From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which

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... turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O 2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted ( 0 ) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family. Cybrid study. 0 HeLa cells were prepared by treating the HeLa cells with ethidium bromide as previously reported (16). 0 HeLa cells were grown in RPMI-1640 with 10% fetal bovine serum (FBS) containing 0.15% NaHCO 3 , gentamicin (50 µg/ml), nystatin (100 U/ml), pyruvate (1 mmol/l), and uridine (20 µmol/l) (17). Exogenous mtDNAs were transferred to 0 HeLa cells in the presence of 50% polyethylene glycol 1500 (Boehringer Mannheim, Mannheim, Germany) by using platelets obtained from the proband or 3 age-matched control subjects as the mtDNA donors (18). The fusion mixture was cultured in the DM 170 selection medium without glucose (Kyokuto Chemical, Tokyo) supplemented with 10% FBS containing 0.1% NaHCO 3 , gentamicin (50 µg/ml), and nystatin (100 U/ml) (17). Cybrid cells were cultured in the selection medium for 2-3 weeks after the fusion. Then, cybrid cells were grown in RPMI-1640 with 10% FBS containing 0.15% NaHCO 3 , gentamicin (50 µg/ml), and nystatin (100 U/ml) for 2-3 weeks and were cloned by the cylinder method. Activities of mitochondrial respiratory chain complexes I (19) and IV (20) and the rate of O 2 consumption (16) were measured as described.