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Biomedical subjects

S D Wilton

Publications and source records attributed to S D Wilton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390-kD protein that is approximately 91% the size of normal dystrophin. This 390-kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse-transcriptase polymerase chain reaction on GRMD muscle has revealed two in-frame dystrophin transcripts which lack either exons 3-9 or exons 5-12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Alternative Splicing↗
PubMed1998

Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations.

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele-specific oligonucleotide (ASO) hybridization or amplification, dideoxy fingerprinting, and direct DNA sequencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time-consuming hybridization, template purification, or digestion steps. The ease and simplicity of the SSCP test make it a popular choice for mutation detection, but a significant limitation is that some DNA changes will not alter the overall conformation of either single strand and are thus not amenable to SSCP typing. We describe Snapback SSCP to genotype normal and mdx mice (an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5' terminus, which were complementary to the normal sequence flanking the mdx mutation and used under the original amplification conditions. Each single strand of these snapback PCR products now had one terminus capable of re-annealing or "snapping back" to the normal sequence but not the mdx mutation. In this manner, a conformation change was engineered into the normal strand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP analysis.

Animals↗
PubMed1997

Dystrophin gene transcripts skipping the mdx mutation.

The mdx mouse, an animal model used to study Duchenne muscular dystrophy, has a nonsense mutation in exon 23 of the dystrophin gene which should result in a truncated protein that cannot be correctly localized at the sarcolemma of the muscle fibers. Immunohistochemical staining with antidystrophin antibodies has shown that while most of the muscle tissue is dystrophin-negative, a small percentage of muscle fibers is clearly dystrophin-positive and has somehow bypassed the primary nonsense mutation. A sensitive nested polymerase chain reaction-based examination of dystrophin gene transcripts around the mdx mutation has revealed several alternatively processed transcripts. Four mRNA species skipped the mutation in exon 23, were in-frame, and could be translated into a shorter but still functional dystrophin protein. Specific tests for these transcripts demonstrated these were also present in normal mouse muscle tissue.

Animals↗
PubMed1997

Revertant fibres: a possible genetic therapy for Duchenne muscular dystrophy?

The mdx mouse, an animal model used to study Duchenne muscular dystrophy (DMD), has a nonsense mutation in exon 23 of the dystrophin gene which should result in a truncated protein that cannot be correctly localized at the sarcolemma of the muscle fibres. Immunohistochemical staining with anti-dystrophin antibodies had shown that while most of the muscle tissue was dystrophin-negative, a small percentage of muscle fibres were clearly dystrophin-positive and had somehow by-passed the primary nonsense mutation. A nested PCR-based examination of dystrophin gene transcripts around the mdx mutation revealed several alternatively processed transcripts, of which four mRNA species skipped the mutation in exon 23, were in-frame and could be translated into a shorter, but still functional dystrophin protein. Specific tests for these transcripts demonstrated these were also present in normal adult and embryonic mouse muscle tissue.

Animals↗
PubMed1997

A splice-site mutation causing ovine McArdle's disease.

McArdle's disease is an autosomal recessive myopathy with symptoms of exercise intolerance caused by deficiency of the enzyme muscle glycogen phosphorylase which releases glucose for contraction during exercise. The human cDNA has been sequenced and disease-causing mutations identified. An ovine equivalent of McArdle's disease has been diagnosed and the mutation responsible identified by PCR-amplification of the ovine glycogen myophosphorylase cDNA in six overlapping fragments followed by single strand conformation polymorphism (SSCP) analysis. Two fragments showed SSCPs in the glycogen myophosphorylase cDNA from affected sheep. The SSCP in fragment one was a silent polymorphism, while that in fragment six, was an eight base deletion at the 5' end of exon 20. This deletion will cause a frame-shift, a premature stop codon and remove the last 31 amino-acid residues from the protein. The cDNA deletion suggested that the genomic mutation most likely involved a splice-site. Sequencing intron 19 identified the mutation as an adenine for guanine substitution at the intron 19 3' splice-site. This eliminated an XbaI site present in normal sheep allowing diagnosis of normal, affected and carrier sheep. This ovine model of McArdle's disease is now available for therapeutic trials.

Amino Acid Sequence↗
PubMed1997

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus.

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.

Amino Acid Sequence↗
PubMed1996

Alzheimer's disease and apolipoprotein E genotype in Western Australia: an autopsy-verified series.

OBJECTIVE: To determine the relationship between the apolipoprotein E epsilon 4 allele and autopsy-verified Alzheimer's disease (AD) in an Australian population. DESIGN: Retrospective case-control study. SETTING: Royal Perth Hospital, Perth, Western Australia (a tertiary referral hospital). SUBJECTS: 50 subjects with "definite" AD (according to the histological and clinical criteria of the Consortium to Establish a Registry for Alzheimer's Disease [CERAD]) and 30 control subjects who had died from a non-neurological disease were randomly selected from the hospital's neuropathology register. OUTCOME MEASURES: Histological grading of brain sections stained with the modified Bielschowsky stain according to the criteria of CERAD; number (burden) of neuritic plaques; apolipoprotein E genotype (APOE). RESULTS: Frequency of the epsilon 4 allele was significantly higher in the AD group (37%) than in the control group (2%) (chi 2 = 25.8; P < 0.00001). In the AD group, 50% of subjects were heterozygous for the epsilon 4 allele and 12% were homozygous, while in the control group one subject was heterozygous for the allele and none were homozygous. No association was seen between the epsilon 4 allele and neuritic plaque burden in the hippocampus, entorhinal cortex, middle frontal gyrus or inferior parietal lobule in subjects with AD. CONCLUSIONS: Our findings confirm an association between the epsilon 4 allele and autopsy-verified AD. The epsilon 4 allele may be an important risk factor for susceptibility to AD in the general Australian population.

Aged↗
PubMed1996

Three novel mutations and two variants in the gene for Cu/Zn superoxide dismutase in familial amyotrophic lateral sclerosis.

Autosomal dominant inheritance is exhibited by about 10% of cases of amyotrophic lateral sclerosis (ALS), a paralytic disorder characterized by the death of motor neurons in the brain and spinal cord. A subgroup of these familial cases are linked to mutations in the gene which codes for Cu/Zn superoxide dismutase (SOD1). We report three additional mutations occurring in the SOD1 gene in ALS patients and two single base pair variant changes. The single base pair change in an ALS family causes a glycine 93 to valine substitution, which is the fifth distinct amino acid change reported for the glycine 93 residue. One missense mutation in exon 5 would substitute neutral valine for the negatively-charged aspartate 124 (aspartate 124 to valine). An individual with an apparently sporadic case of ALS carries a three base pair deletion in exon 5 of the SOD1 gene. These three mutations bring to 38 the total number of distinct SOD1 mutations associated with familial ALS.

Amyotrophic Lateral Sclerosis↗
PubMed1996

Characterisation of the chicken Cu,Zn superoxide dismutase gene.

A PCR product was generated from embryonic chicken spinal cord cDNA using primers designed to conserved regions of the human and bovine amino and carboxyl-terminal coding sequences of the Cu,Zn superoxide dismutase (SOD1, EC 1.15.1.1) gene. DNA sequencing confirmed this product to be the chicken homologue of the SOD1 gene. This sequence was compared to SOD1 from bovine, human and Xenopus laevis. Important structural features of SOD1 are shown to be conserved in the chicken gene.

Amino Acid Sequence↗
PubMed1995

A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy.

Nemaline myopathies are diseases characterized by the presence in muscle fibres of pathognomonic rod bodies. These are composed largely of alpha-actinin and actin. We have identified a missense mutation in the alpha-tropomyosin gene, TPM3, which segregates completely with the disease in a family whose autosomal dominant nemaline myopathy we had previously localized to chromosome 1p13-q25. The mutation substitutes an arginine residue for a highly conserved methionine in a putative actin-binding site near the N terminus of the alpha-tropomyosin. The mutation may strengthen tropomyosin - actin binding, leading to rod body formation, by adding a further basic residue to the postulated actin-binding motif.

Amino Acid Sequence↗
PubMed1995

Assignment of the human a-tropomyosin gene TPM3 to 1q22-->q23 by fluorescence in situ hybridisation.

The human tropomyosin 3 (TPM3) gene was previously localized to chromosome 1. The non-muscle isoform of the TPM3 gene product becomes fused to a gene product from the tyrosine kinase receptor gene (NTRK1), previously localized to 1q23-->q24, to generate an active oncogene. Two sequence tagged sites spanning three exons and two introns in the carboxy coding region of the gene were used to localize TPM3 to 1q22-->q23 by fluorescence in situ hybridization. This localization now places the NTRK1 and TPM3 genes in close proximity, so that a gene fusion rearrangement would not be cytologically detected. The 1q22-->q23 localization of TPM3 is within the NEM1 locus associated with autosomal dominant nemaline myopathy, making TPM3 a candidate for this disorder.

Animals↗
PubMed1995

Assignment of the human skeletal muscle alpha-tropomyosin gene (TPM1) to band 15q22 by fluorescence in situ hybridization.

A sequence-tagged site (STS) was developed for the human skeletal muscle alpha-tropomyosin gene (TPM1) and used to isolate a genomic clone, lambda TPM1.1, containing part of the TPM1 gene. Fluorescence in situ hybridization of this clone to metaphase chromosome spreads localised TPM1 to chromosome band 15q22. This localisation in humans is consistent with that recently described for the mouse.

Animals↗
PubMed1995

Autosomal dominant distal myopathy: linkage to chromosome 14.

We have studied a family segregating a form of autosomal dominant distal myopathy (MIM 160500) and containing nine living affected individuals. The myopathy in this family is closest in clinical phenotype to that first described by Gowers in 1902. A search for linkage was conducted using microsatellite, VNTR, and RFLP markers. In total, 92 markers on all 22 autosomes were run. Positive linkage was obtained with 14 of 15 markers tested on chromosome 14, with little indication of linkage elsewhere in the genome. Maximum two-point LOD scores of 2.60 at recombination fraction .00 were obtained for the markers MYH7 and D14S64--the family structure precludes a two-point LOD score > or = 3. Recombinations with D14S72 and D14S49 indicate that this distal myopathy locus, MPD1, should lie between these markers. A multipoint analysis assuming 100% penetrance and using the markers D14S72, D14S50, MYH7, D14S64, D14S54, and D14S49 gave a LOD score of exactly 3 at MYH7. Analysis at a penetrance of 80% gave a LOD score of 2.8 at this marker. This probable localization of a gene for distal myopathy, MPD1, on chromosome 14 should allow other investigators studying distal myopathy families to test this region for linkage in other types of the disease, to confirm linkage or to demonstrate the likely genetic heterogeneity.

Adolescent↗