Autosomal dominant deletions of the mitochondrial genome in a case of progressive encephalomyopathy.

Multiple deletions of the mitochondrial genome were found in a family in which the proband had ataxia and ketoacidotic comas. A progressive multiorgan involvement appeared in the course of the disease, and histopathological investigation demonstrated mitochondrial myopathy features with ragged red fibers. A defect of oxidative phosphorylation was found in both skeletal muscle and lymphocytes. It is surprising that various mtDNA deletions were detected both in the proband and in his healthy mother and maternal aunt but not in the rest of the maternal progeny. All the deletions were located between Cox II and cytochrome b genes, and short (4-5 bp) repeated sequences were consistently present at the boundaries of the rearrangements in different tissues. Therefore, the deletions appear not to be transmitted per se but to be inherited in a Mendelian manner, being possibly dominant. Both the Mendelian inheritance of the trait and the variety of the deletions in carriers suggest that a nuclearly encoded factor(s) might be involved in the triggering of the deletions. However, the presence of the rearrangements in healthy individuals raises the question of whether mtDNA deletions actually cause the clinical expression of the disease.     

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.     

MITOMAP: a human mitochondrial genome database.

We have developed a comprehensive database (MITOMAP) for the human mitochondrial DNA (mtDNA), the first component of the human genome to be completely sequenced [Anderson et al. (1981) Nature 290, 457-465]. MITOMAP uses the mtDNA sequence as the unifying element for bringing together information on mitochondrial genome structure and function, pathogenic mutations and their clinical characteristics, population associated variation, and gene- gene interactions. As increasingly larger regions of the human genome are sequenced and characterized, the need for integrating such information will grow. Consequently, MITOMAP not only provides a valuable reference for the mitochondrial biologist, it may also provide a model for the development of information storage and retrieval systems for other components of the human genome.     

Detection of deletions in the mitochondrial genome of Caenorhabditis elegans.

We have examined an aging population of Caenorhabditis elegans via a PCR assay to determine if deletions in the mitochondrial genome occur in the nematode. We detected eight such deletions, identified the breakpoints of four of these, and discovered direct repeats of 4-8 base pairs at the site of all four deletions. Six of the eight repeats involved in the deletions are located in or immediately adjacent to tRNAs. Without a biochemical bias, the probability of direct repeats being present at all four breakpoints was 4 x 10(-6).     

Site-specific deletions of the mitochondrial genome in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome is a fatal disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a widespread defect of oxidative phosphorylation. Here, we describe deletions of the mitochondrial (mt) genome between repeated 8- to 13-bp sequences as consistent features of the disease. Studying a series of nine unrelated children, including the patient originally reported by H. Pearson, we found five different types of direct repeats at the boundaries of the mtDNA deletions and we provided evidence for conservation of the 3'-repeated sequence in the deletions. In addition, we found a certain degree of homology between the nucleotide composition of the direct repeats and several structures normally involved in mtDNA replication and mtRNA processing. These results are consistent either with the recognition and cleavage of a particular DNA sequence with a factor of still unknown origin or with a homologous recombination between direct-repeat mtDNA sequences in the Pearson syndrome.     

Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA

Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.     

Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.     

MITOMAP: a human mitochondrial genome database--1998 update.

We have continued to develop MITOMAP (http://www.gen.emory. edu/MITOMAP ), a comprehensive database for the human mitochondrial DNA (mtDNA). MITOMAP uses the mtDNA sequence as the unifying element for bringing together information on mitochondrial genome structure and function, pathogenic mutations and their clinical characteristics, population associated variation, and gene-gene interactions. Over the past year we have increased the degree of interlinking of MITOMAP information available on the web page, by using our generalized information management system, GENOME. As increasingly larger regions of the human genome are sequenced and characterized, the need for integrating such information is growing. Consequently, MITOMAP and GENOME provide a valuable reference for the mitochondrial biologist, in addition to being a model for the development of comprehensive, information storage and retrieval systems for other components of the human genome. This paper documents the changes to MITOMAP which have been implemented over the past year.     

Mapping of a new autosomal dominant spinocerebellar ataxia to chromosome 22.

The autosomal dominant cerebellar ataxias (ADCAs) are a clinically and genetically heterogeneous group of disorders. The clinical symptoms include cerebellar dysfunction and associated signs from dysfunction in other parts of the nervous system. So far, five spinocerebellar ataxia (SCA) genes have been identified: SCA1, SCA2, SCA3, SCA6, and SCA7. Loci for SCA4 and SCA5 have been mapped. However, approximately one-third of SCAs have remained unassigned. We have identified a Mexican American pedigree that segregates a new form of ataxia clinically characterized by gait and limb ataxia, dysarthria, and nystagmus. Two individuals have seizures. After excluding all known genetic loci for linkage, we performed a genomewide search and identified linkage to a 15-cM region on chromosome 22q13. A maximum LOD score of 4.3 (recombination fraction 0) was obtained for D22S928 and D22S1161. This distinct form of ataxia has been designated "SCA10." Anticipation was observed in the available parent-child pairs, suggesting that trinucleotide-repeat expansion may be the mutagenic mechanism.     

Experimental transmission of an autosomal dominant spongiform encephalopathy: does the infectious agent originate in the human genome?

Marmosets inoculated intracerebrally with brain tissue from a woman with Gerstmann-Straussler syndrome (an autosomal dominant dementia associated with spongiform change and amyloid deposition) developed an encephalopathy indistinguishable from that seen in marmosets inoculated with brain tissue from a typical case of Creutzfeldt-Jakob disease. As in Huntington''s disease, in the pedigree of the patient with Gerstmann-Straussler syndrome women who subsequently developed the illness had increased fecundity. The pathogen in human transmissible dementia may arise from a sequence (which itself sometimes confers a selective advantage) located within the human genome.     

Increased frequency of deletions in the mitochondrial genome with age of Caenorhabditis elegans.

We have developed a long-extension-PCR strategy which amplifies approximately half of the mitochondrial genome (6.3 kb) of Caenorhabditis elegans using an individual worm as target. We analyzed three strains over their life span to assess the number of detectable deletions in the mitochondrial genome. Two of these strains are wild-type for life span while the third is mutant in the age-1 gene, approximately doubling its maximum life span. At the mean life span in wild-type strains, there was a significant difference between the frequency of deletions detected in the mitochondrial genome compared with the mean number of deletions in young animals. In addition, deletions in the mitochondrial genome occur at a significantly lower rate in age-1 mutants as compared with wild type. We cloned and identified the breakpoints of two deletions and found that one of the deletions had a direct repeat of 8 bp at the breakpoint. This is the largest single study (over 900 individual animals) characterizing the frequency of deletions in the mitochondrial genome as a function of age yet carried out.     

Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae

Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.     

A new locus for autosomal dominant stargardt-like disease maps to chromosome 4

Stargardt disease (STGD) is the most common hereditary macular dystrophy and is characterized by decreased central vision, atrophy of the macula and underlying retinal-pigment epithelium, and frequent presence of prominent flecks in the posterior pole of the retina. STGD is most commonly inherited as an autosomal recessive trait, but many families have been described in which features of the disease are transmitted in an autosomal dominant manner. A recessive locus has been identified on chromosome 1p (STGD1), and dominant loci have been mapped to both chromosome 13q (STGD2) and chromosome 6q (STGD3). In this study, we describe a kindred with an autosomal dominant Stargardt-like phenotype. A genomewide search demonstrated linkage to a locus on chromosome 4p, with a maximum LOD score of 5.12 at a recombination fraction of.00, for marker D4S403. Analysis of extended haplotypes localized the disease gene to an approximately 12-cM interval between loci D4S1582 and D4S2397. Therefore, this kindred establishes a new dominant Stargardt-like locus, STGD4.     

Age-associated mitochondrial DNA deletions in mouse skeletal muscle: Comparison of different regions of the mitochondrial genome

The abundance of mitochondrial DNA (mtDNA) deletions has been shown to increase with age in a number of species and may contribute to the aging process. Estimating the total mtDNA deletion load of an individual is essential in evaluating the potential physiological impact. In this study, we compared three 5-kb regions of the mitochondrial genome: one in the major arc, one in the minor arc, and a third containing the light strand origin of replication. Through PCR analysis of mouse skeletal muscle, we have determined that not all regions produce equal numbers of age-associated deletions. There are, on average, twofold more detectable deletions in the major arc region than in the minor arc region. Deletions that result in the loss of the light strand origin of replication are rarely detected. Furthermore, the mechanism of deletion formation seems to be similar in both the major and minor arcs, with direct repeats playing an important, although not essential, role. © 1996 Wiley-Liss, Inc.     

A loss-of-function model for cystogenesis in human autosomal dominant polycystic kidney disease type 2.

Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci (PKD1, PKD2, and PKD3) that account for the disease. Mutations in the PKD2 gene, on the long arm of chromosome 4, are expected to be responsible for approximately 15% of cases of ADPKD. Although ADPKD is a systemic disease, it shows a focal expression, because <1% of nephrons become cystic. A feasible explanation for the focal nature of events in PKD1, proposed on the basis of the two-hit theory, suggests that cystogenesis results from the inactivation of the normal copy of the PKD1 gene by a second somatic mutation. The aim of this study is to demonstrate that somatic mutations are present in renal cysts from a PKD2 kidney. We have studied 30 renal cysts from a patient with PKD2 in which the germline mutation was shown to be a deletion that encompassed most of the disease gene. Loss-of-heterozygosity (LOH) studies showed loss of the wild-type allele in 10% of cysts. Screening of six exons of the gene by SSCP detected eight different somatic mutations, all of them expected to produce truncated proteins. Overall, >/=37% of the cysts studied presented somatic mutations. No LOH for the PKD1 gene or locus D3S1478 were observed in those cysts, which demonstrates that somatic alterations are specific. We have identified second-hit mutations in human PKD2 cysts, which suggests that this mechanism could be a crucial event in the development of cystogenesis in human ADPKD-type 2.