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D M Turnbull

Publications and source records attributed to D M Turnbull.

At least 19 recordsLinked to original sources
PubMed2001

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease.

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Binding Sites↗
PubMed2001

Paraplegin gene analysis in hereditary spastic paraparesis (HSP) pedigrees in northeast England.

OBJECTIVE: To identify the frequency and characterize the phenotype of paraplegin mutations in the hereditary spastic paraparesis (HSP) population in the northeast of England. BACKGROUND: HSP is a disorder that shows both clinical and genetic heterogeneity. To date, 13 loci have been associated with an HSP phenotype, with the causative gene having been identified in four of these. Two autosomal genes have been identified, paraplegin and spastin, and two X-linked genes have been identified, L1CAM (cell adhesion molecule) and proteolipid protein. METHODS: Thirty HSP pedigrees from the northeast of England were analyzed for mutation in each of the 17 exons of the paraplegin gene. RESULTS: A single family with a paraplegin mutation was identified in which the paraplegin mutation co-segregates with an HSP phenotype in an apparent dominant manner. The authors also describe frequent polymorphism in the paraplegin gene in both the HSP and control populations. CONCLUSION: Mutations in the paraplegin gene are not a common cause of HSP in the northeast of England. The phenotype of the paraplegin-related HSP family described had several striking features including amyotrophy, raised creatine kinase, sensorimotor peripheral neuropathy, and oxidative phosphorylation defect on muscle biopsy.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age.

Human tissues acquire somatic mitochondrial DNA (mtDNA) mutations with age. Very high levels of specific mtDNA mutations accumulate within individual cells, causing a defect of mitochondrial oxidative metabolism. This is a fundamental property of nondividing tissues, but it is not known how it comes about. To explore this problem, we developed a model of mtDNA replication within single human cells. Using this model, we show that relaxed replication of mtDNA alone can lead, through random genetic drift, to the clonal expansion of single mutant events during human life. Significant expansions primarily develop from mutations acquired during a critical period in childhood or early adult life.

Adult↗
PubMed2001

Leber hereditary optic neuropathy: Does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation?

Leber hereditary optic neuropathy (LHON) is a major cause of inherited blindness in young males. Approximately 1 in 7 individuals with LHON harbor a mixture of mutated and wild-type (normal) mtDNA (heteroplasmy), and the risks of developing blindness in heteroplasmic LHON individuals are not well characterized. MtDNA is inherited exclusively down the maternal line, and although the risks of a relative within a homoplasmic LHON pedigree are relatively well established, the risks of transmission in heteroplasmic LHON pedigrees have not been studied in detail. We analyzed 17 independent pedigrees that harbor the most prevalent LHON mutation: G11778A. The pedigrees were influenced by incomplete ascertainment bias, which was reduced by omitting the affected probands from the analysis. We made the following observations: (1) The frequency of blindness in males was related to the mutation load in that individual's blood. (2) Mothers with < or = 80% mutant mtDNA in blood were less likely to have clinically affected sons than mothers with 100% mutant mtDNA in their blood. (3) Within individual lineages, changes in mutation load from one generation to the next were largely determined by random genetic drift in these pedigrees. This study provides insights into the mutation load, or threshold, necessary for expression of the optic neuropathy, the relationship between mutation load in the mother and the risk of blindness in her children, and the complex inheritance of heteroplasmic mtDNA defects.

DNA, Mitochondrial↗
PubMed2001

Mitochondrial DNA mutations in disease and ageing.

The chronological accumulation of mitochondrial DNA mutations has been proposed as a potential mechanism in the physiological processes of ageing and age-related disease. We discuss the evidence behind this theory and relate some of the ageing mitochondrial changes to mitochondrial DNA disorders. In particular, we describe the aggregation of cytochrome c oxidase-deficient cells in both skeletal muscle and the CNS in normal ageing as seen in the mitochondrial DNA disorders. These mitochondrial enzyme-deficient cells have been shown to occur in significant quantities in both muscle and CNS in patients with mitochondrial DNA disorders. In both ageing and mtDNA disorder muscle these cytochrome c-deficient fibres contain high levels of a single mutant strain of mitochondrial DNA. Whether these mutations are a primary or secondary event in the physiology of ageing remains to be determined.

Aging↗
PubMed2001

The mitochondrial ND6 gene is a hot spot for mutations that cause Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a common cause of bilateral optic nerve disease. The majority of LHON patients harbour one of three point mutations of the mitochondrial DNA (mtDNA) complex I, or NADH:ubiquinone oxidoreductase (ND) genes (G11778A in ND4, G3460A in ND1, T14484C in ND6). As a consequence, screening for these mutations has become part of the routine clinical investigation of young adults who present with bilateral optic neuropathy, and the absence of these mutations is interpreted as indicating there is a low likelihood that an optic neuropathy is LHON. However, there are many individuals who develop the clinical features of LHON but who do not harbour one of these primary LHON mutations. We describe two LHON pedigrees that harbour the same novel point mutation within the mtDNA ND6 gene (A14495G). This mutation was heteroplasmic in both families, and sequencing of the mitochondrial genome confirmed that the mutation arose on two independent occasions. This is the seventh mutation in the ND6 gene that causes optic neuropathy, indicating that this gene is a hot spot for LHON mutations. Protein modelling studies indicate that all of these pathogenic mutations lie within close proximity to one another in a hydrophobic cleft or pocket. This is the first evidence for a relationship between a specific disease phenotype and a specific structural domain within a mitochondrial respiratory chain subunit. These findings suggest that the mtDNA ND6 gene should be sequenced in all patients with LHON who do not harbour one of the three common LHON mutations.

Adolescent↗
PubMed2001

Mitochondrial enteropathy: the primary pathology may not be within the gastrointestinal tract.

BACKGROUND: Mitochondrial DNA (mtDNA) defects are an important cause of disease. Although gastrointestinal symptoms are common in these patients, their pathogenesis remains uncertain. AIM: To investigate the role of the mtDNA defect in the production of gastrointestinal dysfunction. PATIENT: A 20 year old woman who presented at 15 years of age with recurrent vomiting and pseudo-obstruction, who did not respond to conservative management and ultimately had subtotal gastrectomy and Roux-en-y reconstruction. She subsequently presented with status epilepticus and was found to have a mitochondrial respiratory chain disorder due to a pathogenic mtDNA point mutation (A3243G). METHODS: Resected bowel was studied using light and electron microscopy and mtDNA analysed from both mucosal and muscular layers using polymerase chain reaction generated RFLP analysis. RESULTS: Histological and electron microscopic studies revealed no morphological abnormalities in the resected stomach, and molecular genetic analysis failed to identify the genetic defect in either the mucosal or muscle layers. CONCLUSION: This study suggests that in some individuals with gastrointestinal symptoms associated with established mitochondrial DNA disease, the primary pathology of the mitochondrial enteropathy lies outside the gastrointestinal tract.

Adolescent↗
PubMed2000

Point mutations of the mtDNA control region in normal and neurodegenerative human brains.

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.

Aged↗
PubMed2000

Analysis of European mtDNAs for recombination.

The standard paradigm postulates that the human mitochondrial genome (mtDNA) is strictly maternally inherited and that, consequently, mtDNA lineages are clonal. As a result of mtDNA clonality, phylogenetic and population genetic analyses should therefore be free of the complexities imposed by biparental recombination. The use of mtDNA in analyses of human molecular evolution is contingent, in fact, on clonality, which is also a condition that is critical both for forensic studies and for understanding the transmission of pathogenic mtDNA mutations within families. This paradigm, however, has been challenged recently by Eyre-Walker and colleagues. Using two different tests, they have concluded that recombination has contributed to the distribution of mtDNA polymorphisms within the human population. We have assembled a database that comprises the complete sequences of 64 European and 2 African mtDNAs. When this set of sequences was analyzed using any of three measures of linkage disequilibrium, one of the tests of Eyre-Walker and colleagues, there was no evidence for mtDNA recombination. When their test for excess homoplasies was applied to our set of sequences, only a slight excess of homoplasies was observed. We discuss possible reasons that our results differ from those of Eyre-Walker and colleagues. When we take the various results together, our conclusion is that mtDNA recombination has not been sufficiently frequent during human evolution to overturn the standard paradigm.

Africa↗
PubMed2000

Mitochondrial DNA haplogroups and susceptibility to AD and dementia with Lewy bodies.

The authors analyzed the relationship between nuclear genetic risk factors (apolipoprotein E genotype) and mitochondrial DNA (mtDNA) sequence variants in pathologically proved cases of AD (n = 185), dementia with Lewy bodies (DLB; n = 84), and control subjects (n = 179). Specific European mtDNA haplogroups and the A4336G mutation were not associated with an increased risk of AD. mtDNA haplogroup H was overrepresented in the DLB patients when compared with control subjects. Additional studies are needed to clarify the significance of the association.

Aged↗
PubMed2000

The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both?

The mammalian mitochondrial genome (mtDNA) is a small double-stranded DNA molecule that is exclusively transmitted down the maternal line. Pathogenic mtDNA mutations are usually heteroplasmic, with a mixture of mutant and wild-type mtDNA within the same organism. A woman harbouring one of these mutations transmits a variable amount of mutant mtDNA to each offspring. This can result in a healthy child or an infant with a devastating and fatal neurological disorder. Understanding the biological basis of this uncertainty is one of the principal challenges facing scientists and clinicians in the field of mitochondrial genetics.

Animals↗
PubMed2000

Mitochondrial DNA mutations in the pathogenesis of human disease.

The coding sequence for the human mitochondrial genome (mtDNA) was published in 1981. Within a decade, the first pathogenic mtDNA mutations were described in humans with sporadic and maternally inherited disease. The last ten years has seen a profusion of reports describing new pathogenic mutations associated with a diverse range of clinical phenotypes. Although we have seen great advances in our understanding of the molecular mechanisms involved in the pathogenesis of mtDNA disease, we are only just beginning to tackle some of the more difficult questions. In this review we describe recent advances in our understanding of mtDNA disease and highlight ways that this knowledge might lead to novel therapies in the future.

Animals↗
PubMed2000

The spectrum of hearing loss due to mitochondrial DNA defects.

Heteroplasmic mitochondrial DNA (mtDNA) defects are an important cause of neurological disease. Although hearing impairment is common in patients with mtDNA defects, the spectrum and pathophysiology of the hearing loss is not well characterized. We therefore studied the relationship between cochlear and brainstem auditory function in 23 patients harbouring a range of different mtDNA mutations. Based upon the pure tone audiogram, patients fell into three distinct groups: (i) normal hearing, (ii) mild to moderate predominantly high frequency hearing loss, and (iii) severe or profound hearing loss at all frequencies. Within this study group only certain genetic defects were associated with hearing loss, and for individuals harbouring the A3243G point mutation, the severity of the hearing loss correlated with the percentage level of mutated mtDNA (mutation load) in skeletal muscle. The 10 patients who had a moderate hearing loss or less had normal brainstem auditory evoked responses and MRI, but it was not possible to interpret the brainstem auditory evoked responses in 13 patients with severe hearing loss. Otoacoustic emissions were absent in patients with a moderate or more severe hearing loss. These findings are consistent with a predominantly cochlear origin for the hearing deficit, which is determined by the precise genetic defect and the percentage mutation load.

Adult↗
PubMed2000

Transmission of the human mitochondrial genome.

The segregation and transmission of mitochondrial genomes in humans are complicated processes, but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. This review describes three aspects of mitochondrial genetics. First, that the segregation and transmission of mitochondrial (mt)DNA molecules are likely to be determined by their physical association within the organelles and by the dynamics of mitochondrial structure and subcellular organization. Second, that the transmission of heteroplasmic mtDNA sequence changes from one generation to the next often involves rapid shifts in allele frequency. For >20 years, the standard explanation has been that there is a developmental bottleneck in which, at some stage of oogenesis, there is a reduction in the effective number of mitochondrial units of inheritance. The third aspect is that ongoing analyses of the segregation and transmission of pathogenic mtDNA mutations indicate the operation of multiple genetic processes. Thus, the segregation and transmission of mtDNA mutations occurs predominantly, but not exclusively, under conditions of random genetic drift. However, there is also evidence for bias due to incomplete ascertainment of pedigrees and for negative selection of pathogenic mutations in rapidly dividing somatic tissues such as the white blood cell population.

Chromosome Segregation↗
PubMed2000

In-vitro genetic modification of mitochondrial function.

Defects of mitochondrial (mt) DNA cause a diverse group of incurable, progressive diseases that often lead to severe disability and premature death. Most patients with pathogenic mtDNA defects have a mixture of mutant and wild-type mtDNA (heteroplasmy), and the clinical defect is only expressed when the percentage of mutant mtDNA exceeds a critical threshold. Since mtDNA is continually replicating and being turned over, we have proposed an approach to the treatment of these disorders that utilizes sequence-specific antigenomic peptide nucleic acids (PNAs) to hybridize and specifically inhibit the replication of mutant mtDNA under physiological conditions. By allowing the selective propagation of wild-type molecules, it may be possible to correct the cellular biochemical defect and to prevent the progression of disease. This paper summarizes the experimental progress in this area, including the cellular uptake of PNA molecules and their import into mitochondria both in vitro and in cell culture by the addition of a nuclear-encoded mitochondrial targeting sequence. The possibilities of extending this strategy to the treatment of mtDNA deletion disorders are discussed.

DNA Replication↗