Prenatal Diagnosis of Duchenne Muscular Dystrophy (DMD) by the Polymerase Chain Reaction (PCR)

The diagnosis of Duchenne muscular dystrophy (DMD) has been drastically improved by recent advances in DNA analysis. The Southern blot hybridization using the cDNA 8 probe and the restriction enzyme Hind III was conducted in a gravida and her family in blood samples. The diagnosis revealed partial gene deletions in both the gravida and the DMD-affected second child. The prenatal diagnosis was performed by studying the PCR (polymerase chain reaction) for target DNAs of exons 48 and 51 that correspond with cDNA 8 probe. In the affected child, the 506 bp band at exon 48 was detected but 388 bp at exon 51 was missing. On the other hand, both the 506 bp band at exon 48 and the 388 bp band at exon 51 were detected in the cultured amniotic cells. Thus, the fetus was determined to be not affected.     

Twenty years-iowa muscle clinic: Reminiscences and prospects

The activities of the last twenty years of the Iowa clinic for neuromuscular diseases are briefly reviewed. Main emphasis in this paper is on management and guidance of the patient with Duchenne muscular dystrophy (DMD) by the families, the school and the public at large. Guidance and treatment given to our patients is briefly described. It is well known that chronic debilitating disease affects not only the patient but the whole family; management of DMD should therefore include the parents and siblings of the patient. Preventability of DMD is illustrated by the experiences in our clinic. The importance of early carrier detection and genetic counseling is stressed. Possible future developments in DMD research are briefly mentioned. Curriculum vitae. Hans Zellweger, born in Lugano, Switzerland, graduated from the University of Zürich in 1934. After three years at the Kantonspital in Lucerne (Pathology, Microbiology) and two years with Dr. Albert Schweitzer in Lambarene he spent twelve years with Prof. G. Fanconi in the Kinderspital Zurich. He was general secretary of the International Congress of Pediatrics in Zurich, 1950. From 1951 to 1959 he was chairman of the Pediatrics Department of the American University in Beirut, Lebanon. Since 1959 he is Professor of Pediatrics at the University of Iowa, Iowa City, Iowa. He opened one of the first laboratories for cytogenetics in 1960. Thereafter he became director of the Genetic Clinic and founded a clinic for neuromuscular disease. In 1976 he organized with Jane Simpson the Regional Genetic Consultation Service for the whole State of Iowa, with fifteen genetic counseling clinics through the State of Iowa. He became Professor Emeritus in 1977, yet is still active in the genetic clinic. Dr. Victor Ionasescu is Director of the Clinic for Neuromuscular Diseases and Chief of the Laboratory for Muscle Research at the University of Iowa, Department of Pediatrics.Herrn Prof. Dr. H.-R. Wiedemann, gewidmet zu seinem 65. Geburtstag     

The effect of dibromo-dulcitol,diepoxy-dulcitol and various new cytostatic hexitol derivatives on the metabolic activities of nucleic acids and proteins—II

The effects of nine antiblastic, 1,6-disubstituted hexitol derivatives were compared on DNA, RNA and protein synthesis. Each compound reduced ¹⁴C-thymidine incorporation into the DNA in surviving rabbit bone marrow cell culture, in vitro. The rate of inhibition increased as a function of incubation time with the compounds and of their concentration. Among the hexitol skeletons carrying identical substituents dulcitol (galactitol) proved to be the most effective, D-mannitol was less and D-sorbitol the least effective. Comparing the reactive functional groups carried by identical hexitol skeletons, the dianhydride (diepoxide) derivatives were the most effective, dibromo derivatives were less and dimethanesulphonyloxy (dimesyl) derivatives the least effective. The inhibition of DNA synthesis by diiodo-D-sorbitol was anomalously strong. At higher concentrations the hexitols reduced precursor incorporation into RNA and protein of human tonsillar cells, in vitro. At relatively low concentration and short incubation times, however, enhancement could be observed. A good correlation was established between the rate of inhibition of DNA synthesis and the cytotoxic effect. Since the dianhydride derivative of hexitol was always more effective than its corresponding dibromo-or dimethane-sulphonyloxy derivative, the anhydrides may have formed from the latter in vivo and these are probably responsible for a large portion of the cytotoxic effect. The various hexitols, especially, dibromodulcitol and dianhydro-dulcitol, used with success in clinical practice have different properties in vivo (transport, transformation, target). In addition to DNA, the nuclear proteins may also be an important target site.     

Expression and subcellular localization of dystrophin in skeletal, cardiac and smooth muscles during the human development

Dystrophin, the product of the DMD gene, is present in all muscle types in normal individuals. Its function has yet to be elucidated, but its absence or the presence of a truncated version of the protein is responsible for the appearance of Duchenne and Becker muscular dystrophies. Using monoclonal antibodies raised against distinct regions of the dystrophin protein, we have examined its expression and subcellular distribution during the human development in skeletal and smooth muscles. We show that both dystrophin expression and its association to the plasma membrane take place earlier in cardiac and smooth muscles (8 weeks of gestation) than in skeletal muscle. In skeletal muscle, dystrophin is first detected in the cytoplasm, and progressively localizes to the plasma membrane from 10 weeks onwards. Since we have obtained marked differences in staining when using antibodies against either a central region of the protein or the C-terminal part, we suggest that different fetal and adult dystrophin isoforms are expressed, probably differing in their C-terminal domain. These findings are discussed in the context of the pathology of Duchenne muscular dystrophy.     

Characterization and subcellular localization of the dystrophin-protein 71 (Dp71) from brain

In the present study, monoclonal antibodies raised against the C-terminal domain of dystrophin were used to identify and characterize Dp71 from the central nervous system. It was observed that the expression of Dp71 gradually increases from the embryo stage until the adult. Subcellular distribution analysis indicates that Dp71 is mainly recovered in synaptic plasma membranes, microsomes and at a lesser extent in synaptic vesicles and mitochondria. The amino acid composition and N-terminal sequence of bovine brain Dp71 were determined. Moreover, we found that this protein is glycosylated.     

干细胞移植治疗神经遗传疾病中HLA配合的几率分析

为了探讨干细胞移植(Stem Cell Transplantation)对神经系统中预后不良的单基因遗传性疾病之一DMD(假肥大型肌营养不良症)的临床可行性，采用PCR反向序列特异性寡核苷酸杂交技术(polymerase chain reactionreverse sequence specific oligonucleotide，PCR—RSSO)对30例DMD患者进行HLA—ABDR基因分型，并与广东省脐血干细胞库668份和中国造血干细胞捐献者库34910份分别进行HLA—ABDR基因频率比较和无关供者干细胞的配合性分析。结果表明：DMD疾病组的HLA基因呈正态遗传，DMD患者HLA基因表达与广东健康人群无统计学上显著差异；DMD患者中1／4寻找到合适用于移植的干细胞，其中在广东脐血库寻找到≥5个HLA—ABDR基因配合的几率为50％，在中国造血干细胞库寻找到6个HLA—ABDR基因配合的几率为26％。结论：对DMD患者进行非亲缘异基因脐带血/外周血移植治疗具有HLA配合供者高选择几率的可行性基础；干细胞移植不仅将使致死性神经肌肉疾病患者获得临床症状改善，并且为难治性神经系统疾病受益于干细胞移植治疗打造技术平台。     

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.