Ullrich型先天性肌营养不良的临床特点(附1例报告)

目的探讨Ullrich型先天性肌营养不良的临床及病理学特点。方法回顾性分析1例Ullrich型先天性肌营养不良患儿的临床资料,并结合相关文献进行复习。结果患儿自出生起肌张力低下,伴有近端关节挛缩、远端关节弹性过度。生化检查示血磷酸肌酸激酶轻度增高。EMG示肌源性损害(近端肌)肌电改变为主,伴轻度神经源损害(下肢远端肌)。基因二代测序示存在COL6A3基因杂合核苷酸变异,为剪切变异;其父母未见异常。肌肉病理示骨骼肌呈肌营养不良样病理改变。肌肉MRI示双侧小腿及大腿肌肉呈弥漫脂肪浸润伴水肿改变,肌营养不良可能。结论本例患者为杂合子新生突变,是先天性肌营养不良的一个亚型。临床表现以近端关节挛缩、远端关节弹性过度为主要特点。EMG、基因、肌肉病理及肌肉MRI检查有助于本病的诊断。     

免疫组织化学dystrophin染色诊断Duchenne型肌营养不良症的研究

目的 探讨Duchenne型肌营养不良症(DMD)肌萎缩蛋白(dystrophin)表达规律和临床意义.方法 收集我院7例DMD患者作为试验组,7例非DMD患者为对照组.使用抗dystrophin杆状结构域单抗、免疫组织化学染色,观察肌膜dystrophin表达.结果 7例DMD患者肌细胞膜dystrophin阴性,7例非DMD患者dystrophin染色阳性.结论 证实DMD患者肌细胞膜dystrophin表达阴性,揭示dystrophin缺失是其发病机制,可以作为确诊DMD手段,对临床诊断DMD有实际意义.     

dysferlinopathy患者40例临床和病理分析

目的 回顾性分析40例dysferlinopathy患者临床及活检骨骼肌组织化学、免疫组织化学染色病理变化,探讨dysferlinopathy的临床、病理诊断价值.方法 对40例dysferlinopathy患者临床资料进行分析;并对活检骨骼肌进行组织化学、免疫组织化学染色病理分析.结果 患者一般临床表现:进行性加重的肌无力、萎缩;根据病初受累肌群分为:肢带型肌营养不良2B型27例,Miyoshi远端型肌营养不良12例,以及胫骨前肌起病的远端肌病1例;血肌酸激酶水平呈不同程度的升高(134～ 19 795 U/L);全部患者肌电图呈肌源性损害;12例行骨骼肌MRI,其中9例行双侧大、小腿MRI检查,3例行双侧小腿MRI检查;近端肌受累为主4例、腓肠肌受累为主7例、胫骨前肌受累为主1例.骨骼肌活检组织化学染色病理表现:肌纤维变性、坏死和再生活跃,结缔组织不同程度增生,16例可见肌内膜、肌束膜和小血管周围炎性细胞浸润;抗-dysferlin单克隆抗体免疫组织化学染色结果显示,30例dysferlinopathy患者肌纤维膜上dysferlin蛋白完全缺失,10例重度减低.结论 本病典型临床表现为进行性加重肌无力、肌萎缩,根据病初受累肌群区分临床表型;血肌酸激酶显著增高,符合膜蛋白破坏型肌营养不良特点;骨骼肌MRI可清晰判断受累肌群范围、程度,帮助判断临床表型及选择合适的骨骼肌活检部位;病理特点为肌营养不良改变,部分患者有炎性细胞浸润,需要结合临床与炎性肌病相鉴别;肌纤维膜dysferlin蛋白缺失或重度减低,是dysferlinopathy分子病理诊断的重要依据.     

Dystrophin在不同类型肌营养不良症中的变化及诊断价值

目的研究dystrophin在不同类型肌营养不良症中的变化及分型诊断价值.方法用抗dystrophin抗体对107例肌营养不良症患者肌组织标本行免疫组织化学分析.结果Duchenne型肌营养不良(DMD)患者肌细胞膜上无显色,Becker型肌营养不良(BMD)患者肌细胞膜上显色浅淡、不连续或呈斑片状.肢带型肌营养不良(LGMD)患者肌细胞膜上染色正常.结论dystrophin免疫组化染色对于年龄较小临床不易区分的DMD/BMD患者,可区分开来,以早期预测功能影响程度.该方法也有助于区分临床表现相似的成年散发BMD和LGMD患者,对于正确地进行遗传咨询具有重要意义.     

DMD、BMD患者骨骼肌、皮肤立毛肌肌营养不良蛋白同时欠损

目的研究、对比肌营养不良蛋白(dystrophin)在杜兴型肌营养不良(Duchenne muscular dystrophy,DMD)和贝克型肌营养不良(Becker muscular dystrophy,BMD)患者活检骨骼肌、皮肤立毛肌中的表达。方法用肌营养不良蛋白三个不同区域的单克隆抗体(Dystrophin-N、-C、-R)对11例DMD患者,5例BMD患者和3例其他神经肌病患者同时行活检骨骼肌、皮肤免疫组织化学染色分析。结果与对照例相比,11例DMD患者抗Dystro-phin-N、-C、-R单克隆抗体免疫组织化学染色显示:骨骼肌肌纤维膜Dystrophin-N、-C、-R呈完全欠损;皮肤立毛肌Dystrophin-N、-R完全欠损,Dystrophin-C轻微表达。5例BMD患者抗Dystrophin-N、-C、-R单克隆抗体免疫组织化学染色显示:肌营养不良蛋白在骨骼肌肌纤维膜和皮肤均呈不完全欠损。结论DMD和BMD患者肌营养不良蛋白在骨骼肌肌纤维膜、皮肤立毛肌呈完全/不完全欠损,与骨骼肌活检相同,皮肤活检也是分子病理学诊断DMD、BMD简便、易行、可靠的方法。     

针吸型肌肉活检联合免疫荧光在假肥大型肌营养不良诊断中的应用

目的 探讨应用针吸型肌肉活检结合免疫荧光染色诊断假肥大型肌营养不良症的应用价值及意义。方法 应用针吸型活检术取533例假肥大型肌营养不良症患者(415例DMD, 118例BMD)的肌组织,采用HE染色观察肌细胞形态,免疫荧光染色技术检测抗肌营养不良蛋白, 以2 例正常人的肌细胞作为对照。结果 正常人肌细胞膜上抗肌萎缩蛋白染色阳性,可见沿肌细胞膜分布完整的荧光条带; DMD 患者肌膜染色阴性,肌细胞膜完全不显色; BM D患者染色弱阳性, 可见沿肌细胞膜分布的间断斑片状荧光带。结论 应用针吸型活检术联合免疫荧光染色可以有效的检测抗肌营养不良蛋白的表达, 有助于DMD 和BMD 的确诊及鉴别诊断。     

dysferlinopathy患者八例临床及分子病理学特点

目的探讨中国dysferlinopathy患者的临床及分子病理学特点。方法分析已确诊的4例肢带型肌营养不良2B型、4例Miyoshi远位型肌营养不良患者的临床、骨骼肌活体组织检查和免疫组织化学染色病理特点。并以Duchenne肌营养不良4例，多发性肌炎和包涵体肌炎各2例作为对照。结果dysferlinopathy患者均以进行性加重的肌无力、萎缩为主要症状，符合进行性肌营养不良的临床表现。组织化学染色示dysferlinopathy患者出现不同程度的肌纤维变性、坏死、再生，结缔组织增生；多数病例可见炎性细胞浸润；抗dysferlin单克隆抗体免疫组织化学染色显示8例dysferlinopathy患者均出现dysferlin蛋自在肌纤维膜上和胞质内的缺失。结论（1）dysferlinopathy符合进行性肌营养不良的临床、病理表现；（2）抗dysferlin单克隆抗体免疫组织化学染色病理分析是诊断dysferlinopathy的可靠方法，值得临床推广应用。     

DMD/BMD肌细胞抗肌营养不良蛋白免疫荧光组化研究

目的:研究Duchenne/Becker型肌营养不良症(DMD/BMD)患者肌细胞中抗肌营养不良蛋白(dystrophin)的表达及其诊断意义。方法:采用免疫荧光抗体染色技术对5例DMD,2例BMD肌细胞中抗肌营养不良蛋白进行检测,以2例正常人的肌细胞作为以照。结果:对照组肌细胞膜上染色阳性,胞核及胞浆呈阴性;DMD患者肌膜完全无显色;BMD患者染色弱阳性,可见沿肌细胞膜分布的间断斑片状荧光带。结论:抗肌营养不良蛋白缺乏或表达异常是DMD/BMD基本病理基础。应用免疫荧光抗体染色法检测抗肌营养不良蛋白,有助于DMD和BMD的确诊及鉴别诊断     

微量标本Western blot在诊断肢带型肌营养不良2A型中的应用

目的 探讨微量标本Western blot在诊断肢带型肌营养不良2A型中的应用.方法 对73例以肢体近端肌肉受累为首要临床表现的进行性肌营养不良患者行开放式骨骼肌活体组织检查,标本行组织化学染色以及抗dystrophin-N、C、R,α、β、γ、δ-sarcoglycan,dysferlin,caveolin-3单克隆抗体免疫组织化学染色.对其中已经除外dystrophin、sarcoglycans、dysferlin、caveolin-3蛋白异常的29例患者再行骨骼肌Western blot抗calpain-3、caveolin-3单克隆抗体免疫反应.结果 共有10例患者被确诊为肢带型肌营养不良2A型,其临床特点均为肢体近端肌无力起病,血清肌酸激酶不同程度升高,肌电图呈肌源性改变.组织化学染色见肌纤维大小不一,可见不同程度肌纤维变性、坏死和再生,结缔组织增生,6例见分叶状肌纤维;免疫组织化学染色见肌纤维中dystrophin、sarcoglycan、dysferlin、caveolin-3蛋白均正常表达.Western blot发现该10例患者相对分子质量94 000条带(calpain-3)与Duchenne/Becker型肌营养不良对照相比,呈完全(8例)或部分(2例)缺失;30 000区域附加条带均呈弱表达,22 000区域条带(caveolin-3)均正常表达.结论 骨骼肌微量标本Western blot是诊断LGMD2A的有效方法,适用于临床LGMD的分型诊断.     

免疫荧光检测对肌营养不良症临床诊断的价值

目的探讨免疫荧光检测对Duchenne型、Becker型和肢带型肌营养不良症(DMD、BMD和LG-MD)临床诊断的价值。方法对25例肌营养不良症患者(DMD 10例,BMD 4例,LGMD 11例)的骨骼肌冰冻切片标本应用免疫荧光法检测抗肌萎缩蛋白(Dys)中央棒状区(Dys1)、C-末端(Dys2)、N-末端(Dys3)单克隆抗体及α、-β、-γ-肌聚糖蛋白(SG)多克隆抗体在肌膜的表达。结果10例DMD患者Dys染色均为阴性,4例BMD患者呈弱阳性;11例LGMD患者的SG染色中,分别有1例α-SG阴性及1例β-SG阴性。结论Dys免疫荧光检测对DMD/BMD的临床诊断具有特异性价值,是临床诊断的可靠方法之一;SG检测对LGMD的临床诊断还需进一步完善。     

Collagen VI status and clinical severity in Ullrich congenital muscular dystrophy: phenotype analysis of 11 families linked to the COL6 loci

Ullrich's congenital muscular dystrophy (UCMD) is an autosomal recessive myopathy characterised by neonatal muscle weakness, proximal joint contractures and distal hyperlaxity. Mutations in the COL6A1, COL6A2 (21 q22.3) and COL6A3 (2 q37) genes, encoding the alpha 1, alpha 2 and alpha 3 chains of collagen VI, respectively, have been recently identified as responsible for UCMD in a total of 9 families. We investigated in detail the clinical and morphological phenotype of 15 UCMD patients from 11 consanguineous families showing potential linkage either to 21 q22.3 (6 families) or to 2 q37 (5 families). Collagen VI deficiency was confirmed on muscle biopsies or skin fibroblasts in 8 families. Although all patients shared a common phenotype, a great variability in severity was observed. Collagen VI deficiency in muscle or cultured fibroblasts was complete in the severe cases and partial in the milder ones, which suggests a correlation between the degree of collagen VI deficiency and the clinical severity in UCMD. No significant phenotypical differences were found between the families linked to each of the 2 loci, which confirms UCMD as a unique entity with underlying genetic heterogeneity.     

Ullrich myopathy phenotype with secondary ColVI defect identified by confocal imaging and electron microscopy analysis

Ullrich congenital muscular dystrophy (UCMD) is clinically characterized by muscle weakness, proximal contractures and distal hyperlaxity and morphologically branded by absence or reduction of collagen VI (ColVI), in muscle and in cultured fibroblasts. The ColVI defect is generally related to COL6 genes mutations, however UCDM patients without COL6 mutations have been recently reported, suggesting genetic heterogeneity. We report comparative morphological findings between a UCMD patient harboring a homozygous COL6A2 mutation and a patient with a typical UCMD phenotype in which mutations in COL6 genes were excluded. The patient with no mutations in COL6 genes exhibited a partial ColVI defect, which was only detected close to the basal membrane of myofibers. We describe how confocal microscopy and rotary-shadowing electron microscopy may be useful to identify a secondary ColVI defect.     

Congenital muscular dystrophy phenotype with neuromuscular spindles excess in a 5-year-old girl caused by HRAS mutation

We report on a 5-year-old girl who presented with an association of symptoms reminiscent of an Ullrich-like congenital muscular dystrophy including congenital hypotonia, proximal joint contractures, hyperlaxity of distal joints, normal cognitive development, and kyphoscoliosis. There was an excess of neuromuscular spindles on the skeletal muscle biopsy. This very peculiar feature on muscle biopsy has been reported only in patients with mutations in the HRAS gene. Sequence analysis of the subject’s HRAS gene from blood leukocytes and skeletal muscle revealed a previously described heterozygous missense mutation (c.187G>A, p. Glu63Lys). The present report thus extends the differential diagnosis of congenital muscular dystrophy with major “retractile” phenotypes and adds congenital muscular dystrophy to the clinical spectrum of HRAS-related disorders.     

Collagen VI involvement in Ullrich syndrome: a clinical,genetic, and immunohistochemical study

BACKGROUND: Ullrich congenital muscular dystrophy (UCMD) is a form of merosin-positive congenital muscular dystrophy characterized by proximal contractures, distal laxity, rigidity of the spine, and respiratory complications. Recently, a deficiency of collagen VI on muscle and skin biopsy together with recessive mutations in the collagen 6A2 gene were reported in three families with UCMD. However, the clinical spectrum, frequency, and level of heterogeneity of this disorder are not known. Subjects and Methods: The authors studied 15 patients (aged 3 to 23.6 years) with a clinical diagnosis of UCMD. Linkage analysis to the three collagen VI genes was performed in all informative families (n = 7), whereas immunohistochemical analysis of collagen VI expression in muscle was performed in the remaining cases. RESULTS: An immunocytochemical reduction of collagen VI was observed in six patients. Three of the six patients belonged to informative families, and haplotype analysis clearly suggested linkage to the COL6A1/2 locus in two cases and to the COL6A3 loci in the third case. In the remaining nine patients, primary collagen VI involvement was excluded based on either the linkage analysis (four families) or considered unlikely based on normal immunolabeling of collagen VI. Age and presentation at onset, the distribution and severity of weakness and contractures, and the frequency of nonambulant patients were similar in the patients with and without collagen VI involvement. Distal laxity, rigidity of the spine, scoliosis, failure to thrive, and early and severe respiratory impairment were found in all patients by the end of the first decade of life, irrespective of their maximum motor functional ability or their collagen status. CONCLUSIONS: These results suggest that collagen VI involvement is relatively common in UCMD (40%); however, the role of this molecule was excluded in a number of cases, suggesting genetic heterogeneity of this condition.     

A new case of Ullrich's disease

A new case of congenital, hypotonic-sclerotic muscular dystrophy is presented. The patient showed congenital hyperlaxity and looseness of distal joints, muscle weakness, and spur-like protrusion of the calcaneus. Afterwards rapid progressive contractures of both knees and hip joints developed. Muscle biopsies revealed unequivocal dystrophic abnormalities and small atrophic fibers with numerous foldings of basal lamina suggestive of a neurogenic lesion. The disease presents clinical variability but the diagnosis is possible when a newborn shows: no dominant family history, slender body, marked distal joint laxity and hyperflexibility, proximal joint contractures and normal or slightly increased serum enzymes.     

Collagen VI-related muscle disorders

Collagen VI-related muscle disorders include severe Ullrich's disease (Ullrich congenital muscular dystrophy:UCMD) and milder Bethlem myopathy. Mutations in the 3 collagen VI genes, namely, COL6A1, COL6A2, and COL6A3, cause both diseases. UCMD is inherited in an autosomal recessive manner, and de novo dominant mutations are also reported. Bethlem myopathy is usually inherited in an autosomal dominant manner, but a rare autosomal recessive inheritance has recently been reported. Patients with UCMD have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Bethlem myopathy is characterized by a combination of proximal muscle weakness and contractures of finger, elbow, and ankle joints. Because intermediate phenotypes occur, UCMD and Bethlem myopathy should be considered diseases in a continuous spectrum of collagen VI-related muscle disorders. Abnormalities of cell adhesion, regeneration, mitochondrial permeability transition pore, and autophagy have been reported in UCMD. Respiratory surveillance for nocturnal hypoventilation and proper respirator implementation are crinical management considerations in UCMD. Orthopedic assessment in necessary if surgery for Achilles tendon contractures is being considered in patient with Bethlem myopathy. We evaluated the role of nonsense-mediated mRNA decay (NMD) in UCMD associated with a premature termination codon in the COL6A2 gene, which caused the loss of collagen VI. A pharmacological block of NMD caused upregulation of the mutant collagen VI and partially functional extracellular matrix formation. Cyclosporin A has been reported to correct mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies, and a pilot trial of cyclosporin A was carried out.     

Pathogenic variants in COL6A3 cause Ullrich-like congenital muscular dystrophy in young Labrador Retriever dogs

The collagen VI-related muscular dystrophies in people include a broad spectrum of diseases ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Clinical features are attributable to both muscle and connective tissue and include progressive muscle weakness and respiratory failure, hyperlaxity of distal joints, and progressive contracture of large joints. Here we describe two different COL6A3 pathogenic variants in Labrador Retriever dogs that result in autosomal recessive or autosomal dominant congenital myopathies with hyperlaxity of distal joints and joint contracture, similar to the condition in people.     

Merosin-positive congenital muscular dystrophy with early orthopaedic problems in relation to Ullrich's disease

We report three patients with sporadic merosin-positive congenital muscular dystrophy (CMD) with torticollis and/or developmental dislocation of the hip in early childhood. Diagnosis of merosin-positive CMD was based on their clinical and dystrophic muscle biopsy findings. At the age 13 months, patient 1 was found to have developmental dislocation of both hips, which was surgically treated at 5 years. Patient 2 had severe torticollis and contracture of both hip joints which had been present since the neonatal period, and underwent repair of the torticollis at 2 years. Patient 3 was found to have developmental dislocation of the left hip at one month of age. Although she had generalized muscle hypotonia she learned to walk at 23 months. She had no facial muscle involvement nor contracture of joints, but had hyperlaxity of distal joints. Her muscle biopsy showed complete collagen VI deficiency immunohistochemically. In contrast to merosin-deficient CMD, merosin-positive CMD appears to be a group of heterogeneous diseases. Since collagen VI was reported to be defective in Ullrich's disease, patient 3 may be diagnosed as having Ullrich's disease but had no typical clinical characteristics of the disease. Further study is needed to identify the pathogenetic mechanism of congenital muscular dystrophy with early joint abnormalities to determine whether there is a primary abnormality of the connective tissue including collagen VI.     

Homozygous splice variant (c.1741-6G>A) of the COL6A1 gene in three patients with Ullrich congenital muscular dystrophy

The three major collagen VI genes: COL6A1, COL6A2, and COL6A3 encode microfibrillar components of extracellular matrices in multiple tissues including muscles and tendons. Pathogenic variants in the collagen VI genes cause collagen VI-related dystrophies representing a continuum of conditions from Bethlem myopathy at the milder end to Ullrich congenital muscular dystrophy at the more severe end. Here we describe a pathogenic variant in the COL6A1 gene (NM_001848.3; c.1741-6G>A) found in homozygosity in three patients with Ullrich congenital muscular dystrophy. The patients suffered from severe muscle impairment characterised by proximal weakness, distal hyperlaxity, joint contractures, wheelchair-dependency, and use of nocturnal non-invasive ventilation. The pathogenicity was verified by RNA analyses showing that the variant induced aberrant splicing leading to a frameshift and loss of function. The analyses were in line with immunocytochemistry studies of patient-derived skin fibroblasts and muscle tissue demonstrating impaired secretion of collagen VI into the extracellular matrix. Thereby, we add the variant c.1741-6G>A to the list of pathogenic, recessive, splice variants in COL6A1 causing Ullrich congenital muscular dystrophy. The variant is listed in ClinVar as of “uncertain significance” and “likely benign” and may presumably have been overlooked in other patients.     

Variable reduction of caveolin-3 in patients with LGMD2B/MM

Abstract. Mutations in the human dysferlin gene (DYSF) cause autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal muscles: limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Recently, an interaction between caveolin-3 and dysferlin in normal and dystrophic muscle (primary caveolin-3 deficiency; LGMD1C) was shown. In this study, clinical,morphological and genetic analysis was carried out in four independent LGMD2B/MM patients. All patients presented with an adult-onset, slowly progressive muscular dystrophy with variable involvement of proximal and distal muscles. We found complete lack of dysferlin in the four LGMD2B/MM patients. Secondary reduction of caveolin-3 was detected in three out of the four patients. Regular caveolae were detected along the basal lamina in two patients by electron microscopy. We provide further evidence that dysferlin and caveolin-3 interact in human skeletal muscle. It remains to be elucidated whether the loss of this interaction contributes to pathogenic events in muscular dystrophy.M. C. Walter and C. Braun contributed equally.