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1.
Neuromuscul Disord ; 15(5): 364-71, 2005 May.
Article in English | MEDLINE | ID: mdl-15833431

ABSTRACT

We performed detailed clinical, histopathological, biochemical, in vitro translation and molecular genetic analysis in patients from two unrelated families harbouring the tRNA(SerUCN) 7472C-insertion mutation. Proband 1 developed a progressive neurodegenerative phenotype characterised by myoclonus, epilepsy, cerebellar ataxia and progressive hearing loss. Proband 2 had a comparatively benign phenotype characterised by isolated myopathy with exercise intolerance. Both patients had the 7472C-insertion mutation in identical proportions and they exhibited a similar muscle biochemical and histopathological phenotype. However, proband 2 also had a previously unreported homoplasmic A to C transition at nucleotide position 7472 in the tRNA(SerUCN) gene. This change lengthens further the homopolymeric C run already expanded by the 7472C-insertion. These data extend the phenotypic range associated with the 7472C-insertion to include isolated skeletal myopathy, as well as a MERRF-like phenotype.


Subject(s)
DNA, Mitochondrial/genetics , Mitochondrial Encephalomyopathies/genetics , Mutation , RNA, Transfer, Ser/genetics , Adolescent , Adult , DNA Mutational Analysis/methods , Electron Transport Complex IV/metabolism , Electrophoresis/methods , Female , Humans , Male , Microscopy, Electron, Transmission/methods , Mitochondria, Muscle/pathology , Mitochondrial Encephalomyopathies/enzymology , Mitochondrial Encephalomyopathies/pathology , Mitochondrial Encephalomyopathies/physiopathology , Mitochondrial Proteins/metabolism , Muscle, Skeletal/enzymology , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Nucleic Acid Conformation , Phenotype , RNA, Transfer, Ser/chemistry , Serine/metabolism
2.
Mol Hum Reprod ; 8(11): 1046-9, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12397219

ABSTRACT

There is a risk that ICSI may increase the transmission of mtDNA diseases to children born after this technique. Knowledge of the fate and transmission of paternal mitochondrial DNA is important since mutations in mitochondrial DNA have been described in oligozoospermic males. We have used an adaptation of solid phase mini-sequencing to exclude the presence of levels of paternal mtDNA >0.001% in ICSI families. This method is more sensitive than those used in previous studies and is sufficient to detect the likely paternal contribution (approximately 0.1-0.5% from simple calculations of expected dilution during fertilization). Using this method, we were able to detect concentrations as low as 0.001% paternal mtDNA in a maternal mtDNA background. No paternal mtDNA was detected in the embryonic (blood or buccal swabs) tissue of children born after ICSI nor in extra-embryonic tissue (placenta or umbilical cord). In conclusion, we did not detect paternal mtDNA in blood, buccal swabs, placenta or umbilical cord of children born after ICSI. We have found no evidence that ICSI increases the risk of paternal transmission of mtDNA and hence of mtDNA disorders.


Subject(s)
DNA, Mitochondrial/analysis , Extrachromosomal Inheritance , Sperm Injections, Intracytoplasmic , DNA, Mitochondrial/blood , DNA, Mitochondrial/genetics , Extrachromosomal Inheritance/physiology , Fathers , Female , Humans , Male , Mouth Mucosa/chemistry , Mouth Mucosa/cytology , Mouth Mucosa/physiology , Oligospermia/therapy , Placenta/chemistry , Placenta/physiology , Pregnancy , Umbilical Cord/chemistry , Umbilical Cord/physiology
3.
Nat Genet ; 29(3): 261-2, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11687794

ABSTRACT

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.


Subject(s)
DNA, Mitochondrial/genetics , DNA-Directed DNA Polymerase/genetics , Genetic Predisposition to Disease/genetics , Infertility, Male/genetics , Mutation/genetics , Alleles , Asian People/genetics , DNA Polymerase gamma , DNA-Directed DNA Polymerase/chemistry , Homozygote , Humans , Infertility, Male/pathology , Male , Microsatellite Repeats/genetics , Peptides/genetics , Peptides/metabolism , Phenotype , Spermatozoa/enzymology , Spermatozoa/metabolism , Spermatozoa/pathology , White People/genetics
4.
Neuromuscul Disord ; 10(7): 484-7, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10996778

ABSTRACT

Mitochondrial DNA is almost entirely maternally inherited. Thousands of copies of mitochondrial DNA are present in every nucleated cell and in most normal individuals these are virtually identical (homoplasmy). Mitochondrial DNA diseases may be caused by mutations in either mitochondrial (Nature 1988;331:717-719) or nuclear genes (Nature 1989;339(6222):309-311; Br J Hosp Med 1996;55:712-716) and hence give rise to maternal or autosomal patterns of inheritance. Antenatal diagnosis of mitochondrial diseases based on chorionic villus sampling is available for Mendelian disorders and the syndromes caused by mutations at bp 8993 (associated with both Leigh's syndrome or neurogenic weakness ataxia and retinitis pigmentosa). However, prenatal diagnosis of many other maternally inherited mitochondrial DNA diseases is less reliable because it is not possible to predict the way in which heteroplasmic mitochondrial DNA mutations segregate within tissues with confidence. This review focuses on the substantial progress that has been made recently, and on the applicability of prenatal diagnosis to genetic counselling in this field.


Subject(s)
DNA, Mitochondrial/genetics , Genetic Counseling/trends , Mitochondrial Myopathies/diagnosis , Mitochondrial Myopathies/genetics , Prenatal Diagnosis/trends , Female , Humans , Pregnancy
7.
Am J Hum Genet ; 63(3): 769-75, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9718339

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/genetics , Kearns-Sayre Syndrome/genetics , Oocytes/chemistry , DNA Primers , Dimerization , Female , Gene Rearrangement , Genomic Imprinting , Humans , Kearns-Sayre Syndrome/pathology , Oocytes/pathology , Ovary/pathology , Phenotype , Polymerase Chain Reaction , Sequence Deletion
9.
Diabetologia ; 41(1): 54-8, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9498630

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/genetics , Diabetes Mellitus, Type 2/genetics , Genetic Variation , Genomic Imprinting , Glucose Intolerance/genetics , Insulin Resistance/genetics , Point Mutation , Adult , Aged , Base Sequence , Blood Glucose/metabolism , Body Composition , Body Mass Index , Cohort Studies , DNA/blood , England , Female , Glucose Tolerance Test , Humans , Male , Patient Selection , Phenotype , Polymerase Chain Reaction
10.
Am J Hum Genet ; 60(2): 408-16, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9012414

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/genetics , Oocytes/chemistry , Point Mutation , Polymorphism, Genetic , Animals , Cell Line , DNA, Mitochondrial/chemistry , Female , Humans , MELAS Syndrome/genetics , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction
11.
Prenat Diagn ; 16(13): 1247-56, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9061755

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/analysis , DNA, Mitochondrial/genetics , Mutation , Prenatal Diagnosis , Animals , Female , Humans , Pregnancy
12.
Hum Mol Genet ; 5(4): 473-9, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8845839

ABSTRACT

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.


Subject(s)
DNA, Mitochondrial/genetics , Genetic Diseases, Inborn/genetics , Point Mutation , Base Sequence , DNA Primers , Female , Humans , Molecular Sequence Data , Polymorphism, Genetic , RNA, Transfer, Amino Acyl/genetics
13.
Brain ; 117 ( Pt 3): 435-43, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8032855

ABSTRACT

Fibroblast cultures from five patients with early onset severe encephalopathy and lactic acidosis were studied for evidence of pyruvate dehydrogenase (PDH) deficiency. Three males had significantly reduced activity (0.29-0.45 nmol/mg protein/min versus normal controls 0.7-1.1 nmol/mg protein/min); two females had PDH activity within the normal range. However, as the majority of cases of PDH deficiency result from defects in the X-linked E1 alpha subunit and both females had biased patterns of X-inactivation (making it impossible to rule out the possibility that they were heterozygous for an E1 alpha gene defect) molecular genetic studies were performed. cDNA from the male patients was sequenced and mis-sense mutations found: Y243N (T-->A) in exon 7, D315A (G-->A) in exon 10 and R378H (G-->A) in exon 11. Single-strand conformation polymorphism analysis of amplified genomic DNA fragments and sequencing revealed a mis-sense mutation M282L (A-->C) in one female and a frameshift mutation caused by insertion of T (R288ins) in the other. Adding to recent descriptions of new mutations, this report emphasizes the allelic heterogeneity of the condition. The identification of mutations in females with a suggestive clinical phenotype, even when peripheral fibroblasts do not show deficient PDH activity, illustrates the importance of molecular analysis of this disease.


Subject(s)
Pyruvate Dehydrogenase Complex Deficiency Disease/genetics , Base Sequence , Female , Gene Expression Regulation, Enzymologic , Genetic Linkage , Humans , Infant , Infant, Newborn , Male , Molecular Sequence Data , Mutation , Pyruvate Dehydrogenase Complex/genetics , X Chromosome
14.
Ann Neurol ; 33(6): 652-5, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8498846

ABSTRACT

We report a patient with necrotizing encephalomyelopathy (Leigh's syndrome) associated with a deficiency of pyruvate dehydrogenase complex activity. The underlying mutation is an A to C transversion in the pyruvate dehydrogenase complex E1 alpha subunit gene. As the E1 alpha subunit is encoded on the X chromosome, this observation confirms that some patients with Leigh's syndrome may potentially exhibit X-linked inheritance.


Subject(s)
Leigh Disease/genetics , Pyruvate Dehydrogenase Complex/genetics , X Chromosome , Amino Acid Sequence , Aspartic Acid , Base Sequence , Brain Stem/pathology , Cells, Cultured , Cesarean Section , Citrate (si)-Synthase/metabolism , DNA/genetics , DNA/isolation & purification , Exons , Fibroblasts/pathology , Fibroblasts/physiology , Gestational Age , Humans , Infant, Newborn , Leigh Disease/pathology , Leukocytes/physiology , Male , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Molecular Sequence Data , Necrosis , Oligodeoxyribonucleotides , Point Mutation , Pyruvate Dehydrogenase Complex/metabolism , Skin/pathology , Skin/physiopathology
15.
FEBS Lett ; 273(1-2): 91-4, 1990 Oct 29.
Article in English | MEDLINE | ID: mdl-2226870

ABSTRACT

Rat soleus muscle strips cultured for 24 h in medium 199 were well preserved in terms of electron microscopy; ATP and creatine phosphate concentrations; rates of glucose utilization, glycogen and protein synthesis, and effects of insulin thereon. Culture led to modest changes in fluid spaces and intracellular (K+); increased basal glucose utilization up to two-fold; had no effect on the maximum response to insulin; and had no effect on sensitivity to insulin except in the presence of adenosine deaminase. Thus in vitro neither denervation nor absence of insulin had any marked effects in 24 h to decrease responses to insulin.


Subject(s)
Glucose/metabolism , Insulin/pharmacology , Muscles/metabolism , Adenosine Deaminase/pharmacology , Adenosine Triphosphate/metabolism , Animals , Glycogen/metabolism , L-Lactate Dehydrogenase/metabolism , Muscles/cytology , Muscles/drug effects , Organ Culture Techniques/methods , Phosphocreatine/metabolism , Rats , Reference Values
16.
Biochem J ; 267(1): 245-7, 1990 Apr 01.
Article in English | MEDLINE | ID: mdl-2158309

ABSTRACT

The increased activity of pyruvate dehydrogenase (PDH) kinase induced in hearts of rats by starvation for 48 h was maintained following preparation of cardiac myocytes, and it was also maintained, though at a decreased level, after 25 h of culture in medium 199. This loss of PDH kinase activity was not prevented by n-octanoate, dibutyryl cyclic AMP or glucagon. The PDH kinase activity of myocytes from fed rats was increased to that of starved rats after 25 h of culture with n-octanoate, dibutyryl cyclic AMP or both agents together.


Subject(s)
Myocardium/enzymology , Protein Kinases/metabolism , Starvation/enzymology , Animals , Bucladesine/pharmacology , Caprylates/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cells, Cultured , Culture Media , Food , Glucagon/pharmacology , Protein Serine-Threonine Kinases , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Rats
17.
Biochem J ; 257(2): 487-91, 1989 Jan 15.
Article in English | MEDLINE | ID: mdl-2539088

ABSTRACT

The activities of pyruvate dehydrogenase (PDH) kinase and of PDH kinase activator protein (KAP) were increased 2-2.4-fold during 25 h of culture of hepatocytes from fed rats with glucagon plus n-octanoate. PDH kinase activity in hepatocytes from starved rats (initially 2.2 x fed control) fell during 25 h of culture in medium 199 (to 1.5 x fed control), but was maintained by glucagon plus octanoate. Dibutyryl or 8-bromo cyclic AMP increased PDH kinase activity 2-2.2-fold in hepatocytes from fed rats, but phenylephrine and isoproterenol (isoprenaline) were without effect. Insulin blocked the action of glucagon to increase PDH kinase activity and decreased the effect of octanoate and octanoate plus glucagon. It is suggested that the effects of starvation to increase activities of PDH kinase and of KAP in liver are mediated by alterations in circulating concentrations of glucagon, fatty acids and insulin and in hepatic cyclic AMP.


Subject(s)
Mitochondria, Liver/enzymology , Protein Kinases/metabolism , Animals , Caprylates/metabolism , Cells, Cultured , Cyclic AMP/pharmacology , Glucagon/metabolism , Insulin/metabolism , Isoproterenol/pharmacology , Mitochondria, Liver/drug effects , Phenylephrine/pharmacology , Protein Serine-Threonine Kinases , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Rats , Starvation
18.
Biochem J ; 246(1): 233-6, 1987 Aug 15.
Article in English | MEDLINE | ID: mdl-3314865

ABSTRACT

In tissue culture of hepatocytes, insulin (0.1-1 munits/ml for 4 h) reversed completely the effects of starvation of rats to decrease the activity of pyruvate dehydrogenase (PDH) complex and to increase the activities of PDH kinase and PDH kinase activator protein. It had no effect in hepatocytes from fed rats. Significant effects of insulin were detected with 0.01 munit/ml after 4 h, and in 1-2 h with 1 munit/ml.


Subject(s)
Insulin/pharmacology , Liver/enzymology , Protein Kinases/metabolism , Starvation/enzymology , Animals , Cells, Cultured , Liver/cytology , Liver/drug effects , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/enzymology , Protein Serine-Threonine Kinases , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Pyruvate Dehydrogenase Complex/metabolism , Rats , Rats, Inbred Strains
19.
Biochim Biophys Acta ; 756(3): 297-307, 1983 Apr 20.
Article in English | MEDLINE | ID: mdl-6131697

ABSTRACT

The activities of hepatic tyrosine aminotransferase, tryptophan oxygenase and serine dehydratase were increased in obese rats shortly after weaning. Immunotitration experiments showed that the increase in tyrosine aminotransferase activity resulted from an increase in enzyme protein in obese rats. No increase in hepatic tyrosine aminotransferase was observed in suckling pre-obese rats. The post-weaning increase in hepatic tyrosine aminotransferase of obese rats was only observed during the light phase of the diurnal cycle, but was prevented by pair-feeding and by starvation. Tryptophan increased hepatic tyrosine aminotransferase of lean rats to obese levels but had no effect in obese rats until tyrosine aminotransferase levels were reduced by starvation or adrenalectomy. Adrenalectomy abolished the increase in hepatic tyrosine aminotransferase activity in obese rats although serum corticosterone was normal in these animals. Hepatic and brain tyrosine concentrations were decreased in obese rats but normalized after adrenalectomy. The results suggest that the corticosteroid-dependent increase in food and tryptophan intake may be the primary cause of the increased hepatic amino acid catabolism of obese rats.


Subject(s)
Liver/enzymology , Obesity/enzymology , Tyrosine Transaminase/metabolism , Adrenalectomy , Aging , Amino Acids/metabolism , Animals , Corticosterone/blood , Diet , Disease Models, Animal , Enzyme Activation/drug effects , Insulin/blood , Obesity/genetics , Rats , Tryptophan/pharmacology , Tyrosine/blood
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