Histological changes in masticatory muscles of mdx mice

Objective

Duchenne muscular dystrophy (DMD) patients have distorted dentofacial morphology that could be a result of changed force balance of masticatory muscles due to unequal dystrophic changes in various masticatory muscles. Skeletal muscles of DMD patients and those of murine model of DMD - mdx mice - are both characterized by Ca²⁺ induced muscle damage, muscle weakness and characteristic histological changes. Therefore, to determine the pathological changes in this animal model of DMD, we examined the masticatory muscles of the mdx mice for histological abnormalities including nuclei localization, fibre diameters, and collagen expression.

Design

Muscle sections from masseter (MAS), temporal (TEM), tongue (TON) and soleus (SOL) of mdx and control normal mice were stained with hemalaun/eosin or with Sirius Red and morphometrically analysed. Levels of collagen staining in normal and mdx muscles were measured using image analysis and the mean optical density (mod) was determined.

Results

Dystrophin deficient masticatory muscles contained 11-75% fibres with centralised nuclei. In mdx mice an increased mean fibre diameter was observed as compared to the age-matched control muscles (control vs. mdx; MAS: 33.44 ± 0.49 μm vs. 37.76 ± 0.68 μm, p < 0.005; TEM: 32.93 ± 0.4 μm vs. 42.93 ± 0.68 μm, p < 0.005; SOL: 33.15 ± 0.29 μm vs. 40.62 ± 0.55 μm, p < 0.005; TON: 13.44 ± 0.68 μm vs. 15.63 ± 0.18 μm, p < 0.005). Increased expression of collagen was found in MAS (mod control vs. mdx: 1.34 vs. 3.99, p < 0.005), TEM (mod control vs. mdx: 3.11 vs. 4.73, p < 0.01) and SOL (mod control vs. mdx: 2.36 vs. 3.49, p < 0.01).

Conclusion

Our findings revealed that mdx masticatory muscles are unequally affected by the disease process. The masticatory muscles of the mdx mice could present a useful model for further investigating the influence of dystrophin deficiency on muscles function.     

A novel SNaPshot assay to detect the mdx mutation

The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD). In order to evaluate possible treatments and to carry out genetic studies, it is essential to distinguish between mice that carry the dystrophic (mutant) or wild-type (wt) allele(s). The current amplification-resistant mutation system (ARMS) assay is labor intensive and yields false negatives, which reduces its efficiency as a screening tool. An alternate assay based on single-nucleotide polymorphism (SNP) primer extension technology (i.e., SNaPshot) is described. The SNaPshot assay has been optimized to identify both wild-type and mutant alleles, providing a robust, potentially automatable assay for high-throughput analysis.     

X‐linked markers in the Duchenne muscular dystrophy gene associated with oral clefts

As part of an international consortium, case–parent trios were collected for a genome‐wide association study of isolated, non‐syndromic oral clefts, including cleft lip (CL), cleft palate (CP), and cleft lip and palate (CLP). Non‐syndromic oral clefts have a complex and heterogeneous etiology. Risk is influenced by genes and environmental factors, and differs markedly by gender. Family‐based association tests (FBAT) were used on 14,486 single nucleotide polymorphisms (SNPs) spanning the X chromosome, stratified by type of cleft and racial group. Significant results, even after multiple‐comparisons correction, were obtained for the Duchenne muscular dystrophy (DMD) gene, the largest single gene in the human genome, among CL/P (i.e. both CL and CLP combined) trios. When stratified into groups of European and Asian ancestry, stronger signals were obtained for Asian subjects. Although conventional sliding‐window haplotype analysis showed no increase in significance, selected combinations of the 25 most significant SNPs in the DMD gene identified four SNPs together that attained genome‐wide significance among Asian CL/P trios, raising the possibility of interaction between distant SNPs within the DMD gene.     

血清VEGF的表达与肌营养不良症的关系

Objective To detect serum levels of vascular endothelial growth factor (VEGF) in patients with muscular dystrophy and further to examine whether VEGF is associated with muscular dystrophy development. Methods We measured serum levels of VEGF in 32 patients with DMD,9 with Becker muscular dystrophy (BMD),5 with myotonic dystrophy (DM),as well as in 15 healthy and eight disease controls. Results The serum level of VEGF in the DMD patients was (274.7±2.52)pg/ml,while it was(358.8±9.64) pg/ml in the BMD patients, (165.0±6.34) pg/ml in the DM patients, (1 48.3±2.91) pg/ml in the healthy controls,and (153.7±5.42) pg/ml in the disease controls. The level of VEGF in BMD was significantly elevated, as compared with DM and control groups. Further, the level of VEGF in the bedridden sub-group of DMD patients was significantly elevated as compared with chair-bound DMD, DM, and control groups. Conclusion We concluded that VEGF may reflect hypoxic and/or ischemic conditions in muscle tissue, and have a relationship with the process of disease progression in DMD and BMD patients.     

Duchenne和Becker型肌营养不良症的电镜观察

目的 本研究通过观察Duchenne和Becker型肌营养不良症(DMD／BMD)患者超微病理的特征，从亚细胞水平上探讨其病因和发病机制。方法 应用电子显微镜，采用超薄切片和冷冻蚀刻技术观察9例DMD／BMD患者超微结构的改变。结果 (1)DMD／BMD透射电镜发现：“Z”线模糊不清，宽窄不一，间距变窄，肌原纤维明带增宽，肌原纤维过度收缩。血管内皮细胞肿胀，毛细血管闭塞，闭塞的毛细血管内皮细胞中可见大量饮液泡；肌原纤维粗细不等，肌浆网明显扩张，肌浆网侵入肌原纤维间，呈虫食样，肌浆网中可见浓缩的包涵物。肌丝排列紊乱，肌浆网管扩张，肌丝间网聚集；线粒体中间段大空泡，肌丝间网较明显，糖原颗粒聚积。(2)DMD骨骼肌冷冻蚀刻电镜图像示：肌原纤维间存在同心圆状排列的板层体；粗细肌丝排列紊乱，正常的六角形点阵结构消失。结论 (1)DMD／BMD超微结构改变表现为肌纤维的坏死和细胞膜的破坏。(2)肌纤维内毛细血管闭塞，血管内皮细胞肿胀，间质毛细血管血流瘀滞。表明DMD／BMD可能还存在血液循环障碍，有待进一步证实。     

Muscle energy metabolism in female DMD/BMD carriers: a 31P-MR spectroscopy study.

31P nuclear magnetic resonance spectroscopy (31P-MRS) was used to investigate the energy metabolism of the gastrocnemius muscle in 16 DMD/BMD female carriers. No significant difference was found with the controls in the resting muscle, while all carriers showed a decreased ability to perform work, and a higher Pi/PCr ratio for comparable relative levels of steady-state work. The rate of postexercise recovery of PCr/Pi ratio was lower in all carriers. The decreased rate of PCr/Pi recovery appears to be mainly due to a decreased rate of Pi recovery. Our findings show abnormal muscle energy metabolism in DMD/BMD female carriers.     

散发型DMD/BMD家系突变分析及产前诊断研究

目的 分析散发型DMD/BMD家系中患者的致病突变,为部分家系的胎儿进行产前基因诊断,明确该类家庭中女性成员是否致病突变携带者,分析该类家系新发突变的比例.方法 共收集30个散发DMD/BMD家系,应用传统mPCR方法 分析DMD基因缺失热区中的18个外显子;应用MLPA方法,对家系中的30例患者及23个家系中的28位女性成员,进行DMD基因全部79个外显子的定量分析,并为其中19个家系进行20例产前诊断.结果 mPCR检测到19例缺失突变;MLPA检测到21例缺失突变和3例重复突变,并明确缺失突变范围.在检测到突变的家系中,10例母亲为缺失突变携带者,2例为重复突变携带者,9例母亲不是携带者,新发突变比例为37.5%.7例患者的姐妹中5例为携带者(3例缺失,2例莺复),2例不是携带者.经产前诊断,12例男性胎儿中5例为患者,8例女性胎儿中2例为携带者.结论 MLPA方法 可全面检测DMD基因缺失及重复突变、明确女性携带者,为产前诊断提供准确信息.     

用多重PCR快速检测DMD基因缺失

应用两步多重ＰＣＲ，对２０例无亲缘关系的患者进行基因缺失检测，检出９例患者为基因缺失型，占受检患者的４５％。该方法简便、快速，可用于临床基因诊断