Identification of a novel DMD duplication identified by a combination of MLPA and targeted exome sequencing
- Beibei Wu†1,
- Liying Wang†2,
- Ting Dong1,
- Jiahui Jin1,
- Yili Lu1,
- Huiping Wu1,
- Yue Luo1Email author and
- Xiaoou Shan1Email author
© The Author(s). 2017
Received: 1 December 2016
Accepted: 4 January 2017
Published: 23 March 2017
Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle-wasting disease caused by a mutation in the DMD gene. The aim of this study was to identify a de novo mutation of the DMD gene in the family of a 9-month-old Chinese male patient, as well as to describe the phenotypic characteristics of this patient.
The patient was suspected to suffer from DMD according to physical examination, biochemical analyses, and electromyogram. We identified a duplication of exons 4–42 in DMD gene with targeted exome sequencing and multiplex ligation-dependent probe amplification (MLPA). In addition, the patient’s mother was a carrier of the same mutation.
We identified a de novo duplication of exons 4–42 in a patient with early stage DMD. The discovery of this mutation may provide insights into future investigations.
Duchenne muscular dystrophy (DMD), an X-linked recessive muscle-wasting disease, is caused by a mutation in the DMD gene that encodes the dystrophin protein [1, 2]. As the most common muscle disease in children, the incidence of DMD is 1 in 3500 live male births . DMD is a progressive neuromuscular disease characterized by pseudohypertrophy in the calf muscle and the Gowers’ sign . Patients with DMD are usually first diagnosed before 5 years of age and many pass away due to respiratory or cardiac failure at around 20 years of age [5, 6].
DMD is the largest known human gene encompassing 2.2 Mb of nucleotides that contain 79 exons . The large size of the gene increases its susceptibility to mutations as evidenced by the one-third of de novo mutations identified in DMD . Furthermore, intragenic deletions represent 65–70% of all mutations, while duplications are found in 7% of patients and the remaining mutations are either point mutations or small deletions/insertions .
Diagnosis of the DMD mainly depends on the genetics of the patient . Recent studies showed that targeted exome sequencing is a powerful tool for diagnosis of Mendelian diseases , This is especially true for patients who are at the early stage of a disease and show no obvious clinical manifestation. Targeted exome sequencing can screen for mutations in a panel of many genes that are implicated in various inherited diseases, making it a tremendously useful technique for identification of disease associated mutations.
However, targeted exome sequencing is relatively less powerful in testing large intragenic rearrangements. In the case of DMD, deletions found in most patients are followed by duplications, therefore, multiplex PCR, which can detect up to 98% of deletion, was considered as the standard diagnostic method . Nevertheless, multiplex PCR is not the ideal technique for identifying duplications and female carriers due to the presence of a normal copy of the DMD gene . Fortunately, the introduction of a dosimetry method based on multiplex ligation-dependent probe amplification (MLPA) has improved the discovery of large intragenic rearrangements .
In this study, we implemented a combination of MLPA with targeted exome sequencing to comprehensively screen for large fragment duplications in the DMD gene in a 9-month-old Chinese male patient. Using this method, we were able to identify a novel duplication of exons 4–42 in the DMD gene.
This study was in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. A 9-month-old male patient was referred to our hospital in 2015. Written informed consent was obtained from his statutory guardian. Patients’ initial symptoms and complaints was diarrhea. The patient received a comprehensive examination including physical examination, biochemical analyses and electromyogram. Detailed family history was obtained from the patient, and peripheral blood samples were collected.
MLPA analysis was performed using SALSA MLPA kit P034/P035 (MRC-Holland, Amsterdam, the Netherlands) kit according to manufacturer’s instructions. The MLPA samples consisted of approximately 100 ng of genomic DNA. Denaturation, hybridization, ligation and amplification were performed with ABI 2720 PCR thermal cycler. The PCR amplification was performed under the following conditions: 33 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 60 and a final extension step at 72 °C for 20 min to allow for adequate probe hybridization with the two sets of probes. Results were analyzed using Coffalyser 9.4 (MRC Holland).
Targeted exome sequencing
Genomic DNA was extracted from blood samples using a DNA Extraction kit (TIANGEN, Beijing) according to the manufacturer’s instructions. The coding exons of 131 genes related to inherited diseases were selected and captured with a GenCap custom enrichment kit (MyGenostics, Beijing) as previously described . Genomic DNA was fragmented and mixed with GenCap probe (MyGenostics, Beijing) for PCR amplification and hybridization. After washing with MyOne beads (Life Technology), the sample was resuspended in binding buffer, transferred along with MyOne beads, and rotated for 1 h. Then, DNA was eluted with Buffer Elute, followed by amplification in post-capture. The enriched libraries were sequenced on a IlluminaSolexaHiSeq 2000 sequencer. After removing PCR duplicates with Picard program, the clean reads were aligned with SOAP aligner program  according to human genome parameters (hg19). Then, we identified SNPs using the SOAPsnp program, realigned the reads by BWA, and determined the deletions or insertions (InDels) with the GATK software . After annotation of the identified InDels and SNPs using the Exome-assistant program (http://122.228.158106/exomeassistant), candidate SNPs, InDels and the short read alignments were viewed by Magic Viewer  To determine pathogenicity, four algorithms, PolyPhen, SIFT, Mutationtaster and PMut were used to evaluated nonsynonymous variant. Sequencing data were deposited in to NIH Short Read Archive (SRP033329).
Results of patient biochemical test
Results of patient electromyogram
maxium average unite width
Left rectus femoris
Right rectus femoris
In this study, we described a DMD family with a de novo duplication of exons 4–42, first suggested by targeted exome sequencing and subsequently detected by MLPA analysis. The patient was a 9-month-old male that did not show any typical characteristics of DMD, such as calf muscle pseudohypertrophy, Gowers’ sign. Biochemistry tests showed sharply increased CPK and electromyogram suggested myogenic damage, which led us to speculate that the patient suffers from DMD. However, clinical evidence was not sufficient for a formal diagnosis of DMD. In addition, increased level of ALT, AST and LDH suggested liver damage. Therefore, differential diagnosis of diseases that could lead to liver damage, such as viral hepatitis and Wilson’s disease, should be investigated. We, therefore, performed a viral hepatitis serology screen and tested for ceruloplasmin in serum of the patient. The results of viral hepatitis serology screen were negative, which ruled out viral hepatitis. In addition, the level of ceruloplasmin was normal, which did not support a diagnosis of Wilson’s disease.
Genetic testing is the mainstay of diagnosis for DMD . Despite that MLPA is now the most widely used technique for identifying duplication in DMD , targeted exome sequencing is prefered when evidence for a diagnosis of DMD is insufficient and when other metabolic diseases have not been ruled out. Furthermore, targeted exome sequencing is capable of detecting subtle mutations in the 79 exons that cannot be detected by MPLA . In our patient, results from targeted exome sequencing were suggestive of a potential duplication in the DMD gene. We then confirmed this finding using MLPA. A combination of MLPA with targeted exome sequencing can therefore, be a valuable method for identifying DMD gene defects.
Dystrophin, as encoded by the DMD gene, is predominantly expressed in skeletal and cardiac muscles . As a significant component of the dystrophin-glycoprotein complex (DGC), dystrophin, plays a key role in the contraction of sarcomeres . Dystrophin is a structural protein of 427-kD, consisting of a N-terminal actin binding domain (ABD1), a central rod region, a cysteine-rich (CR) globular domain, and a C-terminal tail (CT) . The C-terminus is associated with glycoproteins and the N-terminus is associated with actin or actin-like proteins . Previous study suggested that dystrophinis is a cytoskeletal protein that is potentially involved in linking the extracellular matrix to the interior of muscle fibers. Mutations in the DMD gene, therefore, may disrupt such linkage and ultimately lead to DMD as a result of sarcolemma instability . As the disease progresses, DMD patient may lose the ability to walk without assistance and may eventually die from respiratory or cardiac failure as a result of relative muscle damage .
The only pharmacologic agent proven to be effective for treating DMD is corticosteroids. Long-term studies indicated that long-term daily administration of corticosteroids reduces the decline of cardiorespiratory function, lowers the risk of progressive scoliosis, prolongs ambulation by up to 3 years, and improves life expectancy of DMD patients [15–18]. In addition, novel therapies for DMD, such as stem cell therapy, gene therapy (e.g. viral vectors), and dystrophin restoration approach, have recently been developed . In this study, we demonstrated that a combination of targeted exome sequencing and MLPA is a valuable diagnostic method for detecting DMD at the early stage of the disease, which is critical for early intervention and can ultimately lead to better prognosis of DMD patients. If a patient with rapid increase of CPK, even though does not show any typical characteristics of DMD, such as calf muscle pseudohypertrophy, Gowers’ sign, we should suspect DMD and consider to perform Targeted exome sequencing and MLPA.
In summary, our study identified a de novo duplication of exons 4–42 in the DMD gene in a 9-month-old male Chinese patient using a combination of targeted exome sequencing and MLPA. The discovery of this novel mutation may provide important insights for future investigations.
The authors appreciate the participation of the patient and his family in the present study. The present study was supported by the Zhejiang Provincial Natural Science Foundation of China (no. Y2100530, awarded to X.S.).
The study was part of a project funded by the Zhejiang Provincial Natural Science Foundation of China (no. Y2100530, awarded to X.S.).
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
BBW and LYW analysed the results and wrote the paper; TD, JHJ, YLL, HPW collected clinical data and perform the experiments; YL and XOS revised the paper. All authors reviewed and approved the submitted version of the manuscript.
The authors declare that they have no competing interest with the contents of this article.
Consent to participate for publication
Written informed consent was obtained from the parents of the patient for publication of this paper. A copy of the written consent form is available.
Ethics approval and consent to participate
This study was in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. Written informed consent was obtained.
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