Recently, we showed that homozygosity for the common 677(C-->T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for neural-tube defects (NTDs). We now report on another mutation in the same gene, the 1298(A-->C) mutation, which changes a glutamate into an alanine residue. This mutation destroys an MboII recognition site and has an allele frequency of .33. This 1298(A-->C) mutation results in decreased MTHFR activity (one-way analysis of variance [ANOVA] P < .0001), which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous state is associated with higher plasma homocysteine (Hcy) or a lower plasma folate concentration-phenomena that are evident with homozygosity for the 677(C-->T) mutation. However, there appears to be an interaction between these two common mutations. When compared with heterozygosity for either the 677(C-->T) or 1298(A-->C) mutations, the combined heterozygosity for the 1298(A-->C) and 677(C-->T) mutations was associated with reduced MTHFR specific activity (ANOVA P < .0001), higher Hcy, and decreased plasma folate levels (ANOVA P <.03). Thus, combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677(C-->T) mutation. This combined heterozygosity was observed in 28% (n =86) of the NTD patients compared with 20% (n =403) among controls, resulting in an odds ratio of 2.04 (95% confidence interval: .9-4.7). These data suggest that the combined heterozygosity for the two MTHFR common mutations accounts for a proportion of folate-related NTDs, which is not explained by homozygosity for the 677(C-->T) mutation, and can be an additional genetic risk factor for NTDs.

Background: A reliable and sensitive complex I assay is an essential tool for the diagnosis of mitochondrial disorders, but current spectrophotometric assays suffer from low sensitivity, low specificity, or both. This deficiency is mainly due to the poor solubility of coenzyme-Q analogs and reaction mixture turbidity caused by the relatively high concentrations of tissue extract that are often required to measure complex I. Methods: We developed a new spectrophotometric assay to measure complex I in mitochondrial fractions and applied it to muscle and cultured fibroblasts. The method is based on measuring 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. The assay thus is designed to avoid nonspecific NADH oxidation because electrons produced in these reactions are not accepted by decylubiquinone, resulting in high rotenone sensitivity. Results: The assay was linear with time and amount of mitochondria. The K m values for NADH and 2,6-dichloroindophenol in muscle mitochondria were 0.04 and 0.017 mmol/L, respectively. The highest complex I activities were measured with 0.07 mmol/L decylubiquinone and 3.5 g/L bovine serum albumin. The latter was an essential component of the reaction mixture, increasing the solubility of decylubiquinone and rotenone. In patients with previously diagnosed complex I deficien-

For production of proteins that are encoded by the mitochondrial genome, mitochondria rely on their own mitochondrial translation system, with the mitoribosome as its central component. Using extensive homology searches, we have reconstructed the evolutionary history of the mitoribosomal proteome that is encoded by a diverse subset of eukaryotic genomes, revealing an ancestral ribosome of alpha-proteobacterial descent that more than doubled its protein content in most eukaryotic lineages. We observe large variations in the protein content of mitoribosomes between different eukaryotes, with mammalian mitoribosomes sharing only 74 and 43% of its proteins with yeast and Leishmania mitoribosomes, respectively. We detected many previously unidentified mitochondrial ribosomal proteins (MRPs) and found that several have increased in size compared to their bacterial ancestral counterparts by addition of functional domains. Several new MRPs have originated via duplication of existing MRPs as well as by recruitment from outside of the mitoribosomal proteome. Using sensitive profile–profile homology searches, we found hitherto undetected homology between bacterial and eukaryotic ribosomal proteins, as well as between fungal and mammalian ribosomal proteins, detecting two novel human MRPs. These newly detected MRPs constitute, along with evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.

Oxidative phosphorylation (OXPHOS) has a prominent role in energy metabolism of the cell. Being under bigenomic control, correct biogenesis and functioning of the OXPHOS system is dependent on the finely tuned interaction between the nuclear and the mitochondrial genome. This suggests that disturbances of the system can be caused by numerous genetic defects and can result in a variety of metabolic and biochemical alterations. Consequently, OXPHOS deficiencies manifest as a broad clinical spectrum. Complex I, the biggest and most complicated enzyme complex of the OXPHOS system, has been subjected to thorough investigation in recent years. Significant progress has been made in the field of structure, composition, assembly, and pathology. Important gains in the understanding of the Goliath of the OXPHOS system are: exposing the electron transfer mechanism and solving the crystal structure of the peripheral arm, characterization of almost all subunits and some of their functions, and creating models to elucidate the assembly process with concomitant identification of assembly chaperones. Unravelling the intricate mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease will pave the way for developing adequate diagnostic procedures and advanced therapeutic treatment strategies.