A novel deep intronic variant introduce dystrophin pseudoexon in Becker muscular dystrophy: A case report

Abstract

Most pathogenic DMD variants are detectable and interpretable by standard genetic testing for dystrophinopthies. However, approximately 1∼3% of dystrophinopthies patients still do not have a detectable DMD variant after standard genetic testing, most likely due to structural chromosome rearrangements and/or deep intronic pseudoexon-activating variants. Here, we report on a boy with a suspected diagnosis of Becker muscular dystrophy (BMD) who remained without a detectable DMD variant after exonic DNA-based standard genetic testing. Dystrophin mRNA studies and genomic Sanger sequencing were performed in the boy, followed by in silico splicing analyses. We successfully detected a novel deep intronic disease-causing variant in the DMD gene (c.2380 + 3317A > T), which consequently resulting in a new dystrophin pseudoexon activation through the enhancement of a cryptic donor splice site. The patient was therefore genetically diagnosed with BMD. Our case report further emphasizes the significant role of disease-causing splicing variants within deep intronic regions in genetically undiagnosed dystrophinopathies.

1. Introduction

The dystrophin protein, as the central component of the dystrophin-associated glycoprotein complex, plays a key role in stabilizing muscle integrity and sarcolemma stability in heart and skeletal muscle myofibers [1]. Dystrophin abnormalities result from pathogenic DMD mutations can lead to sarcolemma instability and, as a result, cause the development of X-linked recessive muscle diseases, i.e., dystrophinopathies which include a rare form of DMD-associated dilated cardiomyopathy and two relatively common forms of Duchenne muscular dystrophy and Becker muscular dystrophy (BMD) [2]. The high complexity of the dystrophin gene structure makes the spectrum of pathogenic DMD variants quite perplexing, which ranges from subexonic single-nucleotide variants to large-scale copy number variations or structural chromosome rearrangements [[3], [4], [5], [6]]. Most disease-causing DMD variants occur in DMD exonic regions and/or flanking intronic regions that mainly consist of canonical and non-canonical splice site regions, which refer to the typical disease-causing variants in DMD. Typical disease-causing DMD variants can be discovered and interpreted by exonic DNA-based analyses, including multiplex ligation-dependent probe amplification (MLPA)-analysis of copy number variations in the dystrophin gene followed by sequencing of the entire dystrophin coding and flanking non-coding regions, which are now widely used as the standard genetic testing for dystrophinopathies [3,4]. However, the exonic DNA-based analyses cannot reveal disease-causing variants in some DMD-associated dystrophinopathies patients, such as approximately one percent in the Italians population [7], two percent in the Japanese population [8], and three percent in the Chinese population [3]. Moreover, the exonic DNA-based analyses also cannot confirm the influence of genomic DMD variants on the open reading frame of dystrophin mRNA, which primarily determines disease severity of dystrophinopathies patients.

Most unrevealed pathogenic DMD variants after DNA-based standard genetic testing are structural chromosome rearrangements and deep intronic pseudoexon-activating variants (atypical disease-causing DMD variants), the detection and appropriate interpretation of which requires the combined use of DNA- and mRNA-based sequencing technologies [4]. Bioinformatic and computational analyses are also significant for the identification of atypical disease-causing DMD variants [4]. In this case report, a 10.2-year-old boy with a suspected molecular diagnosis of BMD was enrolled. The exonic DNA-based standard genetic testing did not find disease-causing variants including DMD variants in him. To identify possible atypical disease-causing DMD variants that were undetectable by the exonic DNA-based analyses, we performed analysis of the entire dystrophin mRNA derived from the patient's muscle sample, followed by genomic Sanger sequencing and bioinformatic and computational analyses. By combing these techniques and analyses, we detected a novel deep intronic pseudoexon-activating variant in the dystrophin gene, which was canonically described as NM_004006.2:c.2380 + 3317A > T and determined the molecular diagnosis of BMD in our patient.

2. Case presentation

2.1. Phenotypic characteristics

A 10.2-year-old boy, who was the only son of non-consanguineous couples and presented to our hospital because of an incidental discovery of hyper-creatine kinase-emia, was enrolled. He had obvious fatigue since 9.7-years of age. He also suffered from activity-induced myalgia and/or muscle cramps for half a year. He had bilateral calf hypertrophy and no obvious tendon contractures confirmed by his neurological examination at the first visit. He had no muscle weakness in either proximal or distal extremities; however, he had mild muscle fatty replacement of the gluteus maximus muscle (Fig. 1A). In addition, no significant muscle fatty replacement of his bilateral thigh and lower leg muscles was found by the muscle magnetic resonance imaging (Fig. 1B and C). Histological hematoxylin and eosin staining of his biopsied muscle tissue revealed some pathological myopathic changes, which included a few hypertrophic, hypercontracted and atrophic muscle myofibers, several clusters of necrotic and regenerating muscle myofibers, and some myofibers with internal nuclei (Fig. 1D). Dystrophin expression analysis using the immunohistochemical staining revealed a partial reduced expression of dystrophin-N and dystrophin-C, while a mild reduction in the expression of dystrophin-R (Fig. 1E–G). Histological and immunohistochemical staining of an enrolled healthy control revealed no pathological changes (Fig. 1H–K). The patient had an obviously increased level of serum creatine kinase in his every blood test, ranging from 2205 IU/L to 5589 IU/L. The phenotypic and muscle pathological characteristics as well as the increased creatine kinase level together indicated a suspected molecular diagnosis of BMD in our enrolled boy.

Fig. 1.

Muscle magnetic resonance imaging features and histopathologic characteristics of the enrolled patient. Muscle T1-weighted images of the enrolled patient at the pelvis (A), bilateral thigh (B), and bilateral lower leg levels (C). (D) Hematoxylin and eosin staining revealing some pathological myopathic changes. Dystrophin immunohistochemical staining revealing a partial reduction of dystrophin-N (E) and dystrophin-C (F), and a mild reduction of dystrophin-R (G). Histological and immunohistochemical staining revealing no pathological changes (H) and positive expression of dystrophin-N (I), dystrophin-C (J), and dystrophin-R (K). (A)–(G) The enrolled patient. (H)–(K) An enrolled healthy control.

2.2. Exonic DNA-based standard genetic testing

We therefore initiated exonic DNA-based standard genetic testing for finding disease-causing DMD variants [3,4] because of the suspected BMD diagnosis of our enrolled boy, including whole exome sequencing and MLPA-analysis of copy number variations in the dystrophin gene. Whole exome sequencing detected no disease-causing variants (including DMD variants) in the enrolled boy. Additionally, the MLPA-analysis identified no copy number variations (exonic duplications and/or deletions) in the dystrophin gene.

2.3. Analysis of the entire dystrophin mRNA

We performed analysis of the entire dystrophin mRNA in our enrolled boy after exonic DNA-based analyses, which failed to confirm the molecular genetic diagnosis of our patient. We first extracted total muscle mRNA from the biopsied muscle tissue. We then amplified twenty-two overlapping cDNA fragments of the entire DMD mRNA (GenBank accession number NM_004006.2) from the total muscle mRNA using the reverse transcription polymerase chain reaction method (RT-PCR) as previously described [4]. After agarose gel electrophoresis analysis of the twenty-two amplified cDNA fragments, we observed two aberrant DMD splicing transcripts (two bands with different size) in the fifth cDNA fragment of the dystrophin gene. We found that the lower band was almost the same size as the control's band and the upper band was longer than the control's band shown in the agarose gel image (Fig. 2A). To confirm the exact sequences of two aberrant splicing transcripts, we directly conducted Sanger sequencing of the aberrant RT-PCR products; however, it failed to recognize the overlapping sequences within the two bands (Fig. 2B). TA cloning was then performed and confirmed the exact sequences of the two transcripts, which included the normally spliced DMD transcript (DMD exons 19 to 20) and an aberrant 133-bp insertion transcript between DMD exons 19 and 20 (Fig. 2C and D).

Fig. 2.

Analysis of the entire dystrophin mRNA and genomic DNA sequencing. (A) Agarose gel electrophoresis analysis of the aberrant RT-PCR products of the enrolled patient revealed two bands with different size. (B) Sanger sequencing of the aberrant RT-PCR products failed to recognize the overlapping sequences. TA cloning confirmed the exact sequences of the two transcripts, including the normal splicing of DMD exons 19 to 20 (C) and a 133-bp insertion transcript between DMD exons 19 and 20 (D). (E) Sanger sequencing of genomic DNA around the dystrophin pseudoexon found a novel variant. (F) Graphic representation of the aberrant dystrophin pseudoexon activated by the novel DMD variant. RT-PCR, reverse transcription polymerase chain reaction; MaxEnt, Maximum Entropy; HSF, Human Splicing Finder; 5′ ss, donor splice site; 3′ ss, acceptor splice site; *, premature stop codon.

The homology search using the BLAT Search tool confirmed that the aberrant 133-bp insertion transcript was completely derived from a deep intronic region of dystrophin gene intron 19 (NC_000023.10; chrX:32516557–32516689). We thus interpreted the aberrant 133-bp insertion transcript as a pseudoexon activation or inclusion in DMD (NM_004006.2:r.[ = ,2380_2381ins2380 + 3183_2380 + 3315]), which has not been reported. A frameshift and premature stop codon was occurred within the aberrant transcript (NP_003997.1:p.[ = ,Gly795Metfs*47]). The aberrant pseudoexon-containing transcript was predicted to be degraded result from nonsense-mediated decay. The lower level of normally spliced DMD transcript shown in the lower faint band (Fig. 2A and C) led to the decreased expression of dystrophin-N, –C and –R observed in our patient (Fig. 1E–G).

2.4. Genomic DMD sequencing and bioinformatic interpretation

To identify genomic variant(s) that might activate the dystrophin pseudoexon in our patient, we conducted Sanger sequencing of genomic DNA around the dystrophin pseudoexon and found a novel single-nucleotide variant (Fig. 2E). The novel variant was located within the deep intronic region of dystrophin gene intron 19, with canonical descriptions of NM_004006.2:c.2380 + 3317A > T at cDNA level and NC_000023.10:g.32516555T > A at genomic level. The deep intronic novel variant is a de novo variant and, to our knowledge, it is not reported in the published studies. Moreover, it is not recorded in several public genome databases as well, including Leiden Open Variation Database (unique variants in the DMD gene), ClinVar, and Genome Aggregation Database.

To interpret aberrant splicing mechanisms underlying the novel DMD variant, several bioinformatic splicing algorithms, including the Human Splicing Finder (HSF) [9], SpliceAI [10], and Maximum Entropy (MaxEnt) Scanning [11], were conducted in our study. We found that the c.2380 + 3317A > T variant identified in our patient strengthened a cryptic donor splice site (also refers to 5′ splice site; MaxEnt score, reference sequence: 2.29, variant sequence: 10.47; HSF score, reference sequence: 69.88, variant sequence: 97.02) in a deep intronic region of dystrophin gene intron 19 (Fig. 2F). Moreover, the novel c.2380 + 3317A > T variant also activated a cryptic acceptor splice site (also refers to 3′ splice site; MaxEnt score 0.65, HSF score 79.91; Fig. 2F). Acceptor Site Gain for the cryptic 3′ splice site (134-bp) and Donor Site Gain for the strengthened cryptic 5′ splice site (2-bp) were also predicted by the SpliceAI for the novel c.2380 + 3317A > T variant. We thus concluded that the activation of a cryptic 3′ splice site and enhancement of a cryptic 5′ splice site result from the novel c.2380 + 3317A > T variant consequently caused the dystrophin pseudoexon activation or inclusion detected in the patient (Fig. 2D and F). We classified and interpreted the deep intronic novel variant (c.2380 + 3317A > T in DMD) as a splicing and disease-causing variant according to the interpretation guidelines of genetic variants [12]. The novel DMD variant complied with the following criteria for moderate evidence (PM2), strong evidence (PS2), and very strong evidence (PVS1) specified in the guidelines.

3. Discussion

Molecular genetic diagnosis of patients with single-gene disorders is always of great significance, even though the precise characterization of genetic mutations is sometimes challenging because of the presence of atypical disease-causing variants. The dystrophin or DMD gene, spanning over 2.2 Mb of human genomic DNA, has a high frequency of mutation events (including both typical and atypical variants) due to its large genomic size [2,13]. Of the atypical disease-causing DMD variants, deep intronic DMD variants are relatively common and challenging for detection and pathogenicity interpretation. As per the central dogma, deep intronic variants in protein-coding genes that affect pre-mRNA splicing and result in the formation of aberrant mature transcripts are considered to be disease-causing variants for a single-gene disorder. Thus, the correct characterization and interpretation of disease-causing deep intronic DMD variants requires analyses of the entire dystrophin mRNA to confirm abnormalities in mature dystrophin transcripts caused by deep intronic DMD variants [[3], [4], [5], [6]]. In addition, abnormalities in dystrophin protein are also important evidence to confirm the pathogenicity of deep intronic DMD variants. In this case report, we successfully revealed a deep intronic novel variant in DMD intron 19 (c.2380 + 3317A > T) via the combined application of the entire dystrophin mRNA analysis, Sanger sequencing of genomic DNA, and in silico splicing analysis.

Our study suggests that even in the genomic era, skeletal muscle biopsy remains an important diagnostic investigation for some patients with dystrophinopathies, as their disease-causing variants occur in the deep intronic non-coding regions of DMD that are not covered by the exonic DNA-based standard genetic testing. Skeletal muscle-derived dystrophin mRNA is an ideal source for identifying abnormalities in mature dystrophin transcripts, as the full-length transcript of dystrophin is primarily expressed in myofibers. Moreover, skeletal muscle-derived entire dystrophin mRNA studies are also of significance in patients with uncertain exonic DMD variants in addition to deep intronic DMD variants, like synonymous and missense variants that affect exonic splicing regulatory elements and thus altering DMD pre-mRNA splicing [7]. It is worth noting that skeletal muscle biopsy is also essential for establishing abnormal size or reduced levels of dystrophin protein via qualitative and/or quantitative protein studies (immunohistochemistry and/or western blotting analysis), which determines the clinical severity of a patient with dystrophinopathy. As in our patient, we find that the reduced level of dystrophin protein expression is in line with his BMD phenotype. However, skeletal muscle biopsy-based dystrophin mRNA and protein studies may not always be ideally available for certain pediatric patients, as skeletal muscle biopsy requires a surgical intervention. In addition, the genetic testing strategy adopted in our study is not possible to detect complex structural variants or chromosome rearrangements in DMD, of which the genetic characterization needs long-read sequencing. The correct characterization and interpretation of disease-causing DMD variants is important for both disease management and genetic counseling in dystrophinopathies.

Disease-causing intronic variants in dystrophin gene can activate and induce various aberrant pre-mRNA splicing events of dystrophin gene through causing abnormalities in the essential splicing signals or even the auxiliary splicing regulatory elements, including partial intron inclusion, whole intron retention, exon-skipping, exon-truncation, and pseudoexon activation [14,15]. Among the published and reported aberrant pre-mRNA splicing events caused by disease-causing intronic DMD variants, dystrophin pseudoexon activation has been frequently identified to be associated with Duchenne and Becker muscular dystrophy [15,16]. Dystrophin pseudoexons caused by disease-causing intronic DMD variants are derived from the homologous intronic sequences of DMD and usually located far from the canonical DMD exons [16]. The weak exon-like profile of a putative dystrophin pseudoexon, including weak essential splicing signals and/or splicing regulatory elements, could be enhanced by a genomic pseudoexon-activating mutation. As a result, the putative dystrophin pseudoexon could be aberrantly spliced into the mature full-length dystrophin transcript (Dp427 m) and then cause Duchenne or Becker muscular dystrophy [16,17]. As in our enrolled boy, the deep intronic novel variant in DMD (c.2380 + 3317A > T) strengthened a cryptic donor splice site, which enhanced the exon-like profile (an enhanced essential splicing signal) of a putative dystrophin pseudoexon in intron 19 and thus caused the inclusion of the putative pseudoexon into the mature dystrophin transcript. It is theoretically possible to develop antisense oligonucleotides that induce pseudoexon-skipping in dystrophinopathies, just as it is done in Duchenne muscular dystrophy by skipping different exons to produce dystrophin protein [18].

In conclusion, our case report further emphasizes the significant role of deep intronic pseudoexon-activating variants, i.e., disease-causing splicing variants located within deep intronic regions of dystrophin gene, in genetically undiagnosed dystrophinopathies and the novel intronic DMD variant expands the mutational spectrum of human Becker muscular dystrophy.

Ethics statement

Our case report was approved by the Ethics Committee at Peking University First Hospital (2023 109-002). Informed consent was obtained from the parents of the enrolled patient.

Consent for publication

The parents of the enrolled patient provided consent for participation in this case study and for publishing the obtained results, including clinical characteristics, muscle MRI images and pathological images.

Funding statement

Our work was funded by the National Natural Science Foundation of China (grant number 82201553) and High Quality Clinical Research Project of Peking University First Hospital (grant number 2023HQ10).

Data availability statement

All authors confirm that the data supporting the case report are available within the article.

CRediT authorship contribution statement

Chang Liu: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Yanyu Lu: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Haiyan Yu: Writing – review & editing, Software, Methodology, Data curation. Zhihao Xie: Writing – review & editing, Visualization, Software, Investigation, Data curation, Conceptualization. Chengyue Sun: Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. Xinchao Cheng: Writing – review & editing, Software, Investigation, Data curation. Fangfang Niu: Writing – review & editing, Software, Methodology, Data curation. Yawen Zhao: Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. Jianwen Deng: Writing – review & editing, Software, Methodology, Investigation, Data curation. Lingchao Meng: Writing – review & editing, Validation, Software, Methodology, Investigation, Data curation. Zhaoxia Wang: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. Yun Yuan: Writing – review & editing, Visualization, Validation, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Zhiying Xie: Writing – review & editing, Visualization, Validation, Supervision, Software, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.