Background Up to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing.
Methods RNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of the DMD gene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients.
Results We identified an alteration in the mRNA level in all the patients. We detected three pseudoexons in DMD caused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity.
Conclusion These findings indicate that mRNA analysis of the DMD gene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing.
- Frameshift Mutation
- Neuromuscular Diseases
- Sequence Analysis, RNA
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Duchenne muscular dystrophy (DMD, OMIM #310200) and Becker muscular dystrophy (BMD, OMIM #300376) are X-linked recessive disorders caused by pathogenic variants in the DMD gene (OMIM *300 377), which encodes the dystrophin protein.1 DMD is the most severe form of the disease and the most common neuromuscular disorder in childhood, affecting 1 in 3500 live male births.2 Patients with BMD present a less severe phenotype with a more progressive and variable disease course. Disease severity can be explained by the reading frame rule in 90% of patients: frameshift variants in the DMD gene result in a premature termination codon (PTC) and a non-functional dystrophin leading to DMD. In contrast, in-frame variants usually give place to a partially functional protein and a BMD phenotype.3
Currently, the diagnostic algorithm consists of multiplex ligation-dependent probe amplification (MLPA) followed by exome sequencing (ES) in genomic DNA (gDNA). Exonic deletions and duplications are the most prevalent pathogenic variants (65%–75% of patients), and subsequently, small disease-causing variants are detected in 20%–25% patients.4–6 With this diagnostic approach, up to 7% of patients with dystrophinopathy remain without a precise genetic diagnosis.7 8 As more than one-third of disease-causing variants in the Human Genome Mutation Database are estimated to perturb pre-mRNA splicing,9 patients with undiagnosed DMD/BMD are thought to carry complex structural variants (SVs) or splicing alterations in the DMD gene. Nonetheless, pathogenic variants in non-coding regions potentially disrupting canonical splicing of DMD are missed in routine diagnosis. Identifying and characterising splicing variants in the DMD gene are essential to obtain a precise genetic diagnosis of these patients, to be eligible to mutation-specific therapies, and to offer families appropriate genetic and reproductive counselling.
The aim of this study was to evaluate the utility of mRNA analysis of the DMD gene in patients with dystrophinopathy who remained genetically undiagnosed after MLPA and ES. Through DMD mRNA analysis in seven patients, we detected three pseudoexons (PEs) in the DMD gene, one chromosomal rearrangement between Xp21.2 and 8p22, and three exon skipping events with unclear pathogenicity.
Patients were recruited from several hospitals in Spain, and they were included when (1) DMD and BMD were suspected in the clinical examination, and it was supported by alterations in dystrophin immunohistochemistry and/or western blot; and (2) MLPA of the DMD gene (P034 and P035 Sauce Kit; MRC-Holland, Amsterdam, Netherlands) and ES had negative results. In total, cDNA analysis was performed on seven patients (table 1).
Amplification of DMD cDNA sequence
RNA was extracted from muscle biopsies using the Animal Tissue RNA Purification Kit (Norgen Biotek, Ontario, Canada). RNA was retrotranscribed using Oligo(dT)20 Primer (Invitrogen, California, USA). The whole mRNA DMD was sequenced with overlapping primer pairs (online supplemental table 1). PCR products were sequenced with BigDye Terminator V.1.1 Cycle Sequencing Kit (ThermoFisher) and analysed in an Applied Biosystems 3500 Dx Series Genetic Analyzer (ThermoFisher). Splicing in silico analyses were performed with Alamut Visual software V.2.6.1. Variants have been submitted to the ‘Global Variome shared LOVD’ (https://databases.lovd.nl/shared/references/DOI:10.1136/jmg-2022-108828).
gDNA analysis and whole-genome sequencing (WGS)
gDNA was extracted from total peripheral blood using QIAsymphony SP (Qiagen). The presence of deep intronic variants (in patient 1 (P1), patient 2 (P2) and patient 4 (P4)) and intronic variants near exon boundaries (in patient 3 (P3), patient 5 (P5) and patient 7 (P7)) was evaluated through Sanger sequencing.
WGS was performed in P6. One microgram of fragmented gDNA was used for library preparation with Truseq DNA PCR-Free (Illumina, California, USA). Sequencing was performed in a NovaSeq 6000 (Illumina). Manta V.1.6.0 was used to detect SVs and junction breakpoints.10 SVs passing the default filtering were inspected on IGV (Integrative Genomics Viewer) . Breakpoints were confirmed by Sanger sequencing.
Droplet digital PCR (ddPCR)
Mini Affinity Plus qPCR probes (Integrated DNA Technologies, Iowa, USA) were designed to quantify exon 9 and 71 skipping (online supplemental table 3). ddPCR reactions were performed with 10 ng of cDNA and ddPCR Supermix for Probes (no dUTP (deoxyuridine Triphosphate)) (Bio-Rad Laboratories, California, USA) following manufacturer protocol. Droplets were generated with Droplet Generation Oil for Probes (Bio-Rad) in the QX200 Droplet Generator (Bio-Rad). Thermocycling conditions were as follows (ramping rate 2 °C/s): 95°C 10 min, 40 cycles of 94°C for 30 s and 58°C for 1 min, and 98°C for 10 min. Droplets were loaded in the QX200 Droplet Reader (Bio-Rad). For each sample, four replicates were performed and a non-template control was included in each assay. The data were analysed with the QX Manager Software Standard Edition V.1.2 (Bio-Rad). Mann-Whitney test was performed. Only wells with more than 10 000 droplet reads were considered.
Deep intronic variants and PE inclusion
We identified the inclusion of PEs in dystrophin mRNA in three patients (P1, P2 and P4) due to deep intronic variants (table 1).
In P1, we detected an inclusion of 214 bp from intron 15 (from c.1813–947 to c.1813–734) into the DMD transcript (PE15) (figure 1A). We detected the deep intronic variant g.32584731A>G (c.1813–733A>G) in intron 15, classified as a ‘variant of uncertain significance’ (VUS) following the American College of Medical Genetics guidelines.11 This variant, not present in human genetic variation databases, creates a new donor splice site (SS) in intron 15 near a cryptic acceptor SS (figure 1B and online supplemental table 2). The deep intronic variant c.1813–733A>G results in the disruption of the reading frame of DMD transcripts (p.Val605Ilefs*33), probably targeted by nonsense-mediated decay.
In P2, we observed the presence of an in-frame PE between exons 37 and 38 (PE37) that incorporated a PTC (p.Ala1776Valfs*9) (figure 1C). gDNA analysis revealed the intronic variant g.32371864A>C (c.5326–5219T>G), not previously reported in databases, that results in the retention of 48 bp from intron 37. This variant creates a new cryptic acceptor SS and activates a pre-existing donor SS (figure 1D, (online supplemental table 2)).
In P4, besides residual levels of full-length dystrophin, we detected a PE of 58 bp between exons 62 and 63 (PE62). The deep intronic variant g.31279418T>C (c.9225–285A>G) was identified, inherited from the patient’s mother. PE62 had already been described in patients with BMD.12 The pathogenic deep intronic variant c.9225–285A>G, located in position+5 from the cryptic donor SS, increases complementarity between the pre-mRNA and U1snRNA, and thus increases the splicing efficiency of the cryptic donor SS.12
P6 cDNA sequencing revealed a 64 bp cryptic exon (p.Phe32Asnfs*5) between exons 2 and 5 apart from the wild-type dystrophin transcript. BLAT (BLAST-like alignment tool) analysis of the cryptic exon resulted in 100% identity with LOC101929028 located in 8p22 (chr8:16 369 356–16 369 419). The flanking region of 64 bp inserted in the mRNA of dystrophin probably confers an exon-like profile with an acceptor, donor SS and exonic splicing enhancers that can be recognised by the spliceosome complex (figure 1E), suggesting the presence of a chromosomal rearrangement between chromosome 8 and DMD. WGS was performed in P6 to determine the exact breakpoints of the rearrangement. We detected an insertion of chromosome 8 (chr8: 16 346 710–16 422 443) in intron 2 of the DMD, and a 6 bp deletion in DMD intron 2 (chrX:32 873 009–32 873 014) (figure 1F). IGV showed the junction breakpoints surrounded by chimeric reads aligning both DMD and chromosome 8 (figure 1G). The insertion of 75.7 kb encompassed the LOC101929028 detected in the mRNA analysis and did not contain any protein-coding gene. In-depth analysis of the flanking sequences of the breakpoint junctions revealed the presence of LINE elements in 5′ and 3′ junctions.
Alternative exon skipping events with unclear pathogenicity
In three patients with BMD (patient 3 (P3), P5 and P7) (table 1), no other alteration was detected in dystrophin mRNA apart from transcripts with exon skipping events, together with full-length transcript. In P3, a patient with BMD manifesting only hyperCKemia, we detected exon 9 skipping. In P5, who had mild BMD, exon 71 and 78 skipping was revealed. Finally, in P7, we detected exon 71 skipping. No intronic variants were found near canonical SS that could explain the induction of these exon skipping events. Exon 9 and 71 DMD skipping isoforms were quantified by ddPCR in P3 and P7. Both exon skipping events were present in slightly higher proportion in patients than in control samples (exon 9 skipping: 0.97%±0.2 in controls and 11.15%±0.3 in P3; exon 71 skipping: 1.34%±0.16 in controls and 16.45%±0.9 in P7) (online supplemental figure 1).
Here we show the relevance of performing an mRNA-based analysis of the DMD gene to establish a definite genetic diagnosis in patients with undiagnosed dystrophinopathy after MLPA and ES. Up to 7% of the patients remain genetically undiagnosed after routine genetic testing.7 8 Nonetheless, having a precise genetic diagnosis is essential for these patients to be eligible for mutation-specific therapies and for their relatives to receive an accurate genetic and reproductive counselling. Here we studied seven patients with clinical suspicion of DMD/BMD but no precise genetic diagnosis after clinical genetic testing (table 1). We detected a transcriptional alteration in the DMD in all patients and established a final genetic diagnosis in four of the seven patients. Our findings indicate that variants that alter DMD splicing, such as deep intronic variants and SVs, are an important cause of DMD/BMD in patients with undiagnosed dystrophinopathy.
The implementation of high-throughput technologies in clinical testing has enabled a faster and a more efficient exploration of the human genome. As a result, invasive procedures such as muscle biopsy tend to be avoided. However, this procedure remains an essential tool in the differential diagnosis of several neuromuscular disorders. If in the first approach we had performed WGS instead of mRNA sequencing of DMD, we would not have been able to interpret and validate some of the findings responsible for the pathology in our cohort. For instance, through WGS, we would have detected two novel deep intronic variants in P1 and P2 (figure 1A–D), but we would not have been able to assess their pathogenicity. Computational algorithms for predicting alternative splicing are usually less reliable for deep intronic variants than for variants in canonical SSs.13 In these cases, performing an RNA-based analysis is essential to assess the impact of deep intronic variants in the canonical splicing of the DMD gene and to reclassify intronic variants previously classified as VUS.
The DMD is a complex gene for the spliceosome machinery due to its large protein-coding sequence and intronic regions. In muscle tissue, few alternative splicing events are observed in comparison with other tissues, as a tight splicing regulation is needed in the muscle tissue to maintain dystrophin functionality.14 15 In our cohort, we detected three exon skipping events with unclear pathogenicity that commonly occur in non-muscle tissues (exon 9 skipping in P3, exon 71 skipping in P5 and P7, and exon 78 skipping in P5).16 17 P3, P5 and P7 present mild forms of BMD with hyperCKemia, muscle cramps or rhabdomyolisis (table 1). Similar works have also reported exon skipping events in patients with BMD with no other genomic alteration.18 19 However, further analyses in a larger patient cohort will be needed to clarify the role of these exon skipping events.
In summary, after evaluating splicing alterations in the DMD gene through cDNA sequencing, we found a transcriptional alteration in all patients who remained undiagnosed after clinical genetic testing and established a precise diagnosis in four of the patients. Recently, RNA sequencing (RNA-seq) is being considered as a promising diagnostic approach in undiagnosed patients after ES. It is estimated that RNA-seq increases the genetic diagnostic rate in Mendelian disorders by up to 35%.20 One of the main advantages of this approach is the ability to evaluate not only alternative splicing events but also transcript isoform abundance, monoallelic expression and differential gene expression in the targeted tissue. However, to detect the presence of SVs, RNA-seq can only detect those that alter canonical splicing, such as the insertion identified in P6 (figure 1E–G). SVs that do not have a direct effect in RNA processing would have been missed through an RNA-based analysis. Hence, an integrated approach with transcriptomics and WGS can help to achieve a precise genetic diagnosis in patients with dystrophinopathy and may allow them the accessibility to mutation-specific therapies and genetic and reproductive counselling.
Patient consent for publication
This study involves human participants and was approved by the ethics committee of Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) (REF: IIBSP-CAN-2021-110). Written informed consent was obtained from all patients and signed by parents if the patient was a minor.
We thank the patients and their relatives for their participation in this research study and the European Reference Network for Neuromuscular Diseases. We also thank Carolyn Newey for English language editing.
Twitter @AlbaSegarraC, @lidiagquer
Contributors PG, LG-Q and AS-C: study design, acquisition and interpretation of data, preparation of the draft of the manuscript, and reviewing and editing of the final manuscript. PG and LG-Q: funding acquisition. PG, AH-L, ACa, MTS-C, CD-G, AC-B, MM, ER, ACo, CO, DN-dB, AN and MJR: clinical data collection, data analysis, and reviewing and editing the final manuscript. All authors approved the final manuscript.
Funding This study was supported by Instituto de Salud Carlos III and FEDER, ‘Una manera de hacer Europa’ (grant number FIS PI18/01585, LG-Q and PG) and the Ministerio de Universidades (grant number FPU20/06692, AS-C).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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