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ORIGINAL ARTICLE
Year : 2011  |  Volume : 59  |  Issue : 6  |  Page : 797-802

Duchenne or Becker muscular dystrophy: A clinical, genetic and immunohistochemical study in China


1 Department of Medicine, Senior Profession College; Department of Medical Genetics, China Medical University, Shenyang, China
2 Cell Treatment Center, The Four Six Three Hospital of Liberation Army, Shenyang, China
3 Department of Medical Genetics, China Medical University, Shenyang, China

Date of Submission21-May-2011
Date of Decision29-Jun-2011
Date of Acceptance16-Jul-2011
Date of Web Publication2-Jan-2012

Correspondence Address:
Chunlian Jin
Department of Medical Genetics, China Medical University, Shenyang-110001
China
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DOI: 10.4103/0028-3886.91354

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 » Abstract 

Background and Objective: Duchenne and Becker muscular dystrophies are X-linked diseases caused by mutations in the dystrophin gene, which affect approximately 1 in 3,500 and 1 in 18,000 boys, respectively. The aim of this work was to develop a method to assist the diagnosis and classification of the disease. Materials and Methods: A large data set of dystrophin mutations was detected in 167 Chinese patients by multiplex ligation-dependent probe amplification and sequencing. Muscle biopsy, immunohistochemistry and STR analysis were also carried out in the patients and carriers. Results: One hundred and three deletions, 23 duplications and two-point mutations. The deletion of one or more exons was detected in 103 (61.7%) patients. The region spanning exons 44-55 was the most frequent deletion. The duplication was identified in 23 (13.8%) patients, which was more common than previously reported. Most duplications were found in exons 2-18. Six out of the 45 muscle biopsies analyzed showed the presence of other muscle diseases. Conclusions: This study may be important to enable comparisons of mutation type and the most appropriate analytical approach for samples from different geographical areas and ethnicities.


Keywords: Deletion/duplication, Duchenne/Becker muscular dystrophy, immunohistochemistry, multiplex ligand-dependent probe amplification, mutation


How to cite this article:
Wang Q, Yang X, Yan Y, Song N, Lin C, Jin C. Duchenne or Becker muscular dystrophy: A clinical, genetic and immunohistochemical study in China. Neurol India 2011;59:797-802

How to cite this URL:
Wang Q, Yang X, Yan Y, Song N, Lin C, Jin C. Duchenne or Becker muscular dystrophy: A clinical, genetic and immunohistochemical study in China. Neurol India [serial online] 2011 [cited 2014 Jul 24];59:797-802. Available from: http://www.neurologyindia.com/text.asp?2011/59/6/797/91354



 » Introduction Top


Duchenne and Becker muscular dystrophies (DMD and BMD), the most common lethal X-linked disorders; are caused by mutations in the dystrophin (DMD) gene and affect approximately 1/3,500 and 1/18,000 live male newborns, respectively. [1],[2] DMD is a progressive disease in which boys lose independent ambulation before the age of 16 years. In their third decade, death usually occurs from respiratory failure and cardiac or respiratory complications. BMD has a milder disease course than DMD and the affected individuals remain ambulatory beyond 16 years and may lead near normal lives. [3],[4] The DMD gene, with 79 exons spanning approximately 2.6 million base pairs, is the largest yet identified in the human genome. [5],[6] Approximately 65% of DMD and up to 85% of BMD cases are caused by large deletions of the DMD gene, which have been found to cluster to a major 3΄-hotspot and a minor 5´-hotspot. The remaining cases are caused by duplications (5~10%) or point mutations (25~30%). [7] Mutation detection in the DMD gene has historically been challenging, due primarily to the wide mutation spectrum of the gene. Approximately one-third of DMD/BMD patients occur by new mutations, the others are inherited through mother who are carriers or that arise from germ line mosaicism. [8] Population-specific information on mutations is necessary for appropriate counseling, prenatal diagnosis and also for future molecular therapies. In this report, we describe the mutation spectrum of 167 Chinese patients with DMD/BMD detected by multiplex ligation-dependent probe amplification (MLPA), followed by sequencing and muscle biopsy.


 » Materials and Methods Top


Patients

Chinese DMD/BMD male patients who presented to the Department of Pediatrics at the Shengjing Hospital of China Medical University, Peking Union Medical University or the Shenyang 463 Hospital between January 2000 and October 2010 were referred to the Genetics Department of the China Medical University. The case records were reviewed. Informed consent was obtained from each patient for molecular analyses to be performed. This study was approved by the ethics committees of China Medical University.

The subjects included 143 boys diagnosed with DMD and 24 with BMD, as well as the parents. Patients were clinically diagnosed as DMD/BMD on the basis of the clinical symptoms, family history, EMG data and creatine phosphokinase (CPK) levels. [9],[10],[11] Serum CPK levels were also measured in all the 201 first-degree female relatives at least twice and were considered to be elevated at >200 U/L (normal range, 29-200 U/L).

DNA analysis

Two independent MLPA kits (SALSA P034 and P035), which have been designed to screen all exons of the dystrophin gene, were purchased from MRC Holland, Amsterdam. The 80 probes were divided into two mixtures. All reactions were carried out on a standard thermal cycler as recommended by the manufacturer. The electrophoretic separation was performed by an ABI 3130XL genetic analyzer (Applied Biosystems, Foster City, CA). The data was analyzed by Coffalyser MLPA analysis software (MRC-Holland, Amsterdam, the Netherlands). Apparent single exon deletions detected by MLPA were confirmed with a second method, as small mutations have been found to disturb MLPA amplification. Therefore, defined exons including intron/exon borders were directly sequenced in some patients.

Muscle biopsy

All patients underwent skeletal muscle biopsy from which muscle tissue was obtained according to the research protocol. Muscle biopsies were obtained by the open method from the quadriceps muscles under local anesthetic. Muscle biopsy specimens were embedded in OTC mounting medium (Tissue-Tek; Miles Inc., Elkhart, IN), frozen by immersion in isopentane cooled in liquid nitrogen and stored at −80°C; staining with hematoxylin and eosin (HE) was then performed.

Immunohistochemistry

Immunohistochemistry was performed with monoclonal antibodies to dystrophin (Santa, USA) at a dilution of 1:100, including amino acids 3,200-3,684 located within the deleted region, using standard techniques on consecutive 7-mm sections of snap-frozen muscle tissue. The Miranda immunohistochemistry classification was used to establish the diagnosis. [12]

Microsatellite analysis

Short tandem repeat (STR)-based linkage analysis was performed. In this study, four microsatellite (CA) n repeat markers, including STR44, STR45, STR49 and STR50, were selected. [13]


 » Results Top


The specific clinical findings of the 167 affected boys are presented in [Table 1].
Table 1: Clinical data of features related to DMD or BMD

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Mutations detected by MLPA

A total of 103 deletions and 23 duplications were found in the 167 identified cases after MLPA was performed [Figure 1]. All single exon deletions detected by MLPA were confirmed by PCR. The results are summarized in [Table 2]. The overview of the spectrum of the 126 deletion and duplication mutations is given in [Figure 2].
Figure 1: Electrophoresis analysis: (a) 19 is a proband with a duplication of exons 11, 12, 13, 14 and 15; 18 is the normal reference control. (b) 20 is a proband with a deletion of exons 3, 4, 5, 6 and 7; 18 is the normal reference control

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Figure 2: Graphical overview of the deletions and duplications. (a) Exonic deletions were found in 103 patients. Deleted exon regions are represented by horizontal bars. On the lowest line, exons are numbered 1-79. The number at the end of each bar represents the number of cases with an identical deletion, whereas no number means that the deletion is unique. (b) Exon duplications were found in 23 patients

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Table 2: Results of dystrophin gene analysis in this patient cohort

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Notably, several mutations appeared to be confined to regions of the DMD gene that have been rarely reported to be involved. The longest deletion of the DMD gene was detected in a boy with the clinical phenotype of DMD (#864); this was the largest exon deletion detected in the DMD gene, as all of the 79 exons were undetected by MLPA and further multiplex PCR amplification. It was not clear where the genomic deletion ended. In another patient, MLPA signaled an apparent deletion of exon 23. However, further investigation of this unique deletion suggested that this result was incorrect. Amplification of exon 23 using alternative PCR primers was possible, and the sequence analysis revealed a previously unknown c.3196T°C, p.F1066L mutation. Another 5132del(A) (p.S1630V) mutation in close proximity to the ligation site of the MLPA probe has been reported previously. Considering the localization and size of the duplicated fragments detected in the present sample, it is interesting to note that all the duplications except two were located in the 5΄-hot spot of DMD. These included one boy (#872) with two independent duplications that involved exons 11-13 and 31 and another with duplications of exons 3-7 and 44. [7]

Dystrophin expression

Muscular biopsies were performed in 45 patients, in whom no mutations were found in DMD. These patients were diagnosed as DMD/BMD on the basis of a muscle biopsy, which confirmed the lack of dystrophin expression. Thirty-six DMD patients showed no reaction for the domains with amino acids 3,200-3,684 [Figure 3]. Only three BMD patients had large gaps or punctuated discontinuous immunostaining for dystrophin in the sarcolemma of muscle fibers. Six out of the 45 muscle biopsies showed the presence of other muscle diseases. One showed a severe denervation, compatible with the diagnosis of spinal muscular atrophy (SMA); another had alterations in the muscular fibers that were compatible with congenital myopathy; and four had changes that were compatible with muscular dystrophy, but had normal immunohistochemistry. These were diagnosed under unclassified limb-girdle muscular dystrophy.
Figure 3: (a) Hematoxylin and eosin (HE) staining of normal control sample. (b) Hematoxylin and eosin (HE) staining of 5-year-old patient with DMD with evident extensive necrotic and fibrotic areas. Both are shown at a magnification of ×300. (c) Immunohistochemistry of sections of snap-frozen quadriceps tissue obtained from a normal control. Muscle fibers stained positive by the dystrophin antibody. (d) Immunohistochemistry of sections of snap-frozen quadriceps tissue obtained from 5-year-old patient. Muscle fibers showed no specific immunoreaction

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CPK levels of carriers

The mean CPK value in 201 female relatives was 287.4 ± 432.5 U/L (range, 39-2,856 U/L); in 98 mothers and sisters, the values were above the upper limit of normal (200 U/L). In the 98 first-degree relatives with high CPK values, only one was excluded as a DMD carrier by microsatellite analysis; 31 carriers were found in the other 103 first-degree relatives with normal CPK levels.


 » Discussion Top


The spectrum of clinical severity in the different muscular dystrophies is very broad. To establish the mutation spectrum for the gross arrangements of DMD and BMD in China, we screened 173 patients with DMD and BMD; these diseases were excluded in six patients. The data provides invaluable information for both clinical medicine and basic science. Overall, 75.8% carriers were found to have abnormal CPK levels. Carrier state assessment is therefore essential for appropriate genetic counseling and prenatal diagnosis. The study also provides new insights into the frequency of BMD, which supports a higher birth incidence and frequency of BMD than previously thought. [14],[15],[16]

Over the last 10 years, further analysis of both the dystrophin gene and protein has enhanced the diagnosis of DMD/BMD. Multiplex PCR has been used for a long period of time as the mutation screening method of choice to assess the deletion of selected exons in DMD. [17],[18],[19],[ 20] Our study proved that a combination of MLPA with sequencing of the DMD gene, which could examine all 79 exons for deletion or duplication mutations, enabled a mutation detection rate of 70% in Chinese DMD/BMD cases. In addition, MLPA has been shown to detect mutations in close proximity to the ligation site of the MLPA probe, which has improved the detection rate of duplications of the DMD gene that have long been under-detected in patients. [21],[22]

In China, MLPA identified a higher incidence of deletions (61.7%) and a lower incidence of duplications (13.8%) in this cohort of 167 DMD/BMD patients compared with 36% and 24.7%, respectively, in a study from Taiwan. [23] Other reports have shown a deletion rate of 60%, but a duplication rate of only 5-10%. [24],[25],[26],[27] The distribution of the exon deletions into two 'hot-spot' regions (exons 3-19 and 45-55) is similar with the published literature. [28] However, most of the duplications were of exons 2-18, in contrast to data published by Petr et al., who reported that a duplication of exons 25-55 had the highest frequency. [29] Although differences in the ethnicity of patients should be taken into account, variability in the number of tested exons throughout the study could also affect the diagnostic results.

Patients without detectable deletions or duplications should have muscle biopsy to corroborated the absence of dystrophin by immunolabeling with antibodies to dystrophin. [30] Such cases had altered muscle fibers, including severe denervation, congenital myopathy, unclassified limb-girdle muscular dystrophy and dystrophin or sarcoglycan deficiency. Thus, immunohistochemistry of muscle biopsies remains the gold standard for the diagnosis of DMD/BMD, but it is a more invasive procedure. [31] It should therefore be used in cases when the patient symptoms, family history and clinical findings suggest DMD or BMD, but molecular analysis fails to find any mutations that are specific to the DMD gene.


 » Acknowledgment Top


This study was supported by the Doctoral Program Foundation of Liaoning Province (20111101).

 
 » References Top

1.Almomani R, van der Stoep N, Bakker E, den Dunnen JT, Breuning MH, Ginjaar IB. Rapid and cost effective detection of small mutations in the DMD gene by high resolution melting curve analysis. Neuromuscul Disord 2009;19:383-90.  Back to cited text no. 1
[PUBMED]  [FULLTEXT]  
2.Sura T, Eu-ahsunthornwattana J, Pingsuthiwong S, Busabaratana M. Sensitivity and frequencies of dystrophin gene mutations in Thai DMD/BMD patients as detected by multiplex PCR. Dis Markers 2008;25:115-21.  Back to cited text no. 2
[PUBMED]  [FULLTEXT]  
3.Del Gaudio D, Yang Y, Boggs BA, Schmitt ES, Lee JA, Sahoo T, et al. Molecular diagnosis of Duchenne/Becker muscular dystrophy: Enhanced detection of dystrophin gene rearrangements by oligonucleotide array-comparative genomic hybridization. Hum Mutat 2008;29:1100-7.  Back to cited text no. 3
[PUBMED]  [FULLTEXT]  
4.Carsana A, Frisso G, Intrieri M, Tremolaterra MR, Savarese G, Scapagnini G, et al. A 15-year molecular analysis of DMD/BMD: Genetic features in a large cohort. Front Biosci (Elite Ed) 2010;2:547-58.  Back to cited text no. 4
[PUBMED]    
5.Hoffman EP, Dressman D. Molecular pathophysiology and targeted therapeutics for muscular dystrophy. Trends Pharmacol Sci 2001;22:465-70.  Back to cited text no. 5
[PUBMED]  [FULLTEXT]  
6.Nowak KJ, Davies KE. Duchenne muscular dystrophy and dystrophin: Pathogenesis and opportunities for treatment. EMBO Rep 2004;5:872-6.  Back to cited text no. 6
    
7.Wang Q, Li-Ling J, Lin C, Wu Y, Sun K, Ma H, et al. Characteristics of dystrophin gene mutations among Chinese patients as revealed by multiplex ligation-dependent probe amplification. Genet Test Mol Biomarkers 2009;13:23-30.  Back to cited text no. 7
[PUBMED]    
8.Ferreiro V, Szijan I, Giliberto F. Detection of germline mosaicism in two Duchenne muscular dystrophy families using polymorphic dinucleotide (CA)n repeat loci within the dystrophin gene. Mol Diagn 2004;8:115-21.  Back to cited text no. 8
[PUBMED]  [FULLTEXT]  
9.Hoffman EP, Pegoraro E, Scacheri P, Burns RG, Taber JW, Weiss L, et al. Genetic counseling of isolated carriers of Duchenne muscular dystrophy. Am J Med Genet 1996;63:573-80.  Back to cited text no. 9
[PUBMED]    
10.Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: One gene, several proteins, multiple phenotypes. Lancet Neurol 2003;2:731-40.  Back to cited text no. 10
[PUBMED]  [FULLTEXT]  
11.Neri M, Torelli S, Brown S, Ugo I, Sabatelli P, Merlini L, et al. Dystrophin levels as low as 30% are sufficient to avoid muscular dystrophy in the human. Neuromuscul Disord 2007;17:913-8.  Back to cited text no. 11
[PUBMED]  [FULLTEXT]  
12.Miranda AF, Francke U, Bonilla E, Martucci G, Schmidt B, Salviati G, et al. Dystrophin immunocytochemistry in muscle culture: Detection of a carrier of Duchenne muscular dystrophy. Am J Med Genet 1989;32:268-73.  Back to cited text no. 12
[PUBMED]    
13.Ferreiro V, Giliberto F, Francipane L, Szijan I. The role of polymorphic short tandem (CA)n repeat loci segregation analysis in the detection of Duchenne muscular dystrophy carriers and prenatal diagnosis. Mol Diagn 2005;9:67-80.  Back to cited text no. 13
[PUBMED]  [FULLTEXT]  
14.Mostacciuolo ML, Miorin M, Pegoraro E, Fanin M, Schiavon F, Vitiello L, et al. Reappraisal of the incidence rate of Duchenne and Becker muscular dystrophies on the basis of molecular diagnosis. Neuroepidemiology 1993;12:326-30.  Back to cited text no. 14
[PUBMED]    
15.Bushby KM, Thambyayah M, Gardner-Medwin D. Prevalence and incidence of Becker muscular dystrophy. Lancet 1991;337:1022-4.  Back to cited text no. 15
[PUBMED]  [FULLTEXT]  
16.Peterlin B, Zidar J, Meznaric-Petrusa M, Zupancic N. Genetic epidemiology of Duchenne and Becker muscular dystrophy in Slovenia. Clin Genet 1997;51:94-7.  Back to cited text no. 16
[PUBMED]    
17.Gonzalez-Herrera L, Gamas-Trujillo PA, Garcia-Escalante MG, Castillo-Zapata I, Pinto-Escalante D. Identifying deletions in the dystrophin gene and detecting carriers in families with Duchenne's/Becker's muscular dystrophy. Rev Neurol 2009;48:66-70.  Back to cited text no. 17
    
18.Park Y, Kim J, Choi JR, Song J, Chung JS, Lee KA. Evaluation of multiplex PCR assay using dual priming oligonucleotide system for detection mutation in the Duchenne muscular dystrophy gene. Korean J Lab Med 2008;28:386-91.  Back to cited text no. 18
[PUBMED]  [FULLTEXT]  
19.Bellayou H, Hamzi K, Rafai MA, Karkouri M, Slassi I, Azeddoug H, et al. Duchenne and Becker muscular dystrophy: Contribution of a molecular and immunohistochemical analysis in diagnosis in Morocco. J Biomed Biotechnol 2009;2009:325210.  Back to cited text no. 19
[PUBMED]  [FULLTEXT]  
20.Piko H, Vancso V, Nagy B, Ban Z, Herczegfalvi A, Karcagi V. Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families - detection of carrier status in symptomatic and asymptomatic female relatives. Neuromuscul Disord 2009;19:108-12.  Back to cited text no. 20
    
21.Gatta V, Scarciolla O, Gaspari AR, Palka C, De Angelis MV, Di Muzio A, et al. Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Genet 2005;117:92-8.  Back to cited text no. 21
[PUBMED]  [FULLTEXT]  
22.Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics 2005;6:29-35.  Back to cited text no. 22
[PUBMED]  [FULLTEXT]  
23.Hwa HL, Chang YY, Chen CH, Kao YS, Jong YJ, Chao MC, et al. Multiplex ligation-dependent probe amplification identification of deletions and duplications of the Duchenne muscular dystrophy gene in Taiwanese subjects. J Formos Med Assoc 2007;106:339-46.  Back to cited text no. 23
[PUBMED]  [FULLTEXT]  
24.White S, Kalf M, Liu Q, Villerius M, Engelsma D, Kriek M, et al. Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am J Hum Genet 2002;71:365-74.  Back to cited text no. 24
[PUBMED]  [FULLTEXT]  
25.Lalic T, Vossen RH, Coffa J, Schouten JP, Guc-Scekic M, Radivojevic D, et al. Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 2005;13:1231-4.  Back to cited text no. 25
[PUBMED]  [FULLTEXT]  
26.Schwartz M, Duno M. Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method. Genet Test 2004;8:361-7.  Back to cited text no. 26
    
27.Zeng F, Ren ZR, Huang SZ, Kalf M, Mommersteeg M, Smit M, et al. Array-MLPA: Comprehensive detection of deletions and duplications and its application to DMD patients. Hum Mutat 2008;29:190-7.  Back to cited text no. 27
[PUBMED]  [FULLTEXT]  
28.Hallwirth Pillay KD, Bill PL, Madurai S, Mubaiwa L, Rapiti P. Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal. J Neurol Sci 2007;252:1-3.  Back to cited text no. 28
[PUBMED]  [FULLTEXT]  
29.Brabec P, Vondracek P, Klimes D, Baumeister S, Lochmuller H, Pavlik T, et al. Characterization of the DMD/BMD patient population in Czech Republic and Slovakia using an innovative registry approach. Neuromuscul Disord 2009;19:250-4.  Back to cited text no. 29
    
30.Muntoni F. Is a muscle biopsy in Duchenne dystrophy really necessary? Neurology 2001;57:574-5.  Back to cited text no. 30
[PUBMED]  [FULLTEXT]  
31.Freund AA, Scola RH, Arndt RC, Lorenzoni PJ, Kay CK, Werneck LC. Duchenne and Becker muscular dystrophy: A molecular and immunohistochemical approach. Arq Neuropsiquiatr 2007;65:73-6.  Back to cited text no. 31
[PUBMED]  [FULLTEXT]  


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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