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Biomedical subjects

J Poulton

Publications and source records attributed to J Poulton.

At least 19 recordsLinked to original sources
PubMed1999

Mitochondrial DNA polymerase gamma deficiency and mtDNA depletion in a child with Alpers' syndrome.

Deficiency of mitochondrial DNA polymerase gamma activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase gamma was undetectable.

Ataxia↗
PubMed1998

A common mitochondrial DNA variant is associated with insulin resistance in adult life.

Mitochondrial DNA is maternally inherited. Mitochondrial DNA mutations could contribute to the excess of maternal over paternal inheritance of non-insulin-dependent diabetes mellitus (NIDDM). We therefore investigated the relationship between this variant, insulin resistance and other risk factors in a cohort which had been well characterised with respect to diabetes. Blood DNA was screened from 251 men born in Hertfordshire 1920-1930 in whom an earlier cohort study had shown that glucose tolerance was inversely related to birthweight. The 16189 variant (T--> C transition) in the first hypervariable region of mitochondrial DNA was detected using the polymerase chain reaction and restriction digestion. DNA analysis showed that 28 of the 251 men (11%) had the 16189 variant. The prevalence of the 16189 variant increased progressively with fasting insulin concentration (p < 0.01). The association was independent of age and body mass index and was present after exclusion of the patients with NIDDM or impaired glucose tolerance. We found that insulin resistance in adult life was associated with the 16189 variant. This study provides the first evidence that a frequent mitochondrial variant may contribute to the phenotype in patients with a common multifactorial disorder.

Adult↗
PubMed1998

Evidence from human oocytes for a genetic bottleneck in an mtDNA disease.

We have examined oocytes from a patient with Kearn-Sayre syndrome caused by mtDNA rearrangements. In mtDNA diseases, mutant and wild-type mtDNA frequently coexist in affected individuals (the condition of heteroplasmy). The proportion of mutant mtDNA transmitted from mother to offspring is variable because of a genetic bottleneck, and the "dose" of mutant mtDNA received influences the severity of the phenotype. The feasibility of prenatal diagnosis is critically dependent on the nature and timing of this bottleneck. Significant levels of rearranged mtDNA were detectable in the majority of the patient's oocytes, by use of multiplex PCR, with wide variation, in the levels of mutant and wild-type molecules, between individual oocytes. We also used length variation in a homopolymeric C tract, which is often heteroplasmic in normal controls, to identify founder subpopulations of mtDNAs in this patient's oocytes. We present direct evidence that the number of segregating units (n) is three to five orders of magnitude less than the number of mitochondria in the human female oocyte. In some cases, the best estimate of n may correspond to a single mitochondrion, if it is assumed that intergenerational transmission of mtDNA can be treated as a single sampling event. The bottleneck appears to contribute a major component of the variable transmission from mother to oocyte, in this patient and in a control. That this bottleneck had occurred by the time that oocytes were mature advances the prospects for prenatal diagnosis of mtDNA diseases.

DNA Primers↗
PubMed1997

Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck.

While mtDNA polymorphisms at single base positions are common, the overwhelming majority of the mitochondrial genomes within a single individual are usually identical. When there is a point-mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Length polymorphisms in homopolymeric tracts have been demonstrated in the large noncoding region of mtDNA. We have developed a new method, T-PCR (trimmed PCR), to quantitate heteroplasmy for two of these tracts (D310 and D16189). D310 variation is sufficient to indicate clonal origins of tissues and single oocytes. Tissues from normal individuals often possessed more than one length variant (heteroplasmy). However, there was no difference in the pattern of the length variants between somatic tissues in any control individual when bulk samples were taken. Oocytes from normal women undergoing in vitro fertilization were frequently heteroplasmic for length variants, and in two cases the modal length of the D310 tract differed in individual oocytes from the same woman. These data suggest that a restriction/amplification event, which we attribute to clonal expansion of founder mtDNA(s), has occurred by the time oocytes are mature, although further segregation may occur at a later stage. In contrast to controls, the length distribution of the D310 tract varied between tissues in a patient with heteroplasmic mtDNA rearrangements, suggesting that these mutants influence segregation. These findings have important implications for the genetic counselling of patients with pathogenic mtDNA mutations.

Animals↗
PubMed1996

Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin.

Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.

Base Sequence↗
PubMed1996

Prospects for DNA-based prenatal diagnosis of mitochondrial disorders.

Mitochondria have their own DNA which is maternally inherited. Mitochondrial DNA (mtDNA) diseases are extremely variable because of the genetics of mtDNA and the unique pathogenesis of these disorders. This makes predicting the prognosis and the transmission of mtDNA disorders difficult. While mtDNA polymorphisms at a single base position are common, the overwhelming majority of the mitochondrial genomes within a single human individual are usually identical. When there is a point mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Autosomally inherited disorders of mitochondrial function may be caused by mutations in genes for the components of the respiratory chain and for the machinery of mitochondrial biogenesis, which are nuclear-encoded. Accurate diagnosis of these disorders is important as prenatal diagnosis is available in a minority of cases.

Animals↗
PubMed1996

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non-coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.

Base Sequence↗
PubMed1996

Leber's hereditary optic neuropathy: heteroplasmy is likely to be significant in the expression of LHON in families with the 3460 ND1 mutation.

AIM: To assess the effect of heteroplasmy on the expression of Leber's hereditary optic neuropathy (LHON) in a large family with the 3460 LHON mutation. METHODS: Mutation detection was performed by restriction enzyme digestion of polymerase chain reaction (PCR) products. Heteroplasmy was estimated by quantitation of wild type:mutant product ratios. RESULTS: There is a significant association between levels of mutant mtDNA and manifestation of the disease phenotype. CONCLUSION: As a high proportion of families with the 3460 mutation demonstrate heteroplasmy; this is likely to be a significant factor in disease expression.

Adolescent↗
PubMed1995

Duplications of mitochondrial DNA in Kearns-Sayre syndrome.

mtDNA duplications were detectable in 10 of 10 patients with mtDNA deletions and Kearns-Sayre syndrome (KSS) and in none of 8 patients with chronic progressive external ophthalmoplegia (CPEO). Thus, duplications of mtDNA seem to be a distinctive feature of KSS, including patients where Pearson's syndrome is the first manifestation. Diabetes mellitus was identified in 4 of 7 patients with high or moderate levels of mtDNA duplications. The balance of mtDNA rearrangements may be central to the pathogenesis of this unique group of disorders.

Adult↗
PubMed1995

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA.

Studies in vitro have shown that a respiratory-deficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A-->G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G-->A) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (rho0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.

Adolescent↗