Here we described patients with a history of DMD or BMD diagnosed by genetic examination and a total of 48 individuals derived from 20 families were included in the study. We analyzed the clinical manifestations, serum biochemical tests of 20 patients, and also detected gene mutations of 20 probands together with their parents and siblings. We found that the high prevalence of the first consultation among patients is in the age range of 4-7-year-old individuals with progressive irreversible muscle weakness, gastrocnemius muscle pseudohypertrophy, and elevated serum CK complicated with a rise of AST and ALT serum concentration. However, using liver and myocardium sensitive biochemical indicators, such as transaminase enzyme and myocardial enzyme, might lead to misdiagnosed as myocarditis, rhabdomyolysis, and hepatocellular injury11. Because of the diverse clinical manifestations, insufficient specificity, and difficulties in diagnosis of progressive muscular dystrophy (PMD), thus, we need to diagnose the disease with the help of EMG, muscle biopsy, and genetic testing to reduce the rate of clinical misdiagnosis. In our study, the clinical manifestations of patients conformed to the natural course of BMD/DMD, and several patients had the family history with the typical gastrocnemius muscle pseudohypertrophy, reduction or loss of tendon reflexes, positive Gower sign with the myogenic damage and abnormal echocardiography. All children’s genetic testing results finally confirmed the clinical diagnosis.
4.1 Pathogenesis studies
Full-length dystrophin is a large rod-shaped protein in association with the formation of DGC which connects the subsarcolemmal F-actin cytoskeleton to the basal lamina of the extracellular matrix, stabilizing membrane during repeated cycles of contraction and relaxation12, 13. Besides, The DGC also has an essential role in cell signaling apart from mechanical function. The dysfunction of dystrophin protein, which plays a crucial role in structural and functional maintenance, leads to the permeabilization of the cell membrane of skeletal muscle, the loss of sarcolemmal integrity, the leakage of intracellular creatine kinase, the degeneration and necrosis of muscle tissue, and the proliferation of adipose and fibrous tissue14–16 which is consistent with the clinical manifestations (progressive muscle damage and weakness, gastrocnemius muscle pseudohypertrophy, decreased muscle ton) and laboratory examination (elevated serum CK) results in our patients. Besides, the protein also expresses in the central nervous system and localizes to postsynaptic neurons in the cerebral cortex and hippocampus which are critical for memory and cognitive functioning17, 18. Dystrophin colocalizes with GABAA receptors subunit clusters and it plays an essential role in the stabilization of GABAA receptors in the subset of central inhibitory synapses19. According to recent reports, no severe abnormality has been reported in the brain of DMD patients. However, detailed microscope results indicate that abnormal dendritic development and arborization may contribute to intellectual impairment20. We can find mental retardation, movement disorders, language disorders, and panic attacks in some patients in our study which may be related to the abnormal organization of the structures and functions in the brain.
4.2 Molecular studies
As the largest gene in humans, DMD gene spans 2.5 million bases and consists of 79 exons, and a variety of mutations occur within the DMD gene. The large rearrangement including deletions and duplications were detected in 15 patients (11 deletions and 4 duplications), and point mutations were found in 5 patients including nonsense, frameshifts, and splice mutations. The result was consistent with the early reports that large deletions are the most common mutations in the DMD gene accounting for 55–65% and duplications represent up to 5–15%, while, point mutations account for 30%21, 22. There are two hot spots in DMD gene deletion mutations, exons 1–22 located in the proximal region, and exons 43–55 in the distal region 23–25. Deletions found in 11 patients in our study having its first and last exon within the proximal and distal hot spot made up 27% and 64% of the total deletions. Further, point mutations were identified in our patients using NGS and verified by Sanger Sequencing. This study reported five mutations (c.354G > A, c.6913-1G > T, c.2702delG, c.8672delT, c.9014dupT) detected in patients with a premature stop code in mRNA that eventually led to the production of truncated proteins.
A nonsense mutation was identified in the patient, c.354G > A, which was not found in the DMD gene of patient’s father. The variant was predicted to lead to amino acid deletion p.Trp118Ter. The pathogenicity of this mutation has been reported in the literature and is related to DMD26. The splice sites point mutation, c.6913-1G > T, was identified in the DMD gene of our patient. The pathogenicity of this mutation has not been reported in the literature, but the pathogenicity of the mutation c.6913-1G > A in the same position as the mutation has been reported in the literature and is related to myopathy27. A nucleotide variation of c.2702delG was found in the DMD gene of the subject, resulting in translation termination of amino acids (p.Gly901AspfsTer48). This variation is classified as “likely pathogenic” according to rules for combining criteria to classify sequence variants published by the American College of Medical Genetics and Genomics (ACMG)28. The subject's father showed no abnormalities at this site, while his mother and her sister were heterozygous. The frameshift mutation which led to the No. 8672 nucleotide T deletion in coding region and caused to the production of truncated proteins (p.Leu2891ArgfsTer28) was identified in the patient and his mother was heterozygous. We also found another frameshift mutation of the DMD gene (c.9014dupT, p.Leu3005PhefsTer9) among the point mutations. The evidence of pathogenicity of this mutation was weighted as very strong (PVS1).
To our best knowledge, it is the first time that the four-point mutations (c.6913-1G > T, c.2702delG, c.8672delT, c.9014dupT) were reported and linked to DMD/BMD, which expand knowledge of the DMD variant spectrum. The carriers were proved to be DMD/BMD through pathological examination and clinical follow-up.
4.3 Current and future therapies
To date, no curative therapies are available for BMD or DMD patients. Corticosteroids, which was first suggested by Drachman29, is the gold standard therapy to improve children’s muscle strength and slow down disease progression. Some new therapies are emerging as a future method to systematically treat dystrophin deficiency, such as using stem cells as the alternative approach for muscle regeneration, gene therapy for restoring the dystrophin protein, stop-codon read-through for expressing a full-length functional dystrophin protein, utrophin up-regulation to replace the mutated dystrophin and splicing therapy for skipping exons with mutations. Here, another emerging treatment for the disease focus on the receptor P2RX7. P2RX7 is an ATP-gated ion channel permeable to small cations, including Ca2+, and its expression and function are up-regulated by ATP which would release when tissue damage occurs. In DMD, due to the dysfunction of dystrophin protein, the cell membrane of skeletal muscle is damaged and the ATP in very large amounts is released into the extracellular space, creating an environment consistent with increased activation of P2RX730. Over-activation of P2RX7 could contribute to damage both directly by causing the death of dystrophic muscles and indirectly by stimulating harmful inflammatory responses. Recent research has found that selective ablation or blockade of P2RX7 ameliorates the MDX dystrophic process both short and long term and does not cause detectable side effects in this DMD mouse model31. Therefore, the single treatment that takes P2RX7 as the therapeutic target for improving muscle function both short and long term, and also rescues cognitive impairment and bone loss could potentially be purposed for this lethal disease.