Immunohistochemistry And Western Blotting With Micro-sample In The Diagnosis Of Muscular Dystrophy

[Background]The muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness of variable distribution and severity.They can be subdivided into several groups,in accordance with the distribution of predominant muscle weakness:Duchenne; Becker;limb-girdle;facioscapulohumeral;oculopharyngeal;congentital and Emery-Dreifuss.Duchenne muscular dystrophy(DMD) is the commonest form of the muscular dystrophies.The form is X-linked disorders.BMD is a rapidly progressive myopathy.The symmetry of limb weakness is mainly proximal.The most patients have enlarged calves.The patient may lose the ability to walk independently before the age of 12.The other clinical features have been described,such as dilated cardiomyopathy,mental retardation,night blindness.Most deaths between 15-25 yesrs,due to respiratory or cardiac failure.The serum creatine kinase(CK) is elevated. The course of BMD is more benign,the disease has a slower rate of progression,with age of onset is more than 7 years old.The distribution of muscle wasting and weakness is closely similar to that in DMD.The calf may be pain on exercise.The patients also have calf hypertrophy.The ageof failure to walk is 16-80yesrs.The serum CK is elevated,too.As a result of dystrophin protein and gene mutations,the clinical features have not only DMD/BMD,but also the cardiomyopathy,cramp/myalgia syndromes, female carriers,mental retardation and chromosome X21 contiguous gene syndromes.All of these can be referred to dystrophinopathy.Limb-girdle muscular dystrophies are a heterogeneous group of the disorders characterized by weakness and wasting of the pelvic and shoulder girdle muscles.The clinical course of LGMD ranges from severe forms with disease onset and rapid progression within the first decade of life,to milder forms with later onset and slower progression.The classical grouping of the LGMDs into autosomal dominant-LGMD(AD-LGMD or LGMD1) and autosomal recessive-LGMD(AR-LGMD or LGMD2) forms is being complemented by a classification based on the involved proteins and the underlying genetic defects.Tagawa found the frequency of LGMD2B was 19%,LGMD2A was estimated at 21%of LGMDpatients.LGMD2B will be the second frequent type of LGMD in Japan.LGMD2B will be the second frequent type ofLGMD in Japan.With the rapid development of Molecular Biology,people have been increasingly recognized a group of disorder that the protein and gene involved in are identified. The Molecular Biology increasingly used in diagnosis.At present,It has been little reseach in our coutry.[Objective]1,We want to evaluate their own advantages of IHC and WB in the diagonosis of dystrophinopathy,and optimize the protein level of diagnostic methods.2,The cases that the clinical manifestations are not typical dystrophinopathyand it is difficult to judge to rely on IHC loss of dystrophin protein,need to been detected the dystrophin protein on WB,in order to have a clear diagnosis.3,The suspected LGMD cases need to be used the Multiplex Western blowing system for the analysis of muscular dystrophy proteins. 4,We want to analysis the clinical features and differential diagnosis of dystrophinopathy and LGMD.[Methods]The cases generated in PUMCH's neuropathological institution. In the first part,according to the clinical features and the pathological changes in muscle,37 cases with clinically and pathologically suspected dystrophinopathies on muscle biopsy were screened,on which IHC detection of dystrophin were performed(the three antibody are dys-R,dys-N,dys-C), 25 cases in which had IHC detection ofα-sarcoglycan,β-sarcoglycan,caveolin3 were performed.36 cases(1 case did not extract enough protein) had WB detection of dystrophin and dysferlin.In the second part,30cases with clinically and pathologically suspected LGMD on muscle biopsy were screened,on which IHC detection ofα-sarcoglycan,β-sarcoglycan,caveolin3 were performed,26cases had multiplex WB detection of dystrophin, dysferlin,calpain3 and caveolin3.We collected these cases history and clinical data.[Results]In the first part,4 cases had only a single antibody no reaction, other cases had varying degrees of immunostains with three antibodies in the sarcolemma,with 10 cases of abnomal immunostains of other proteins. Western blot,compared with control,confirmed that the band was absent, abnomal molecular weight,intensity decreased,a single antibody no reaction and normal in 36 cases.4 cases in which had dysferlin abnormalities.We integrated these cases information of clinical features,muscle biopsy,IHC and WB analysis,32 cases were diagnose dystrophinopathoes.In the second part,1 case mild weakened immunostains withβ-sarcoglycan,1 case mild weakened immunostains withα-sarcoglycan and caveolin3.Western blot,compared with control,confirmed that the dysferlin was absent and intensity decreased in 9 cases,the calpain3 was absent or intensity decreased in Icases,the dystrophin was intensity decreased in 1 cases,the dystrophin was suspicious dual-band and intensity decreased in 1 case.11 cases are nomal,and the remaining cases with at least 2 different antibodies reduce band intensity.We diagnose dysferlinopathies 9 cases,LGMD2A 1 cases.[Conclusion]In the first part,we have the conclusions:1,The relationship of IHC and WB in the detection of dystrophin protein:1) IHC shows the absence of three antibodies or absence of a single antibody:WB and IHC results are consistent,in future,such cases can be done only IHC;2) IHC showed mild deficiency:these cases need to been done WB to detect the dystrophin protein;3)The cases have a high degree of suspected clinical diagnosis,IHC showed normal:these cases need to been done WB to detect the dystrophin protein.2,The clinical features,differential diagnosis of the dystrophinopathy:1) It is difficult to identify adult-onset BMD and LGMD in the clinical features:it need to been detected the dystrophin protein;2) no clinical symptoms or mild symptoms,serum CK increased significantly, gastrocnemius hypertrophy:it need to been detected the dystrophin protein;3) cardiomyopathy associated with significantly increased serum CK:it need to been detected the dystrophin protein.3,dystrophin protein-gene-clinical type has a certain relevance,but there are a lot of heterogeneity.The second part of the conclusion of the study:1,LGMD patients with diverse clinical manifestations,it is difficult clinical diagnosis.2, Dysferlinopathy were found in suspected LGMD patients.The clinical manifestations are LGMD2B and MM.The individual case is the simple increased serum CK.3,One LGMD2A was found in this study.The clinical phenotype of this case was Leyden-Mobius type.4,When we analysed multiplex WB,we found there may be a number of abnormal protein.We need pay attention to the secondary deficiencies.If necessary,we can have genetic testing.