Analysis of Human Clinical Mutations of Mitochondrial ND1 in a Bacterial Model System for Complex I

Simple Summary Genetic diseases can now be detected directly by determining the DNA sequence of genes, through recent advances in technology. Of the more than 20,000 human genes, several dozen are located in the mitochondria of cells, rather than the nucleus. These genes are essential for energy production and have the unique feature of being passed directly from the mother to her children. After identification of mutations in mitochondrial genes of patients, it is not always simple to determine if the disease is caused by the identified mutation. In this study we have analyzed nine mutations in a mitochondrial gene called ND1 that were reported in the medical literature. We have modeled those mutations in a closely related gene found in bacteria, because it is currently not possible to do this in human cells. We confirmed that five of the mutations cause significant loss of function in the bacterial system, consistent with a severe disease state in humans. The other four mutations had milder effects, and this suggests that additional factors might be necessary for severe disease in humans. Such information can be useful in genetic counseling of women carrying mitochondrial mutations. The most common causes of mitochondrial dysfunction and disease include mutations in subunits and assembly factors of Complex I. Numerous mutations in the mitochondrial gene ND1 have been identified in humans. Currently, a bacterial model system provides the only method for rapid construction and analysis of mutations in homologs of human ND1. In this report, we have identified nine mutations in human ND1 that are reported to be pathogenic and are located at subunit interfaces. Our hypothesis was that these mutations would disrupt Complex I assembly. Seventeen mutations were constructed in the homologous nuoH gene in an E. coli model system. In addition to the clinical mutations, alanine substitutions were constructed in order to distinguish between a deleterious effect from the introduction of the mutant residue and the loss of the original residue. The mutations were moved to an expression vector containing all thirteen genes of the E. coli nuo operon coding for Complex I. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity and proton translocation were measured. Samples were also tested for assembly by native gel electrophoresis and for expression of NuoH by immunoblotting. A range of outcomes was observed: Mutations at four of the sites allow normal assembly with moderate activity (50-76% of wild type). Mutations at the other sites disrupt assembly and/or activity, and in some cases the outcomes depend upon the amino acid introduced. In general, the outcomes are consistent with the proposed pathogenicity in humans.

Automated summary

Recent advances in technology have made it possible to directly detect genetic diseases by determining the DNA sequence of genes. Researchers have simulated mitochondrial gene mutations in human cells and found that some of these mutations result in loss of function, which is valuable for genetic counseling.