A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J x CAST/Ei) x A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ x CAST/Ei) x NOD/LtJ and (SKH2/J x CAST/Ei) x SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.     

SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination

The Escherichia coli gene recQ was identified as a RecF recombination pathway gene. The gene SGS1, encoding the only RecQ-like DNA helicase in Saccharomyces cerevisiae, was identified by mutations that suppress the top3 slow-growth phenotype. Relatively little is known about the function of Sgs1p because single mutations in SGS1 do not generally cause strong phenotypes. Mutations in genes encoding RecQ-like DNA helicases such as the Bloom and Werner syndrome genes, BLM and WRN, have been suggested to cause increased genome instability. But the exact DNA metabolic defect that might underlie such genome instability has remained unclear. To better understand the cellular role of the RecQ-like DNA helicases, sgs1 mutations were analyzed for their effect on genome rearrangements. Mutations in SGS1 increased the rate of accumulating gross chromosomal rearrangements (GCRs), including translocations and deletions containing extended regions of imperfect homology at their breakpoints. sgs1 mutations also increased the rate of recombination between DNA sequences that had 91% sequence homology. Epistasis analysis showed that Sgs1p is redundant with DNA mismatch repair (MMR) for suppressing GCRs and for suppressing recombination between divergent DNA sequences. This suggests that defects in the suppression of rearrangements involving divergent, repeated sequences may underlie the genome instability seen in BLM and WRN patients and in cancer cases associated with defects in these genes.     

Recombinant expression of perchloric acid-soluble protein reduces cell proliferation

Perchloric acid-soluble protein (PSP) may play an important role in the regulation of cellular physiological functions because it has been highly conserved throughout evolution; however, this role has not been well elucidated. In previous reports, we suggested that PSP regulates cell proliferation. In this study, we examined the effect of PSP expression on proliferation of the normal rat kidney cell line NRK-52E, the rat hepatocyte cell line RLN-10, and the rat hepatoma cell line dRLh-84. Cells transfected with pcDNA-sense-PSP (pcDNA-S-PSP) over-expressed PSP mRNA and protein, and cell proliferation of the transfected cells was suppressed compared with that of cells transfected with pcDNA-empty (pcDNA-E). Cell viability of pcDNA-S-PSP-transfected cells was similar to that of pcDNA-E-transfected cells. Thus, over-expression of PSP suppresses cell proliferation without any influence on cell viability. These findings are the first to report an inhibitory activity of PSP on cell proliferation. Received 27 April 2001; received after revision 8 June 2001; accepted 8 June 2001     

High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection

Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.     

Failure to confirm NOTCH4 association with schizophrenia in a large population-based sample from Scotland

The NOTCH4 gene was recently reported to be associated with schizophrenia based on TDT analysis of 80 British trios. The strongest evidence for association derived from two microsatellites. We genotyped both loci in a large sample of unrelated Scottish schizophrenics and controls, but failed to replicate the reported association, finding instead that each putative schizophrenia-associated allele had a somewhat lower frequency in schizophrenics than in controls.     

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.     

Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains

Factor V is a plasma protein essential for blood coagulation. This protein is involved in activated protein C resistance, the most common inherited thrombotic disorder known. We utilized the polymerase chain reaction to clone the porcine factor V gene by generating overlapping clones amplified with primers chosen by comparison with known nucleotide sequences. The porcine factor V cDNA contig encodes a predicted 2258-amino acid protein, making it the largest in comparison to the bovine, human, and murine proteins. Porcine factor V has the highest level of homology with bovine factor V, but also has high levels of conservation of important residues with all the species. Radiation hybrid mapping assigned the porcine factor V gene to chromosome 4. Three-dimensional models of factor V were generated and used to analyze membrane-binding sites in terms of conserved, and therefore likely important residues. Received 3 October 2000; revised 23 November 2000; accepted 6 December 2000