ABSTRACT
BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is caused by expansions of the poly (A) binding protein 2 (PABP2) gene. Previous histological analyses have revealed mitochondrial abnormalities in the muscles of OPMD patients but their significance remains uncertain. OBJECTIVE: We had the rare opportunity to study monozygotic twins with identical expansions of the PABP2 gene but with markedly different severities of OPMD. Both had histological features of mitochondrial myopathy. We determined whether mitochondrial DNA abnormalities underlay these changes. METHODS: Clinical information was obtained by history and examination. Muscle biopsies were obtained from each subject and genetic analysis was performed using long-range PCR and Southern blotting. RESULTS: We demonstrate, for the first time, the presence of mitochondrial DNA (mtDNA) deletions by Southern blotting in individuals with OPMD. This correlates with the presence of mitochondrial myopathy in both twins. Moreover, both twins had different mtDNA deletions, which might explain their phenotypic differences. CONCLUSION: We hypothesise that mitochondrial dysfunction may occur as a consequence of PABP2 gene mutations, and that this dysfunction may affect the phenotypic manifestations of OPMD.
Subject(s)
DNA, Mitochondrial/genetics , Gene Deletion , Muscular Dystrophy, Oculopharyngeal/genetics , Point Mutation/genetics , Poly(A)-Binding Protein II/genetics , Twins, Monozygotic/genetics , Aged , Biopsy , Blotting, Southern , DNA Mutational Analysis , Humans , Male , Muscle, Skeletal/pathology , Muscular Dystrophy, Oculopharyngeal/pathology , Phenotype , Polymerase Chain ReactionABSTRACT
Approximately 98% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11-13, uniparental disomy for chromosomes 15 (UPD15), or mutations affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of mutation depends upon the parent of origin. Both disorders are characterised at the molecular level by abnormal methylation of imprinted genes at 15q11-q13 including the small nuclear ribonucleoprotein N gene (SNRPN). Current diagnostic strategies include high resolution cytogenetics, fluorescence in situ hybridisation (FISH), Southern blot hybridisation, or microsatellite typing. We have developed a novel and rapid diagnostic test for PWS and AS based on differential digestion of expressed (paternally imprinted) SNRPN sequences by the methylation sensitive endonuclease NotI or repressed (maternally imprinted) SNRPN sequences by the methylation requiring nuclease McrBC, followed by PCR amplification of the SNRPN promoter. We have evaluated this test by blinded analysis of 60 characterised DNA samples (20 PWS, 20 AS, and 20 unaffected controls). SNRPN sequences could not be amplified from PWS patient DNA which had been digested with McrBC, nor from AS patient DNA which had been digested with NotI. We were able to make a correct diagnosis of PWS, AS, or unaffected in all 60 samples tested. This novel test is rapid and has a high specificity and sensitivity for deletion and UPD15 cases. These features make this new test suitable as the initial step in a molecular diagnostic strategy for PWS/AS.
Subject(s)
Angelman Syndrome/diagnosis , Autoantigens/genetics , Chromosomes, Human, Pair 15 , Polymerase Chain Reaction/methods , Prader-Willi Syndrome/diagnosis , Adolescent , Adult , Angelman Syndrome/genetics , Blotting, Southern , Child , Child, Preschool , Chromosome Deletion , Diagnosis, Differential , Female , Genomic Imprinting , Humans , In Situ Hybridization, Fluorescence , Infant , Male , Microsatellite Repeats , Middle Aged , Prader-Willi Syndrome/genetics , Ribonucleoproteins, Small Nuclear/genetics , snRNP Core ProteinsABSTRACT
Abnormal CAG expansions in the IT-15 gene are associated with Huntington disease (HD). In the diagnostic setting it is necessary to define the limits of the CAG size ranges on normal and HD-associated chromosomes. Most large analyses that defined the limits of the normal and pathological size ranges employed PCR assays, which included the CAG repeats and a CCG repeat tract that was thought to be invariant. Many of these experiments found an overlap between the normal and disease size ranges. Subsequent findings that the CCG repeats vary by 8 trinucleotide lengths suggested that the limits of the normal and disease size ranges should be reevaluated with assays that exclude the CCG polymorphism. Since patients with between 30 and 40 repeats are rare, a consortium was assembled to collect such individuals. All 178 samples were reanalyzed in Cambridge by using assays specific for the CAG repeats. We have optimized methods for reliable sizing of CAG repeats and show cases that demonstrate the dangers of using PCR assays that include both the CAG and CCG polymorphisms. Seven HD patients had 36 repeats, which confirms that this allele is associated with disease. Individuals without apparent symptoms or signs of HD were found at 36 repeats (aged 74, 78, 79, and 87 years), 37 repeats (aged 69 years), 38 repeats (aged 69 and 90 years), and 39 repeats (aged 67, 90, and 95 years). The detailed case histories of an exceptional case from this series will be presented: a 95-year-old man with 39 repeats who did not have classical features of HD. The apparently healthy survival into old age of some individuals with 36-39 repeats suggests that the HD mutation may not always be fully penetrant.
Subject(s)
Huntington Disease/genetics , Minisatellite Repeats , Phenotype , Trinucleotide Repeats , Adult , Aged , Aged, 80 and over , Female , Humans , Huntington Disease/diagnosis , Male , Middle Aged , Mutation , Polymerase Chain Reaction , Reference ValuesABSTRACT
To date, 32 cases of partial 7p monosomy have been described, 14 of which have been associated with craniosynostosis (CRS). There is considerable variation in the size and location of the deleted segment. However, CRS appears to be consistently associated with either a deletion or partial deletion 7p21-->7p22 or more rarely a deletion of 7p13-->7p14. Analysis of a panel of six 7p deletion cases (three with CRS) was undertaken using informative DNA probes, in order to characterize and define the extent of the deletions at the molecular level. There were five de novo deletions and one resulting from the unbalanced product of a paternal balanced insertion. The putative proximal CRS locus at 7p13-->7p14 does not appear to be allelic with Greig cephalopolysyndactyly syndrome. Three probe positions have been refined: pJ5.11 (D7S10) previously mapped to 7p14-->pter does not appear to map proximal to p15; TM102L (D7S135) does not map distal to p22; CRI-P137 (D7S65) maps distal to 7p13.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 7 , Craniosynostoses/genetics , Syndactyly/genetics , Chromosome Banding , Deoxyribonuclease HpaII , Deoxyribonucleases, Type II Site-Specific , Female , Humans , Male , Polymorphism, Restriction Fragment Length , Restriction Mapping , Skull/abnormalities , SyndromeABSTRACT
Craniosynostosis or premature closure of the cranial sutures is a common abnormality occurring in about 1 in 2500 children. There is evidence of mendelian inheritance in some 20% of cases. Published reports of patients with structural alterations of the short arm of chromosome 7 have suggested that two or more genes for craniosynostosis may be situated in this region. The Saethre-Chotzen syndrome (acrocephalosyndactyly type III) is one of the most common autosomal dominant craniosynostosis syndromes. Results of molecular genetic linkage studies provide evidence for localisation of the gene responsible to distal chromosome 7p.
Subject(s)
Acrocephalosyndactylia/genetics , Chromosomes, Human, Pair 7 , Genes, Dominant/genetics , Genetic Linkage/genetics , Chromosome Mapping , DNA Probes/genetics , Female , Humans , Male , PedigreeABSTRACT
Acrocallosal syndrome is an autosomal recessive form of polysyndactyly associated with mental retardation and agenesis of the corpus callosum. There have been suggestions that it is allelic to the Greig cephalopolysyndactyly syndrome. Linkage analysis, using flanking markers, shows this suggestion is unlikely to be correct.
Subject(s)
Abnormalities, Multiple/genetics , Agenesis of Corpus Callosum , Chromosomes, Human, Pair 7 , Fingers/abnormalities , Hand Deformities, Congenital/genetics , Intellectual Disability/genetics , Alleles , DNA Mutational Analysis , Genetic Markers , Humans , Lod Score , Pedigree , Phenotype , SyndromeABSTRACT
Familial multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroid glands, the pancreas, and the pituitary gland. Pancreatic tumors have previously been shown to be associated with the loss of alleles on chromosome 11; we therefore looked for similar genetic alterations in specimens of parathyroid tumors, which are the most common feature of MEN-1. We obtained parathyroid tumors and peripheral-blood leukocytes from six patients with MEN-1; 18 cloned human DNA sequences from chromosome 11 were then used to identify restriction-fragment-length polymorphisms. A loss of heterozygosity was detected in parathyroid tumors from three of the six patients with MEN-1; this finding demonstrated that allelic deletions on chromosome 11 are involved in the monoclonal development of parathyroid tumors in patients with MEN-1. In addition, studies of three affected families (with 17 affected members and 51 unaffected members) established linkage with the oncogene INT2 (peak lod score, 3.30, at 0 percent recombination); the MEN-1 gene was thus mapped to the pericentromeric region of the long arm of chromosome 11 (11q13). Our location of the MEN-1 gene at 11q13 is close to the location previously reported. We conclude that a single inherited locus on chromosome 11, band q13, causes MEN-1 and that the monoclonal development of parathyroid and pancreatic tumors in patients with MEN-1 involves similar allelic deletions on chromosome 11.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 11 , Multiple Endocrine Neoplasia/genetics , Parathyroid Neoplasms/genetics , Adolescent , Adult , Alleles , Child , DNA Probes , Female , Genetic Linkage , Humans , Male , Middle Aged , Pancreatic Neoplasms/genetics , Pituitary Neoplasms/genetics , Polymorphism, Restriction Fragment LengthABSTRACT
The manipulation of gene sequences between cells is a fundamental technique in genetics. Mammalian cells will take up and express genes when they are exposed to either metaphase chromosomes or naked genomic or recombinant DNA. In each case the uptake and expression is enhanced by the formation of a DNA-calcium phosphate precipitate (1,2). Alternatively, cloned recombinant DNA sequences may be introduced directly into mammalian cells by fusion with bacterial protoplasts containing recombinant plasmids (3) or by microinjection of purified DNA directly into the cell's nucleus (4). In many cases the transforming DNA is stably expressed, and consequently alters the genotype of the recipient cell. Since long-term transformation is a relatively rare event, identification of this altered genotype requires the use of genes coding for selectable functions or for proteins easily assayable by single cell antibody or related techniques.
ABSTRACT
Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.
Subject(s)
Alkaloids/metabolism , Mannosidases/antagonists & inhibitors , Alkaloids/pharmacology , Biological Transport/drug effects , Cells, Cultured , Humans , Hydrogen-Ion Concentration , Lysosomes/metabolism , Mannose/pharmacology , Mannosephosphates/pharmacology , Swainsonine , TemperatureABSTRACT
Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.
Subject(s)
Carbohydrate Metabolism, Inborn Errors/veterinary , Cat Diseases/metabolism , Mannose/metabolism , Animals , Carbohydrate Metabolism, Inborn Errors/metabolism , Cats , Chromatography, DEAE-Cellulose , Female , Hydrogen-Ion Concentration , Lysosomes/enzymology , Mannosidases/deficiency , Mannosidases/metabolism , Oligosaccharides/urineABSTRACT
Residual acidic alpha-D-mannosidase, corresponding to 5-16% of the activity in controls, was present in cultured fibroblasts from three unrelated patients with mannosidosis. The residual activity in all three cases had a higher value of Km and a marked lower thermal stability than the normal enzyme and was activated rather than inhibited by Co2+. A higher proportion of alpha-mannosidase B, separable on DEAE-cellulose, was present in the residual activity than in the normal activity. Acidic alpha-D-mannosidase that binds and activity that does not bind to concanavalin A were present in normal cells but both were deficient in mannosidosis. The residual activity in all three patients failed to cross-react with antiserum raised against the normal enzyme and no enzymically inactive cross-reacting material was detected. These results suggest that the genetic defect in the three cases is a mutation in the structural gene for acidic alpha-D-mannosidase. However, it was not possible to distinguish the mutations in the enzyme in these three cases by the biochemical techniques employed. Differences between the mutant activity in these three patients and that in other cases provide further evidence for genetic heterogeneity in mannosidosis.