[Diagnosis of Muscular Dystrophy Using Western Blot with Micro-sample of Muscle].

OBJECTIVE: To diagnose muscular dystrophy using Western blot (WB) by improving the method of the protein extraction. METHOD: Firstly,we compared the effect of different sample buffer solutions and processing Methods on the extraction of muscle protein in rats,then selected the appropriate extracting method and the process of the muscular protein. RESULTS: We put the selected sample buffer into the micro-sample,then mixed. The concentration of the extracting protein was much more,and the loss during the process was much less. We extracted enough protein in 62 cases. The protein bands were showed clearly by WB,and the abnormal protein bands were shown in some patients. Compared with the Results of immunohistochemical staining detected the severe abnormal expressions of Dys-R,Dys-C,and Dys-N in the specimens,we did not detect the corresponding target band in WB. We detected the target protein band of the specimens were abnormal position,light or normal staining in WB,while Dys were mildly expressed in immunohistochemical staining. CONCLUSIONS: The improved protein extraction method can save the muscle tissue,and the protein bands can be used for diagnosing the muscular dystrophy. For clinically suspected patients with dystrophinopathy,if normal or mild deficiency is shown by immunohistochemistry,WB should be applied to detect the dystrophin protein band.

[The clinical and muscular pathological study of dermatomyositis with perifascicular atrophy changes].

OBJECTIVE: To investigate the clinical and pathological characteristics of dermatomyositis with muscular perifascicular atrophy (PFA). METHODS: A series of 104 consecutive patients clinically and pathologically diagnosed as dermatomyositis by muscle biopsy in our laboratory from December, 2003 to August, 2011, were enrolled in this study. Muscle biopsy of all the enrolled patients had shown PFA of muscle fibers. RESULTS: Among the 104 patients, 34 were males and 70 were females with a mean age of 45 years old. Among them, 8 cases had normal electromyogram; 42 had normal serum creatine kinase level; 11 were diagnosed as carcinoma; 75 were found to be combined with interstitial lung disease (ILD). Based on morphologic changes of muscle biopsy, they were divided into pure PFA group with 54 cases and PFA plus focal damage group with 50 cases. Compared with the pure PFA group, there was prominent mononuclear cell infiltration into perimysial intermediate sized vessels and membrane attack complement (MAC) deposition in the intramuscular capillaries in the PFA plus group. Skin biopsy had been taken in 12 cases together with muscle biopsy and had shown the "border effect" of both PFA and interface dermatitis in muscle and skin. CONCLUSIONS: Our study suggests that chronic immune vascular damage and insufficiency in dermatomyositis may cause ischemia and focal myofiber damage in "watershed" regions. The incidence of ILD in our dermatomyositis patients with PFA is high.

[Clinical features and genetic diagnosis of Kennedy disease].

OBJECTIVE: To outline the clinical features of Kennedy disease in Chinese patients. METHODS: The peripheral blood was collected from the male lower motor neuron disease patients of our inpatients and outpatients from July 2005 to September 2008. Then the genome DNA was extracted and the target gene amplified by polymerase chain reaction and sequenced. The clinical data of positive samples were analyzed and summarized. RESULTS: The number of expanded CAG repeats of 12 patients ranged from 43 to 57. And the number of CAG repeats was inversely correlated with the age of onset (r = -0.756, P < 0.005). The first symptom of all of these patients was extremity weakness. The progression of disease was slow. One of the patients died from pneumonia. And the whole disease course lasted for 14 years. CONCLUSION: As an adult onset degenerative disease with a slower clinical progression, Kennedy disease has its own characteristics of inheritance pattern and natural course. It can be accurately diagnosed by androgen receptor gene analysis.

A Comprehensive Analysis of 2013 Dystrophinopathies in China: A Report From National Rare Disease Center.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular disorders caused by mutations in DMD. A high-quality database of DMD/BMD is essential not only for clinical practice but also for fundamental research. Here, we aimed to build the largest Chinese national dystrophinopathy database using the National Rare Diseases Registry System of China. Peking Union Medical College Hospital (PUMCH) was the National Rare Diseases Center of China. This research involved 2013 patients with dystrophinopathies, whose diagnoses were confirmed; they were registered and followed up at PUMCH from March 2011 to December 2018. Family history, clinical signs, and treatment data were reported for patients with DMD and BMD at different rates. All six serum biochemical indexes could accurately distinguish between DMD and BMD patients. Copy number variations were the most frequent mutation type (79.2% in DMD and 84.3% in BMD), of which large deletions accounted for 88.4 and 88.6%, large duplications accounted for 11.6 and 11.4% in DMD and BMD, respectively. An exon deletion hotspot, located in exons 45-54, was observed in DMD, and intron 44 was the most frequent deletion starting point (26.5%). Duplication and single nucleotide variations appeared to be uniformly distributed among all exons. Eleven patients were identified to have ultrarare mutation types. Eleven other patients suffered from two separate mutations simultaneously, some of which may have taken place via dependent mechanisms. Thus, we have established the largest hospital-based Chinese dystrophinopathy database via the National Rare Diseases Registry System. This study provides valuable information for further diagnostic and therapeutic studies of dystrophinopathy.

[Clinical and pathological features of glycogen storage disease type III].

OBJECTIVES: To summarize the clinical and pathological features of glycogen storage disease (GSD) type III. METHODS: The clinical data of 12 GSD type III, 8 males and 4 females, aged 2 - 27, were collected. The biopsy specimens of quadriceps muscle of thigh underwent HE and histochemical staining and light and electron microscopy. RESULTS: The main clinical feature were hepatomegaly and hypoglycemic symptoms, slow growth, and microsome since childhood, while myopathy was mild. Laboratory findings included low plasma glucose (n = 12), high liver transaminases (n = 12), increased CK (n = 11), mild metabolic acidosis (n = 11), hyperlipemia (n = 9), elevation of blood lactate (n = 5), high uric acid (n = 1), and decrease of serum carnitine level (n = 1). One patient had echographic evidence of cardiomyopathy. 11 patients were postprandial adrenalin stimulation test positive. Raw corn starch therapy was used on all patients and showed effective on liver manifestations. Muscle biopsy showed vacuolar myopathy, PAS positive glycogen granules in muscle fibers, small foci of intense ACP reactivity, and deposit of lipid droplets. CONCLUSION: GSD type III exhibits a clinical heterogeneity. Besides hepatic symptoms, myopathy and cardiomyopathy should be addressed adequately. The degree of pathological change of muscles is not significantly related to the degree of functional impairment, duration of disease, and level of CK.

[Effect of pregnancy and spontaneous delivery on the morphology of levator ani muscle and expression of vaginal nerve fibers].

OBJECTIVE: To investigate the effect of pregnancy and spontaneous delivery on the morphologic characteristics of the levator ani muscle and innervation of the vaginal mucosa. METHODS: Eight nullipara without pelvic floor dysfunction (PFD) and 64 normal primipara undergoing spontaneous delivery were enrolled in this study during July to December 2006 in Peking Union Medical College Hospital. Biopsy specimens of levator ani muscle (LAM) and anterior and posterior vaginal walls were obtained from the puerpera as well as from the 8 nullipara undergoing vaginal operation. The structures of LAM were examined with histological techniques. Vaginal mucosa specimens were examined using immunohistochemistry staining for protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide (VIP) and ne uropeptide Y (NPY), and the positive stained nerve fibers were calculated respectively. RESULTS: The LAMs of the puerpera undergoing spontaneous delivery presented myogenetic and neurogenetic changes, both acute and chronic. Type I muscular fibers were predominant (79%) with both types increasing in diameters [(86 +/- 9) microm and (79 +/- 15) microm]. Significantly different (P < 0.05) innervation of PGP 9.5, VIP, and NPY nerve fibers was observed between epithelial lamina of anterior vaginal wall (5.9 +/- 3.3, 7.6 +/- 3.1 and 8.2 +/- 3.2, respectively) and that of posterior vaginal wall (3.8 +/- 2.9, 5.9 +/- 3.1 and 6.0 +/- 3.0, respectively), with the nerve fibers being more in epithelial lamina of anterior vaginal wall, while no difference in the innervation of nerve fibers was observed in the lamina propria. Significantly different (P < 0.05) innervation of PGP 9.5 and VIP nerve fibers was observed in the lamina propria of the anterior vaginal wall in puerperal undergoing vaginal delivery (6.9 +/- 3.2 and 4.9 +/- 2.1) compared with those in nullipara (3.9 +/- 3.6 and 3.1 +/- 1.2). CONCLUSIONS: Pathologic changes occur in LAMs and pelvic floor nerves during labor and delivery. LAM fibers become hypertrophy to adapt to the physiological changes during pregnancy. Richer innervation of PGP 9.5 and VIP nerve fibers in the lamina propria of the anterior vaginal wall in puerpera undergoing spontaneous delivery is beneficial for dilation of the blood vessels and smooth muscles and makes preparation for delivery.

[Study of morphological changes in levator ani muscle of patients with stress urinary incontinence].

OBJECTIVE: To observe the the histological changes of levator ani muscle in patients with stress urinary incontinence (SUI) or pelvic organ prolapse (POP) and to determine the alteration that contributed to pathogenesis. METHODS: Biopsy specimens of levator ani muscle were obtained from 15 patients with SUI, 19 patients with POP and 3 asymptomatic control with rectal cancer during operation. Levator ani muscle's structure was examined with routine histological techniques. At the same time, fiber distribution and diameters were also measured. RESULTS: The morphological features of levator ani muscle in SUI and POP group included the muscular fiber density decreased and separated by large amounts of dense connective tissue. The mean diameter of levator ani muscle in 4 SUI patients containing striated muscle fibers was (24 +/- 9) micro m, the mean diameter in 3 POP patients was (24 +/- 5) micro m, and the mean diameter in 3 control cases was (54 +/- 11) micro m. There was no diameter difference between SUI and POP group (P > 0.05), and the mean fiber diameters of SUI and POP patients were significantly smaller than that of the control group (P < 0.05). The proportion of type I fibers in SUI and POP patients had the tendency to increase (79.6%, 97.2% and 77.2%), with relative decrease of type II fibers. In control group the mean diameter of muscle fiber decreased significantly with age and menopausal time (P = 0.000). CONCLUSIONS: There was obvious fibrosis and the muscle fiber atrophy in SUI and POP group, which resulted in the weakness of pelvic floor. The prevalence of type I fibers might be disadvantage to forceful contraction during straining. Aging also contributed to neuromuscular degeneration and then associated with urinary incontinence in elderly women.

[Study of morphological changes in levator ani muscle of patients with stress urinary incontinence or pelvic organ prolapse].

OBJECTIVE: To investigate the morphological characteristics of levator ani muscle in patients with stress urinary incontinence (SUI) or pelvic organ prolapse (POP) and to explore whether the alterations could contribute to pathogenesis of the diseases. METHODS: Biopsy specimens of levator ani muscle were obtained from 15 patients with SUI, 19 patients with POP and 3 asymptomatic controls with rectal cancer during operation. The structure of levator ani muscle was examined with routine histological techniques: HE staining, modified Gomori trichrome staining, NSE staining, ACP staining, and adenosine triphosphatase (ATPase) staining. RESULTS: There was no significant difference in age, parity, menopausal time, disease severity and leak point pressure between SUI patients with or without muscle fibers (P > 0.05). The muscular fiber density of levator ani muscles in SUI and POP groups was decreased, fibers were arranged in disorder and separated by large quantities of dense connective tissues with infiltrating inflammatory cells. The muscle fiber fascicles showed obvious grouped denervative atrophy, fiber type grouping and angular in shape. And also there was myopathic degeneration such as centrally located nuclei, peripheral phagocytosis and vacuolated necrosis. CONCLUSIONS: There are both neurogenic and myopathic alterations in levator ani muscle's structure in patients with SUI and POP. The presence of both acute and chronic abnormalities indicates that the weakness of pelvic floor is a consequence of prolonged denervation.

Association between DPP6 polymorphism and the risk of sporadic amyotrophic lateral sclerosis in Chinese patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease characterized by the loss of motor neurons in the spinal cord, brainstem, and cerebral cortex, which results in muscle weakness, atrophy. Sporadic ALS (SALS) accounts for about 90% of ALS cases, but the etiology is largely unknown. Most of the researchers consider it to be a complex disease. There have been several genome-wide association (GWA) studies reporting several single nucleotide polymorphisms (SNPs) which are susceptible to ALS, but no data of Asians (including Chinese) yet. We investigate whether the polymorphism of rs10260404 in DPP6 gene is associated with SALS in Chinese Han origin to compare the ethnic differences between Chinese Han origin and other populations. METHODS: The genomic DNA was extracted from the leukocytes of whole blood samples in 58 Chinese Han patients with SALS and 52 healthy controls. The asymmetric PCR was processed in the presence of an unlabeled probe that contained the rs10260404 locus. The product was genotyped on a light scanner using high resolution melting method and some were confirmed with sequencing. RESULTS: The rs10260404 polymorphism was in Hardy-Weinberg equilibrium in patients and controls. The CC genotype and the C allele were similar in patients compared with healthy subjects and not associated with an increased risk of Chinese SALS patients (chi(2) = 0.29, OR = 1.26, 95% CI 0.55 - 2.87, P > 0.05). CONCLUSIONS: The rs10260404 is not associated with ALS susceptibility in Chinese people with Han origin which may be due to ethnic differences. More study with large number of cases in Chinese population is really necessary.