Duchenne or Becker muscular dystrophy: A clinical, genetic and immunohistochemical study in China
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
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. , 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. , The DMD gene, with 79 exons spanning approximately 2.6 million base pairs, is the largest yet identified in the human genome. , 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%).  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.  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.
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. ,, 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).
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.
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 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. 
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. 
The specific clinical findings of the 167 affected boys are presented in [Table 1].
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].
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. 
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.
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.
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. ,,
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. ,,,[ 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. ,
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.  Other reports have shown a deletion rate of 60%, but a duplication rate of only 5-10%. ,,, The distribution of the exon deletions into two 'hot-spot' regions (exons 3-19 and 45-55) is similar with the published literature.  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.  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.  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.  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.
This study was supported by the Doctoral Program Foundation of Liaoning Province (20111101).
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]