese men. The frequency of the Trp64Arg allele was 0.19, and serum triglyceride was significantly higher in Arg64 homozygotes versus Trp64 homozygotes. The frequency of the G allele was 0.51, and the body mass index (BMI) was significantly higher in subjects with the G allele (GG homozygotes and AG heterozygotes) versus those without it (AA homozygotes). The beta3AR mutation and UCP1 polymorphism were not found to have additive effects, and they were not related to glucose tolerance patterns and insulin resistance. Our results suggest that the beta3AR mutation is associated with hypertriglyceridemia and the UCP1 polymorphism may be a weak contributing factor to obesity in Japanese men.

We examined genetic mutations in the coding regions of the uncoupling protein 2 (UCP2) gene in 100 patients with non-insulin-dependent diabetes mellitus (NIDDM). The sequences of each exon-intron boundary were detected by polymerase chain reaction (PCR) using specific primer pairs designed in the cDNA sequence of UCP2 and a cycle-sequence method. Using the specific primer pairs in the intron 5'- or 3'-untranslated region, each exon with its exon-intron boundaries was amplified with the PCR method, and the PCR products were analyzed using a single-strand conformation polymorphism (SSCP) method. One nucleotide substitution in exon 4 was found, which exchanged Ala (gcc) at position 55 of the amino acid sequence for Val (gtc), previously reported in Denmark by Urhammer et al in 1997. The polymorphism was reanalyzed in all patients and 120 normal subjects using a PCR-restriction fragment length polymorphism method. There was no difference in the genotype distribution between patients and normal subjects, and our genotype distribution was similar to the Danish study. Furthermore, there were no clinical differences between genotype groups among the patients. No other mutation including the exon-intron boundary was found in these patients. Genetic mutations of UCP2 may not be commonly associated with obesity or diabetes in Japanese subjects.