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O Hurko

Publications and source records attributed to O Hurko.

At least 37 records · Page 2Linked to original sources
PubMed1994

Yeast kar1 mutants provide an effective method for YAC transfer to new hosts.

Yeast artificial chromosome (YAC) clones propagate large segments of exogenous DNA in a host organism with well-developed classical and molecular genetics. Most extant YAC clones are from libraries created in a single yeast host (AB1380). The application of techniques allowing the manipulation and/or restructuring of these cloned DNA segments often requires a change in the yeast genetic background to introduce desirable genetic markers. Transfer methods in current use require extremely high yeast transformation efficiencies or require access to equipment for yeast tetrad analysis. We have developed an alternative method for moving YAC clones from one yeast strain to another, taking advantage of the properties of kar1 mutants altered in a gene required for normal karyogamy (nuclear fusion) during mating. Transfer by this method requires generally accessible methods, including yeast cell culture, replica plating, and pulsed-field gel electrophoresis. We present data demonstrating efficient transfer of nine different YACs from their original host (AB1380) to a kar1 recipient strain (YPH925) with genetic markers that facilitate the use of existing homologous recombination-based modification methods. The enhanced ability to transfer clones to this new host will accelerate the pace of refinement and fine-structure mapping of the YAC contigs currently under construction and facilitate gene manipulation on YACs for subsequent functional analysis.

Chromosomes, Artificial, Yeast↗
PubMed1994

Linkage mapping of the spinal muscular atrophy gene.

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder resulting in loss of motor neurons. We have performed linkage analysis on a panel of families using nine markers that are closely linked to the SMA gene. The highest lod score was obtained with the marker D5S351 (Zmax = 10.04 at theta = O excluding two unlinked families, and Zmax = 8.77 at theta = 0.007 with all families). One type III family did not show linkage to the 5q13 markers, and in one type I consanguineous family the affected individual did not show homozygosity except for the marker D5S435. Three recombinants were identified with the closet centromeric marker, D5S435, which position the gene telomeric of this marker. These recombinants will facilitate finer mapping of the location of the SMA gene. Lastly, two families provide strong evidence for a remarkable variability in presentation of the SMA phenotype, with the age at onset in one family varying from 17 months to 13 years.

Adolescent↗
PubMed1994

Total craniospinal decompression in achondroplastic stenosis.

We describe our experience with total craniospinal decompression along the entire neuraxis, extending from the brain stem to the cauda equina, in seven patients with achondroplasia. These patients presented with clinically significant compression at multiple levels. In these patients, there were focal areas of complete myelographic block, typically at the cervicothoracic or thoracolumbar junction, as well as diffuse narrowing of the entire spinal subarachnoid space. In some, there were further complications of basilar impression, Arnold-Chiari malformation, or syringomyelia. Total craniospinal decompression was completed in either one or two stages. Only a small minority of our patients with achondroplasia had critical stenosis over this many levels, requiring total craniospinal decompression. However, with proper preparation and technique, we found that patients can tolerate even such an extensive decompressive procedure and benefit from surgery without suffering postoperative spinal instability.

Achondroplasia↗
PubMed1994

Association between Ag1-CA alleles and severity of autosomal recessive proximal spinal muscular atrophy.

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has been mapped to an 850-kb interval on 5q11.2-q13.3, between the centromeric D5S823 and telomeric D5S557 markers. We report a new complex marker, Ag1-CA, that lies in this interval, whose primers produce one, two, or rarely three amplification-fragment-length variants (AFLVs) per allele. Class I chromosomes are those which amplify a single AFLV allele, and class II chromosomes are those which amplify an allele with two or three AFLVs. Ag1-CA shows highly significant allelic association with type I SMA in both the French Canadian (Hôpital Sainte-Justine [HSJ]) and American (Ohio State University [OSU]) populations (P < .0001). Significant association between the Ag1-CA genotype and disease severity was also observed. Type I patients were predominantly homozygous for class I chromosomes (P = .0003 OSU; P = .0012 HSJ), whereas the majority of type II patients were heterozygous for class I and II chromosomes (P = .0014 OSU; P = .001 HSJ). There was no significant difference in Ag1-CA genotype frequencies between type III patients (P = .5 OSU; P = .25 HSJ) and the paired normal chromosomes from both carrier parents. Our results indicate that Ag1-CA is the most closely linked marker to SMA and defines the critical candidate-gene region. Finally, we have proposed a model that should be taken into consideration when screening candidate SMA genes.

Alleles↗
PubMed1993

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by multicolor in situ hybridization.

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) results in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. The smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%).

Amniocentesis↗
PubMed1992

Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.

The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho 0) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.

Cell Division↗
PubMed1992

Ophthalmic involvement in myo-neuro-gastrointestinal encephalopathy syndrome.

We studied the clinical, histopathologic, neuroradiologic, biochemical, and genetic profile of a patient with the myo-neuro-gastrointestinal encephalopathy syndrome, a recently described multisystem mitochondriopathy characterized by blepharoptosis and ophthalmoparesis. The patient had severe intestinal pseudo-obstruction and a mixed demyelinating and axonal neuropathy. Abnormal collections of mitochondria in nerve and muscle as well as diffuse white matter disease were present. Cytochrome oxidase activity in muscle mitochondria was reduced. No mitochondrial DNA deletions were detected.

Adult↗
PubMed1992

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.

Adolescent↗
PubMed1992

MELAS mutation in mtDNA binding site for transcription termination factor causes defects in protein synthesis and in respiration but no change in levels of upstream and downstream mature transcripts.

The pathogenetic mechanism of the mitochondrial tRNA(LeuUUR) gene mutation responsible for the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) syndrome was investigated in transformants obtained by transfer of mitochondria from three genetically unrelated MELAS patients into human mitochondrial DNA (mtDNA)-less (rho 0) cells. Marked defects in mitochondrial protein synthesis and respiratory activity were observed in transformants containing virtually pure mutant mtDNA, as compared to the parent of the rho 0 cells (the 143B cell line) or to transformants containing exclusively wild-type mtDNA, derived from one of the patients or a maternally related asymptomatic individual. A striking protective effect against the mutation was exerted in the transformants by levels of residual wild-type mtDNA above 6%. The MELAS mutation occurs within the mtDNA binding site for a protein factor (mTERF) that promotes termination of transcription at the 16S rRNA/tRNA(LeuUUR) gene boundary. A marked decrease in affinity of purified mTERF for the mutant target sequence was observed in in vitro assays. By contrast, RNA transfer hybridization experiments failed to show any significant change in the steady-state amounts of the two rRNA species, encoded upstream of the termination site, and of the mRNAs encoded downstream, in the transformants carrying the MELAS mutation.

Acidosis, Lactic↗
PubMed1992

Confusion as the presenting manifestation of vertebral osteomyelitis: a case report.

A 44-year-old patient presented with increasing confusion. He was first diagnosed as having intermittent pressure hydrocephalus but a further evaluation showed CSF pleocytosis and hypoglycorrhachia. Five weeks later, his physical examination was unrevealing. Nuclear imaging techniques were conflicting, with negative gallium- and indium-labelled white blood cells scans but a Tc scan pointing towards a vertebral infection. A well-demarcated lesion in the T9 vertebral body, demonstrated by CT scan, confirmed the diagnosis of vertebral osteomyelitis. Although we were unable to recover the causative organism, antibiotic treatment for presumed staphylococcal osteomyelitis resulted in full recovery. This case indicates that vertebral osteomyelitis may cause significant meningeal inflammation even in the absence of epidural or subdural abscess. We recommend that in patients with meningitis without a clear etiology vertebral osteomyelitis should be considered and pursued with CT scannings of the vertebrae, a procedure that can yield positive findings even when other scanning modalities are negative.

Adult↗
PubMed1990

Heteroplasmy in chronic external ophthalmoplegia: clinical and molecular observations.

Chronic progressive external ophthalmoplegia (CPEO) describes a recognizable clinical syndrome frequently associated with variable dysfunction in other organ systems. Histochemical and biochemical studies suggested primary dysfunction of oxidative phosphorylation. This has recently been confirmed by demonstration of partially deleted as well as normal mitochondrial DNA--heteroplasmy--in some of these patients, most of them sporadic. In the six heteroplasmic CPEO patients that we have examined to date, the partially deleted species has been detected in all tissues tested, albeit in vastly different proportions. We report here detection of physiologically significant proportions of partially deleted mitochondrial DNA in several organs taken at autopsy from a CPEO patient with severe multisystem disease. We discuss the relationship of CPEO to several other clinical phenotypes associated with mitochondrial dysfunction, and discuss the possible implications of heteroplasmy for the development of variable phenotypes.

Child↗
PubMed1990

Craniocervical decompression for cervicomedullary compression in pediatric patients with achondroplasia.

The congenital osseous abnormalities associated with achondroplasia include stenosis of the foramen magnum and the upper cervical spinal canal. In the pediatric achondroplastic patient, such stenosis may lead to cervicomedullary compression with serious sequelae, including paresis, hypertonia, delayed motor mile-stones, and respiratory compromise. Using a standardized protocol the authors have treated 15 young achondroplastic patients with documented cervicomedullary compression by craniocervical decompression and duroplasty. Following this procedure, significant improvement in presenting neurological or respiratory complaints was noted in all patients. The mortality rate in this series was zero. The major cause of morbidity associated with this procedure was perioperative cerebrospinal fluid (CSF) leakage from the surgical wound, presumably related to coexisting abnormalities of CSF dynamics. This problem was successfully managed by temporary or, when necessary, permanent CSF diversion procedures. It is concluded that craniocervical decompression is an effective and safe treatment for young achondroplastic patients with cervicomedullary compression.

Achondroplasia↗
PubMed1989

Preferential amplification and molecular characterization of junction sequences of a pathogenetic deletion in human mitochondrial DNA.

Deletions of mitochondrial DNA have been detected in skeletal muscle of some patients with mitochondrial encephalomyopathies, but their junctions have been defined only approximately. We developed a procedure, using widely spaced primers for the polymerase chain reaction, that amplifies preferentially the sequences bracketing the deletion. This procedure permits detection of minor proportions, not detectable by Southern analysis, of deleted mitochondrial DNA species in a heteroplasmic mixture. Different proportions of intact mitochondrial DNA and species deleted from nucleotide 8708 to 13,722 were found in skeletal muscle, blood, and urinary epithelial cells from a patient with chronic progressive external ophthalmoplegia. These data indicate that the mutation occurred at or before early embryonic development and provide the first definition at the nucleotide level of a human disease caused by a deletion of mitochondrial DNA.

Adenosine Triphosphatases↗