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J Poulton

Publications and source records attributed to J Poulton.

At least 19 recordsLinked to original sources
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

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↗
PubMed1995

Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome.

Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with 35S[dCTP] and a reference nuclear DNA probe labelled with [32P]dCTP. The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by 'normal' quadriceps muscle. The clinical features to two of these patients were similar to earlier case reports of mtDNA depletion. In three patients the clinical course was relatively benign compared to cases that have previously been described. Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect.

Adolescent↗
PubMed1995

Mitochondrial DNA, diabetes and pancreatic pathology in Kearns-Sayre syndrome.

Mitochondrial DNA (mtDNA) mutations are associated with diabetes mellitus but their role in the onset of hyperglycaemia is unclear. A patient presented with diabetes requiring insulin therapy at the age of 7 years, followed by diagnosis of Kearns-Sayre syndrome (KSS). Beta-cell function was absent at age 19 years as shown by lack of glucose-stimulated C-peptide secretion. Following development of a cardiac conduction defect the patient died aged 21 years. Analysis of mtDNA in blood and several tissues revealed related re-arranged deletions, duplications and deletion dimers in addition to normal mtDNA with the highest levels of duplications in kidney and blood. Pancreatic tissue from the KSS patient was compared with tissue from an insulin-dependent diabetic patient with a similar clinical history of diabetes. Islets in KSS were small, regular in shape and contained predominantly glucagon-containing cells with no evidence of beta cells. In comparison, a small number of beta cells were present in some of the larger more irregularly-shaped islets from the insulin-dependent diabetic patient. These data together suggest that in KSS the loss of beta cells at the onset of diabetes is less disruptive to islet architecture: a small proportion of beta cells or their gradual destruction over a long period would allow retention of islet shape. Abnormal function of the re-arranged mtDNA could affect both development and function of pancreatic islet cells since glucose-stimulated insulin secretion is energy dependent.

Adult↗
PubMed1995

Pancreatic pathology in non-insulin dependent diabetes (NIDDM).

NIDDM is a heterogeneous disease and subgroups of NIDDM include MODY (Maturity Onset Diabetes of the Young), Malnutrition-related diabetes (MRDM) and Fibrocalculus pancreatic diabetes (FCPD). Endocrine cell population is relatively unchanged in NIDDM: B-cells are reduced by up to 30% and A-cells increased by 10%. Islet amyloid is found in 96% of subjects occupying up to 80% of the islet associated with a reduction in B-cells. Amyloid formation is unlikely to cause diabetes but progressive accumulation increases the severity of the disease. Islet amyloid is formed from the islet amyloid polypeptide (IAPP), a normal constituent of B-cells, co-secreted with insulin. The causal factors for IAPP fibrillogenesis are unknown but abnormal synthesis or overproduction could be involved: stimulation of B-cell secretion in NIDDM by obesity, hyperglycaemia or suphonylurea therapy may promote amyloidosis and further aggravate islet pathology. A mutation of the glucokinase gene in MODY leads to diminished B-cell secretion but not amyloid formation. Diabetes and mutations of mitochondrial DNA is associated with poorly developed islet structure. Exocrine pancreatic size is reduced and there is evidence of sub-clinical chronic pancreatitis in NIDDM. In MRDM and FCPD, chronic pancreatitis and exocrine necrosis is associated with reduced insulin secretion. Unlike cystic fibrosis where islet amyloid is present in diabetic individuals, amyloid is absent from subjects with FCPD. Pathological changes in the exocrine and endocrine pancreas in NIDDM results from and contributes to the pathophysiology of insulin secretion in NIDDM.

Amyloid↗
PubMed1995

Leber's hereditary optic neuropathy: implications of the sex ratio for linkage studies in families with the 3460 ND1 mutation.

Leber's hereditary optic neuropathy (LHON), which is associated with mutations in mitochondrial DNA (mtDNA), is commoner in males than females. A study of over 30 LHON families with a mutation at position 3460 of mtDNA demonstrates a significantly decreased male excess from that generally quoted, with evidence for a marked bias in the ascertainment of males over females. This has implications for the analysis of those factors which give rise to the male bias.

Adult↗
PubMed1995

Mitochondrial DNA does not appear to influence the congenital onset type of myotonic dystrophy.

Neither the maternal inheritance pattern nor the early onset of congenital myotonic dystrophy are fully explained. One possible mechanism is that mitochondrial DNA (mtDNA) mutations might interact with the DM gene product, producing an earlier onset than would otherwise occur. We have used Southern hybridisation to show that high levels of major rearrangements of mtDNA are not present in muscle of five and in blood of 35 patients with congenital myotonic dystrophy. We used sequence analysis to show that no one particular mtDNA morph appears to cosegregate with congenital onset. A minor degree of depletion of mtDNA compared with nuclear DNA was present in the muscle of five patients with congenital DM, but we propose that this is not the primary cause of the muscle pathology but secondary to it. We have not found evidence that mtDNA is involved in congenital myotonic dystrophy.

Blotting, Southern↗