杜氏肌营养不良症(DMD)及其动物模型研究进展

杜氏肌营养不良症（Duchenne muscular dystrophy,DMD）属于X连锁隐性遗传病.DMD基因是人类最大基因,突变机制复杂.随着分子生物学的研究进展,对DMD的基因和其编码的抗肌萎缩蛋白（dystrophin）及抗肌萎缩蛋白相关蛋白（utrophin）的认识不断深入.本文就DMD的病理学特点,Dys基因结构、表达、功能,DMD突变及其相关检测技术,DMD实验动物模型及相关治疗的研究进展进行综述.     

Allosteric transition of erythrocyte alkaline phosphatase from Duchenne muscular dystrophy (DMD) patients and Duchenne muscular dystrophy carriers (Homo sapiens)

1. The kinetic properties of the p-nitrophenylphosphatase (EC 3.1.3.1) from erythrocytes was investigated in DMD-patients and DMD-carriers. 2. A different allosteric behaviour in the p-nitrophenylphosphatase from DMD-patients and DMD-carriers compared to controls is supported by the following findings: (a) values of n altered in F- inhibition of (K+)-activated p-nitrophenylphosphatase with Hill coefficients -1.5, -2.2 and -3.1; (b) heterotropic effect of increased concentration of Mg2+ on F- inhibition which is reverted by K+ in DMD-carriers and in control, but not in DMD-patients. 3. Evidence is presented showing that in DMD-patients and in DMD-carriers the interaction membrane-enzyme is different from the corresponding controls.     

Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi children

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene (DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe. In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion regions (HFDR’s) in the DMD gene mutations in KSA.     

Estimation of the male and female mutation rates in Duchenne muscular dystrophy (DMD)

Summary We present the results of an international collaborative study aimed at estimating the ratio of male to female mutation rates in Duchenne muscular dystrophy based on the method of C. Müller and T. Grimm. With a sample size of 295, this ratio is found to be very close to 1, thus giving evidence for equal mutation rates in males and females in Duchenne muscular dystrophy.     

Detection of Duchenne muscular dystrophy carriers by dosage analysis using the DMD cDNA clone 8

Deletion screening in 11 unrelated DMD patients has been performed using DMD cDNA clones 1-8. Of these 11 patients, 6 exhibit deletions of the cDNA clone 8. The carriership of 18 female relatives from these six DMD families has been investigated by dosage analysis. It is shown that dosage analysis is an available method to determine the carrier status of the female relatives of DMD patients showing a deletion within a DMD cDNA clone.     

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.     

Long-range genomic map of the Duchenne muscular dystrophy (DMD) gene: Isolation and use of J66 (DXS268), a distal intragenic marker

By cloning the endpoints of a DMD-associated deletion, we have "jumped" 1100 kb from pERT87-1 (DSX164) to a new locus designated J66 (DXS268), mapping distally within the Duchenne muscular dystrophy (DMD) gene. Both J66 and JBir are mapped by field-inversion gel electrophoresis and detect abnormal SfiI fragments in DMD patients and distal DMD-associated X; autosome translocations. Our long-range map extends the physical map of the DMD gene from 800 to 2000 kb (2 Mb) and increases the mapped portion of Xp21 to approximately 8 Mb. The position of the glycerol kinase gene and the adrenal hypoplasia locus are further confined to the region between J66 and the nearest distal probe L1-4. This region spans at least 1.5 Mb. The multiallelic J66 polymorphism has immediate application in the diagnosis of DMD and generally appears to be distal to DMD mutations.     

Preliminary study on the molecular structure of 3'''' region of Duchenne muscular dystrophy (DMD) gene in Chinese

Number and order of HindⅢ exon-containing fragments (Hd) at 3' region of DMD gene were studied systematically using 16 partly-overlapping cDNA subprobes which were produced from dystrophin cDNA 9- 14 with each of 9 restriction endonudeases. There are 25 Hd fragments corresponding to cDNA 9 -14 in DMD gene. Since then, the exact length and the new order of Hd fragments are established. A new 2.1 kb fragment (Hd 55) is revealed, a 5.2 kb fragment (formely designated as Hd 59) is excluded and the existence of a controversial 3.2 kb fragment (Hd 64) is confirmed. Besides, three new exons were revealed by comparing the PvuⅡ and the XbaⅠ hybridization patterns with the Hindlll hybridization patterns for these cDNA subprobes. It is concluded that there are at least 66 Hd fragments, or 79 exons in DMD gene basing on the discovery of three additional exons. The corresponding relationship between the 66 Hd fragments and the SfiⅠ large scale physical map has been studied, and at least 17 Hd fragments or 19 exo     

Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene

Eighty unrelated individuals with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) were found to have deletions in the major deletion-rich region of the DMD locus. This region includes the last five exons detected by cDNA5b-7, all exons detected by cDNA8, and the first two exons detected by cDNA9. These 80 individuals account for approximately 75% of 109 deletions of the gene, detected among 181 patients analyzed with the entire dystrophin cDNA. Endpoints for many of these deletions were further characterized using two genomic probes, p20 (DXS269; Wapenaar et al.) and GMGX11 (DXS239; present paper). Clinical findings are presented for all 80 patients allowing a correlation of phenotypic severity with the genotype. Thirty-eight independent patients were old enough to be classified as DMD, BMD, or intermediate phenotype and had deletions of exons with sequenced intron/exon boundaries. Of these, eight BMD patients and one intermediate patient had gene deletions predicted to leave the reading frame intact, while 21 DMD patients, 7 intermediate patients, and 1 BMD patient had gene deletions predicted to disrupt the reading frame. Thus, with two exceptions, frameshift deletions of the gene resulted in more severe phenotype than did in-frame deletions. This is in agreement with recent findings by Baumbach et al. and Koenig et al. but is in contrast to findings, by Malhotra et al., at the 5' end of the gene.     

Diphenoloxidases in X-linked recessive (Duchenne) muscular dystrophy

Summary In extracts derived from whole blood, a high molecular weight fraction of the diphenoloxidase enzymes has a significantly diminished specific activity in patients and definite carriers (heterozygotes) of the X-linked, recessive (Duchenne) form of muscular dystrophy. This anomaly was studied using spots of blood which had been collected on absorbent paper and stored at 4°C for variable periods of time. Fractions enriched in the enzymes were obtained by subjecting aqueous extracts of the spots to treatment with an anion exchange resin (DEAE Sephadex A 50) followed by gel filtration on Sephadex G-25. It is of interest that this anomaly was observed in some definite carriers of the mutant gene who had had on several occasions a serum creatine kinase level in the normal range. The significance of these observations is discussed.     

DNA-diagnosis of carriers of the Duchenne muscular dystrophy gene

Duchenne muscular dystrophy carrier detection has been performed by using probes XJ1.1 (intragenic probe) and probe 754 for a girl. The carrier probability was estimated by means of a computer program GenRisk combining pedigree and DNA-probe data and turned out to be 95%.     

Nebulin and titin expression in Duchenne muscular dystrophy appears normal

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5'-region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.     

A deletion hot spot in the Duchenne muscular dystrophy gene

We have made a detailed study of a deletion hot spot in the distal half of the Duchenne muscular dystrophy (DMD) gene, using intragenic probe P20 (DXS269), isolated by a hybrid cell-mediated cloning procedure. P20 detects 16% deletions in patients suffering from either DMD or Becker muscular dystrophy (BMD), in sharp contrast to the adjacent intragenic markers JBir (7%) and J66 (less than 1%), mapping respectively 200-320 kb proximal and 380-500 kb distal to P20. Of the P20 deletions, 30% start within a region of 25-40 kb, the majority extending distally. P20 was confirmed to map internal to a distal intron of the DMD gene. This region was recently shown by both cDNA analysis (M. Koenig et al., 1987; Cell 50: 509-517), and field inversion electrophoresis studies (J.T. Den Dunnen et al., 1987, Nature (London) 329: 640-642) to be specifically prone to deletions. In addition, P20 detects MspI and EcoRV RFLPs, informative in 48% of the carrier females. Together, these properties make P20 useful for carrier detection, prenatal diagnosis, and the study of deletion induction in both DMD and BMD.