Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients.
(9/1324)
The distal part of the human dystrophin gene is characterised by particular features and seems to play an important functional role. Additionally in recent years several data have implicated minor mutations in this gene region in some patients with mental retardation (MR). In order to screen for pathogenic mutations at the distal part of the human dystrophin gene we have used single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) in 35 unrelated male Greek DMD/BMD patients with no detectable deletions. Seven patients also had severe mental retardation. Direct sequencing of samples demonstrating a shift of SSCA mobility revealed six different and pathogenic minor changes, five in DMD and one in a BMD patient. Four of the mutations were found in DMD patients with severe MR. Three of these mutations were localised in exon 66, which presents an interesting similarity with part of the 3' end of the genome of eastern equine encephalomyelitis virus (EEEV). The present data from Greek DMD/BMD patients give further information about the phenotypic effects consequent on mutations in exons at the distal part of the human dystrophin gene. (+info)
Bethlem myopathy: a slowly progressive congenital muscular dystrophy with contractures.
(10/1324)
Bethlem myopathy is an early-onset benign autosomal dominant myopathy with contractures caused by mutations in collagen type VI genes. It has been reported that onset occurs in early childhood. We investigated the natural course of Bethlem myopathy in five previously published kindreds and two novel pedigrees, with particular attention to the mode of onset in 23 children and the progression of weakness in 36 adult patients. Our analysis shows that nearly all children exhibit weakness or contractures during the first 2 years of life. Early features include diminished foetal movements, neonatal hypotonia and congenital contractures which are of a dynamic nature during childhood. The course of Bethlem myopathy in adult patients is less benign than previously thought. Due to slow but ongoing progression, more than two-thirds of patients over 50 years of age use a wheelchair. (+info)
Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family.
(11/1324)
Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified in a mother of two sons affected with Duchenne muscular dystrophy. It has been determined that she is a somatic mosaic with the mutation present in 25% of lymphocyte DNA. Further tissue lineages have been tested and the time at which the mutation arose was determined to be before the cellular differentiation into the bilaminar disc at approximately eight days after fertilisation. We suggest that the incidence of mosaicism for dystrophin point mutations may be higher than current data suggest. (+info)
ERG phenotype of a dystrophin mutation in heterozygous female carriers of Duchenne muscular dystrophy.
(12/1324)
PURPOSE: Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD). DMD is associated with an abnormal electroretinogram (ERG) if the mutation disrupts the translation of retinal dystrophin (Dp260). Our aim was to determine if incomplete ERG abnormalities would be associated with heterozygous carriers of dystrophin gene mutations. METHODS: Ganzfeld ERGs were obtained under scotopic and photopic testing conditions from a family which includes the heterozygous maternal grandmother, the heterozygous mother, and her children, two affected boys and dizygotic twin sibs, an unaffected male and heterozygous female. Southern blot analyses were done to characterise the dystrophin mutation. RESULTS: The dystrophin gene was found to contain a deletion encompassing exon 50. The ERGs in the two affected boys were abnormal, consistent with the DMD ERG phenotype. Serial ERGs of the heterozygous females were abnormal; however, they were less severely affected than the DMD boys. The ERG of the female sib showed a greater abnormality than her mother and maternal grandmother. The unaffected twin had a normal ERG. CONCLUSIONS: The ERG shows abnormalities associated with carrier status in this family with a single exon deletion. A large study of confirmed obligate carriers is planned to clarify further the value of the ERG in detecting female heterozygous carriers of dystrophin gene mutations. (+info)
Retroactive DNA analysis for sex determination and dystrophin gene by polymerase chain reaction with archived cytogenetic slides.
(13/1324)
We describe a rapid and efficient diagnostic method for sex determination and the dystrophin gene by the polymerase chain reaction (PCR) using archived cytogenetic slides. Archived cytogenetic slides stored for about 4 years at room temperature were used. To confirm whether DNA analysis is possible using the archived cytogenetic slides, we extracted the DNA from the slides and amplified the Y centromeric region (DYZ3), the X centromeric region (DXZ1) and the exon 46 of the dystrophin gene. Of the 50 cases, 24 were peripheral bloods, 13 were amniotic fluid cells, 5 were chorionic villus samplings and 8 were cord bloods. The PCR related sex determination in 22 females and 28 males, showed 100% concordance with the results of chromosome analysis, and all cases showed positive band for the exon 46 of the dystrophin gene. Of the 50 cases of the archived cytogenetic slides, we were fortunate enough to obtain the fresh blood sample from one fetus whose karyotype showed 45,X[34]/46,X,+mar[145] to compare the results of the gDNA with that from archived cytogenetic slide. To confirm whether the marker chromosome was derived from Y chromosome, we studied the six loci (PABY, SRY, RPS4Y (SY16, 17), ZFY, DYS14) on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm. Of the 8 loci studies, all PCR related Y chromosome showed positive band from both gDNA obtained from cord blood and archived cytogenetic slides. We could conclude from the above results that the marker chromosome was derived from the Y chromosome. We believe our experiment is rapid and efficient for studies of over 10 independent loci from a single slide which has been kept in storage for up to 4 years and that archival Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for clinical and forensic studies. (+info)
Multiplex Western blotting system for the analysis of muscular dystrophy proteins.
(14/1324)
A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers. (+info)
Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein.
(15/1324)
Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures. (+info)
Studies on the creatine kinase MM isoforms of normal and Duchenne muscular dystrophic patients.
(16/1324)
OBJECTIVE: To study the changes of creatine kinase MM (CK-MM) isoforms in Duchenne muscular dystrophy (DMD) patients. METHODS: Serum samples from 49 DMD patients and 40 control subjects were collected for CK-MM isoforms measurement. CK-MM isoforms were separated within 30 minutes by electrophoresis on agarose gel with a discontinuous buffer system at constant current of 30 mA and low voltage of 200-300 V, then measured by fluorescence scanning. RESULTS: Significant differences of MM2/MM1 ratio were found between DMD patients and control subjects (P < 0.05) as well as among the three different age groups of DMD patients (P < 0.05). CONCLUSIONS: CK-MM isoforms may present useful information for the early diagnosis and evaluation of DMD and the ratio of MM2/MM1 can be considered as a specific indicator of the degree of seriousness for DMD patients. (+info)