Background
Duchenne muscular dystrophy (DMD, OMIM: 310200), the most common X-linked recessive inherited muscle disease, affects approximately 1 in 3600–6000 live male births [
1‐
3]. The age of diagnosis is approximately 5 years when early symptoms occur [
4]. Affected children rely on wheelchairs approximately 12 years of age with progressive muscle weakness, and they often die of respiratory or cardiac failure around the second decade of life. Compared with DMD, BMD (OMIM: 300376) is milder, with later symptom occurrence, slower disease progression, and fewer effects on survival; however, it results in decreased quality of life. DMD is caused by structural and functional changes of dystrophin induced by mutations of the
DMD gene (OMIM: 300377), which is located on Xp21.1 and represents the largest known gene in humans. The
DMD gene spans approximately 2.4 Mb of genomic DNA and contains 79 exons and 78 introns, generating a 14 kb mRNA transcript, which may explain its high mutation rate [
5]. Approximately 60–70% of DMD/BMD cases are caused by deletions or duplications of one or more exons in the
DMD gene. In this study, we analysed the genetic mutations of 1051 unrelated Chinese DMD/BMD families, clarified the distribution characteristics of
DMD gene mutations in the Chinese Han population, and explored the detection strategy of
DMD gene mutations. In clinical practice, MLPA has been widely used to detect such mutations. The remaining 25–35% of small mutations, including missense, splice site, nonsense, and frameshift mutations, require Sanger sequencing or NGS for diagnosis. Because of its high throughput and low cost, which compensates for the deficiency of Sanger sequencing, NGS has prominent advantages in detecting
DMD gene mutations [
6].
Discussion
The diagnosis of
DMD gene mutations can support its treatment. In this study, we analysed 1051 Chinese families with DMD/BMD; 1029 (97.91%) patients were identified to have genetic mutations, which should be considered as the largest
DMD gene mutation report in China. Among these families, 740 (70.41%) probands were large deletions, which occupied the most mutation proportions, and 87 (8.28%) were duplications, which corroborated the results of the Leiden database (
http://www.dmd.nl/) (large deletions, 72%; large duplications, 8%) and TREAT-NMD
DMD Global Database [
7] (large deletions, 68%; large duplications, 11%). According to the
DMD genomic structure, it is possible to treat DMD by restoring the ORF of an out-of frame deletion by splicing out the exon. Eteplirsen, developed by Sarepta to skip exon 51, was recently granted accelerated approval by the Food and Drug Administration (FDA). Based on our results of exon deletions, we can easily obtain a clear message about its applicability (Table
4).
De novo mutations cannot be ignored when performing genetic counselling. In this study, a de novo mutation rate of 39.45% (187/474) was obtained. The de novo mutation rate for large deletions (49.53%, 157/317) was highest compared with other mutations, corroborating a study on prenatal diagnosis in 131 Chinese families with
DMD/BMD in 2017 [
8] (51.1% of probands with large
DMD gene deletions had de novo mutations). The mechanism of de novo mutations is not yet fully understood, but germline mosaicism could be one possible reason. Therefore, an effective and expeditious diagnosis method and a systematic pedigree analysis are necessary for genetic counselling of DMD.
MLPA is one of the most widely used methods. It can accurately and rapidly detect large deletion and duplication mutations of the DMD gene. The main limitation of MLPA is its inability to detect non-deletion and non-duplication mutations. Gene deletion or duplication is analysed by MLPA based on probe amplification, but the probe cannot be combined with DNA with small mutations, resulting in loss of amplification and yielding false-positive results. NGS can detect all mutation types and has the advantages of high throughput, short time and abundant data. However, the cost is higher when facing exon deletions/duplications of the DMD gene. Therefore, the combination of MLPA and NGS is the most economical and efficient method for diagnosis. In our study, the patient’s MLPA data were first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing were then applied to exclude MLPA-negative samples. Meanwhile, PCR was applied for detection of single exon deletions to exclude false-positives in MLPA caused by point mutations.
DMD is a serious X-linked, recessive, inherited, fatal disease but often shows mild symptoms prior to the age of 5. Therefore, the diagnosis of female
DMD mutation carriers and children is considered very important. The majority of female
DMD mutation carriers have no significant clinical signs. Symptomatic female
DMD carriers show symptoms in childhood. In recent years, multiple studies have explored the possible pathogenetic mechanisms of symptomatic DMD in female carriers, including skewed X-inactivation [
9], X/autosomal translocation [
10], germline mosaicism [
11], uniparental disomy in the X chromosome [
12], and Turner syndrome with
DMD [
13], with the daughter having female skewed X chromosome inactivation. Overall, 29 DMD female patients were involved in this study, but no heredity predisposition to skewed X chromosome inactivation was found. Therefore, we believe that skewed X chromosome inactivation is likely to occur randomly. In addition, 92.65% (63/68) of asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia enrolled in this study were diagnosed with
DMD mutations. Therefore, serum CK screening for newborns is an effective screening method to identify suspicious patients.
DMD is one of the largest human genes and has several mutation types, including large fragment deletions or duplications (≥1 exon) and small mutations. Therefore, it is difficult to unify the clinical diagnosis methods of DMD/BMD patients. At present, the methods of genetic testing for the
DMD gene include PCR amplification, multiplex PCR, Sanger sequencing, real-time PCR, MAPH, MLPA, and NGS [
14,
15].
As for small mutations, there were no differences between our results and those of Mariko Okubo et al. [
16] in Japan, who demonstrated that there are no racial differences between
DMD mutations. Unlike the hotspot of exon deletions/duplications, there were no mutation hotspots, which means individualized treatment strategies are needed.
In this study, mutations could not be detected in 22 families, which may be due to rearrangements in introns or the 3’or 5′ untranslated regions (UTRs). Further consideration should be given to whole genome sequencing and muscular biopsy, and possible clinical symptoms caused by other neuromuscular diseases cannot be ruled out.
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