When a mid-intronic variation of DMD gene creates an ESE site

Duchenne and Becker muscular dystrophy are X-linked allelic disorders caused by mutations in the DMD gene. The majority (65%) of these mutations are intragenic deletions/duplications that often lead to frameshift errors. Among the remaining ones, we find the mid-intronic mutations that usually create cryptic exons by activating potential splice sites. In this report, we identified, in a Becker muscular dystrophy patient, a mid-intronic variation that creates two ESE sites in intron 26 of DMD gene resulting in the insertion of a new cryptic exon in mRNA. Despite the out of frame character of this mutation, we observed the production of a reduced amount of full-size dystrophin which could be explained by the alternation between normal and altered splicing of dystrophin mRNA in this patient.To our knowledge, this is the first case report describing this novel pathogenic mechanism of mid-intronic variations of DMD gene.     

Coinheritance of Noonan syndrome and Becker muscular dystrophy

We describe for the first time a case of a 9-year old boy with co-existence of dystrophinopathy and Noonan syndrome (NS). Although the patient has a severe muscular clinical phenotype, consistent with Duchenne muscular dystrophy (DMD), the diagnosis of Becker muscular dystrophy (BMD) was proposed based on family history (brother with BMD) and confirmed by muscle immunohistochemistry, and molecular study shown an in-frame DMD gene mutation. The patient also fulfilled the clinical criteria of NS and he harbors a hotspot mutation on PTPN11 gene. This genetic combination may be an explanation for the variability of clinical expression in the family.     

Combining genetics,neuropsychology and neuroimaging to improve understanding of brain involvement in Duchenne muscular dystrophy - a narrative review

Duchenne muscular dystrophy is a multifactorial disease including a cognitive phenotype. It is caused by mutations in the X-chromosomal DMD gene from which dystrophin is synthesized. Multiple isoforms of dystrophin have been identified. The full length dystrophin isoform Dp427_m is expressed predominantly in muscle. Other isoforms include: Dp427_c, Dp427_p, Dp260, Dp140, Dp116, Dp71 and Dp40. The majority of these isoforms are expressed in brain and several hypotheses exist on their role in subtypes of neurons and astrocytes. However, their function in relation to cognition remains unclear. Unlike progressive muscle wasting, cognitive involvement is not seen in all DMD patients and the severity varies greatly. To achieve a better understanding of brain involvement in DMD, a multidisciplinary approach is required. Here, we review the latest findings on dystrophin isoform expression in the brain; specific DMD-associated learning and behavioural difficulties; and imaging and spectroscopy findings relating to brain structure, networks, perfusion and metabolism. The main challenge lies in determining links between these different findings. If we can determine which factors play a role in the differentiation between severe and minor cognitive problems in DMD in the near future, we can both provide better advise for the patients and also develop targeted therapeutic interventions.     

Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.     

Neurocognitive profiles in Duchenne muscular dystrophy and gene mutation site

The presence of nonprogressive cognitive impairment is recognized as a common feature in a substantial proportion of patients with Duchenne muscular dystrophy. To investigate the possible role of mutations along the dystrophin gene affecting different brain dystrophin isoforms and specific cognitive profiles, 42 school-age children affected with Duchenne muscular dystrophy, subdivided according to sites of mutations along the dystrophin gene, underwent a battery of tests tapping a wide range of intellectual, linguistic, and neuropsychologic functions. Full-scale intelligence quotient was approximately 1 S.D. below the population average in the whole group of dystrophic children. Patients with Duchenne muscular dystrophy and mutations located in the distal portion of the dystrophin gene (involving the 140-kDa brain protein isoform, called Dp140) were generally more severely affected and expressed different patterns of strengths and impairments, compared with patients with Duchenne muscular dystrophy and mutations located in the proximal portion of the dystrophin gene (not involving Dp140). Patients with Duchenne muscular dystrophy and distal mutations demonstrated specific impairments in visuospatial functions and visual memory (which seemed intact in proximally mutated patients) and greater impairment in syntactic processing.     

Clinical and genetic characterization of manifesting carriers of DMD mutations

Manifesting carriers of DMD gene mutations may present diagnostic challenges, particularly in the absence of a family history of dystrophinopathy. We review the clinical and genetic features in 15 manifesting carriers identified among 860 subjects within the United Dystrophinopathy Project, a large clinical dystrophinopathy cohort whose members undergo comprehensive DMD mutation analysis. We defined manifesting carriers as females with significant weakness, excluding those with only myalgias/cramps. DNA extracted from peripheral blood was used to study X-chromosome inactivation patterns. Among these manifesting carriers, age at symptom onset ranged from 2 to 47 years. Seven had no family history and eight had male relatives with Duchenne muscular dystrophy (DMD). Clinical severity among the manifesting carriers varied from a DMD-like progression to a very mild Becker muscular dystrophy-like phenotype. Eight had exonic deletions or duplications and six had point mutations. One patient had two mutations (an exonic deletion and a splice site mutation), consistent with a heterozygous compound state. The X-chromosome inactivation pattern was skewed toward non-random in four out of seven informative deletions or duplications but was random in all cases with nonsense mutations. We present the results of DMD mutation analysis in this manifesting carrier cohort, including the first example of a presumably compound heterozygous DMD mutation. Our results demonstrate that improved molecular diagnostic methods facilitate the identification of DMD mutations in manifesting carriers, and confirm the heterogeneity of mutational mechanisms as well as the wide spectrum of phenotypes.     

The expression of the distal dystrophin isoforms Dp140 and Dp71 in the human epileptic hippocampus in relation to cognitive functioning

Dystrophin is an important protein within the central nervous system. The absence of dystrophin, characterizing Duchenne muscular dystrophy (DMD), is associated with brain related comorbidities such as neurodevelopmental (e.g., cognitive and behavioural) deficits and epilepsy. Especially mutations in the downstream part of the DMD gene affecting the dystrophin isoforms Dp140 and Dp71 are found to be associated with cognitive deficits. However, the function of Dp140 is currently not well understood and its expression pattern has previously been implicated to be developmentally regulated. Therefore, we evaluated Dp140 and Dp71 expression in human hippocampi in relation to cognitive functioning in patients with drug‐resistant temporal lobe epilepsy (TLE) and post‐mortem controls. Hippocampal samples obtained as part of epilepsy surgery were quantitatively analyzed by Western blot and correlations with neuropsychological test results (i.e., memory and intelligence) were examined. First, we demonstrated that the expression of Dp140 does not appear to differ across different ages throughout adulthood. Second, we identified an inverse correlation between memory loss (i.e., verbal and visual memory), but not intelligence (i.e., neither verbal nor performance), and hippocampal Dp140 expression. Finally, patients with TLE appeared to have similar Dp140 expression levels compared to post‐mortem controls without neurological disease. Dp140 may thus have a function in normal cognitive (i.e., episodic memory) processes.     

Magnetic resonance spectroscopy and molecular studies in ornithine transcarbamylase deficiency novel mutation c.802A>G in exon 8 (p.Met268Val)

Ornithine transcarbamylase (OTC) deficiency, an X-linked, semidominant disorder, is the most common inherited defect in ureagenesis, resulting in hyperammonaemia type II. The OTC gene, localised on chromosome X, has been mapped to band Xp21.1, proximate to the Duchenne muscular dystrophy (DMD) gene. More than 350 different mutations, including missense, nonsense, splice-site changes, small deletions or insertions and gross deletions, have been described so far. Almost all mutations in consensus splicing sites confer a neonatal phenotype. Most mutations in the OTC gene are ‘private’ and are distributed throughout the gene with a paucity of mutation in the sequence encoding the leader peptide (exon 1 and beginning of exon 2) and in exon 7. They have familial origin or occur de novo. Even with sequencing of the entire reading frame and exon/intron boundaries, only about 80% of the mutations are detected in patients with proven OTC deficiency. The remainder probably occur within the introns or in regulatory domains. The authors present a 4-year-old boy with the unreported missense mutation c.802A>G. The nucleotide transition leads to amino acid substitution Met to Val at codon 268 of the OTC protein.     

Duchenne muscular dystrophy caused by a complex rearrangement between intron 43 of the DMD gene and chromosome 4

Deletions/duplications of exons in the DMD gene cause about 70% of all cases of Duchenne muscular dystrophy (DMD). Most remaining mutations are point mutations or small insertion-deletions located mainly in the coding but also in deep intronic regions of the DMD gene. We describe a novel complex rearrangement in a patient affected with DMD that was undetectable using standard molecular diagnostic methods. Analysis of the proband’s mRNA from a muscle biopsy revealed the insertion of an 80 bp cryptic exon from chromosome 4 between exons 43 and 44 of the dystrophin gene. Array comparative genomic hybridization and breakpoint junction sequence analysis indicated this cryptic exon originated from a complex genomic 90 kb insertion of non-coding chromosome 4 into intron 43 of the dystrophin gene. This rearrangement was also detectable in the patient’s mother. The genomic characterization of this novel complex mutation was essential for accurate carrier and genetic counseling of this family and emphasizes the need for comprehensive molecular diagnosis of patients with clinical signs of DMD and clear pathological changes.     

A symptomatic male carrier of Duchenne muscular dystrophy with Klinefelter's syndrome mimicking Becker muscular dystrophy

Duchenne and Becker muscular dystrophy (DMD/BMD) are commonly inherited muscle disorders. We report a 31-year-old male who had muscle symptoms with left-right differences and intellectual disability. He was diagnosed with BMD at age 15 primarily based on muscle biopsy findings. A few years later, DMD gene analysis revealed that he was a heterozygous carrier of a normal copy of the gene and a mutated copy with an exon 45–54 deletion, which is expected to result in an out-of-frame mutation. A karyotype analysis was compatible with XXY Klinefelter's syndrome. The analysis of X-chromosome inactivation (XCI) using his skeletal muscle sample revealed a skewed XCI pattern. This is the first reported case of a symptomatic male carrier of DMD caused by skewed XCI in Klinefelter's syndrome with a genetically proven heterozygous mutation of the DMD gene. The skewed XCI pattern could also explain the left-right differences in skeletal muscle symptoms observed in this patient.     

Large in-frame 5′ deletions in DMD associated with mild Duchenne muscular dystrophy: Two case reports and a review of the literature

Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype. In this study, we describe two individuals with large in-frame 5′ deletions (exon 3–23 and exon 3–28) that remove the majority of the N-terminal region, including part of the actin binding and central rod domains. Both patients had progressive muscle weakness during childhood but are observed to have a relatively mild disease course compared to typical Duchenne. We show that in muscle biopsies from both patients, truncated dystrophin is expressed at the sarcolemma. We have additionally developed a patient-specific fibroblast-derived cell model, which can be inducibly reprogrammed to form myotubes that largely recapitulate biopsy findings for the patient with the exon 3–23 deletion, providing a culture model for future investigation of this unusual case. We discuss these mutations in the context of previously reported 5′ in-frame DMD deletions and relevant animal models, and review the spectrum of phenotypes associated with these deletions.     

Pig models for Duchenne muscular dystrophy – from disease mechanisms to validation of new diagnostic and therapeutic concepts

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.     

Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system

The Duchenne muscular dystrophy (DMD) gene encodes dystrophin, which is a protein defective in DMD patients, as well as a number of shorter isoforms, which have been shown to be expressed in various non-muscle, primarily neural, tissues. As of yet, the physiological function of the various dystrophin isoforms is not fully understood. In the present study, we investigated the neurological phenotype that arises in the DMD-null mice, where expression of all dystrophin isoforms had been disrupted. We demonstrate that vomeronasal axons in the DMD-null mice are defasciculated, and some of the defasciculated vomeronasal axons aberrantly entered into the main olfactory bulb, which indicates that the product(s) of the DMD gene plays an important role in vomeronasal nerve organization. Through western blot and immunofluorescence analyses, we determined that the dystrophin isoform Dp71 was exclusively expressed in the mouse olfactory system: mainly in the olfactory ensheathing cells (OECs), an olfactory system-specific glia cell that ensheaths fascicles of the olfactory nerve. In the OECs, Dp71 was co-localized with β-dystroglycan, utrophin, laminin, and perlecan. Since β-dystroglycan and perlecan expression was decreased in the OECs of DMD-null mice, we hypothesize that Dp71 expressed in the OECs participates in fasciculation of the vomeronasal nerve, most likely through interactions with extracellular matrix.     

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.     

Small mutation screening in the DMD gene by whole exome sequencing of an Argentine Duchenne/Becker muscular dystrophies cohort

Dystrophinopathies are neuromuscular X-linked recessive diseases caused by mutations in the DMD gene. This study aimed to identify DMD gene small mutations by Whole Exome Sequencing (WES), in order to confirm clinical diagnosis, identify candidates for Ataluren treatment and perform carrier status testing. Furthermore, was our goal to characterize the DMD sequence variants and identify ancestral haplotypes. We analyzed 40 non-related individuals (38 affected boys with dystrophinopathy presumptive clinical diagnosis and 2 at-risk women) with negative MLPA results. Pathogenic DMD variants were found in 32 boys. Surprisingly, in another 4 patients with absence/deficiency of dystrophin in muscle biopsy, pathogenic variants were found in Limb-girdle muscular dystrophy genes. Therefore, the WES detection rate resulted ∼94% (36/38). We could identify 15 Ataluren candidates and exclude 2 at-risk women. The characterization of the occurrence and diversity of DMD sequence variants from our cohort and from LOVD database, revealed no hotspots but showed exons/introns unlikely to carry small molecular alterations and exons presenting a greater mutagenic abundance than others. Also, we have detected the existence of 2 co-segregating haplotypes blocks. Finally, this work represents the first DMD gene small mutations screening applying WES in an argentine cohort, contributes with the characterization of our population and collaborates with the DMD small mutation’s knowledge.     

Dystrophin isoforms Dp71 and Dp427 have distinct roles in myogenic cells

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C₂C₁₂ myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 16–27, 1999