Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle

doi:10.1016/S0022-510X(01)00502-0

Journal of the Neurological Sciences

Volume 186, Issues 1–2, 1 May 2001, Pages 51-57

https://doi.org/10.1016/S0022-510X(01)00502-0 Get rights and content

Abstract

Despite promoter tissue specificity, up-regulation of the brain and Purkinje cell type dystrophin isoforms was described in skeletal muscle of X-linked dilated cardiomyopathy (XLDCM) and BMD affected individuals. An extended population of 11 Duchenne muscular dystrophy (DMD) and 11 Becker muscular dystrophy (BMD) patients was investigated to determine whether ectopic muscle expression of the two full-length non-muscular isoforms is a common event in dystrophinopathies and if it has functional significance. Up-regulation of the two non-muscle-specific isoforms was detected in four DMD patients but in none of the BMD affected individuals or non-dystrophic controls. This is the first report of an expression of these two isoforms in DMD skeletal muscle. Ectopic expression is not confined to regenerating or revertant fibers and does not correlate with age at biopsy, clinical phenotype, cardiac involvement, deletion size or location.

We consider that muscle ectopic expression of the brain and Purkinje cell-type isoforms has no favorable prognostic significance in DMD and BMD patients.

Introduction

Duchenne, and the milder phenotype, Becker muscular dystrophies (DMD and BMD) are caused by mutations in the huge dystrophin gene. The gene is located on Xp21.2, spans three megabases and consists of at least 79 exons.

Dystrophin mRNA expression is one of the most highly regulated in the cytoskeletal protein family [1]. Numerous dystrophin isoforms driven by various promoters have been described but only three (the brain, muscle and Purkinje cell type) were found to be the result of the transcription of a full-length dystrophin cDNA.

The muscle promoter (M) is mainly transcribed in muscle tissue [2], [3] but it is also active in glial cells [4]. The brain promoter (B) has been mapped ∼250 kb upstream of the muscle exon 1 [5], [6] and it has been shown to have transcriptional activity in cerebral and cerebellar neurons. The brain isoform can also be detected in heart, while its expression in skeletal muscle is still controversial. The Purkinje cell-type isoform is specific for cerebellar Purkinje neurons [7]. Holder et al. [8] also reported its expression in cardiac and adult skeletal muscle but this observation has never been confirmed. The Purkinje cell-type promoter (P) localizes to the large 280-kb first intron of the dystrophin gene [7].

Each promoter drives expression from a unique first exon, which is spliced directly to the common second exon of the dystrophin gene. The differential promoter utilization is associated with differences in the dystrophin N-terminus. It is still unknown whether such differences have functional significance or are merely a consequence of tissue-specific promoter activation. Despite promoter tissue specificity, up-regulation of the B and P isoforms in skeletal muscle was described in two brothers affected by X-linked dilated cardiomyopathy (XLDCM) [9].

More recently, Nakamura et al. [10] reported transcriptional activation of the two non-muscle isoforms in the skeletal muscle of two BMD patients with hotspot deletions and typical skeletal muscle involvement. The same authors proposed that these transcripts might be activated compensatively in response to reduced dystrophin expression.

At present, it is still unknown whether ectopic expression of the brain and Purkinje-cell dystrophin isoforms in skeletal muscle plays a role in the modulation of the clinical course of dystrophinopathy.

So far, no investigation on extended populations has been carried out in order to assess the actual frequency of activation of the two non-muscle-specific promoters in DMD and BMD skeletal muscle.

To determine whether up-regulation of the brain and Purkinje-cell isoforms is a common event and if it has functional significance, we analyzed the pattern of dystrophin transcription in the skeletal muscle of 11 DMD and 11 BMD patients. Skeletal muscle tissue from three healthy controls, five polymyositis and four metabolic myopathy patients were taken as controls. We also investigated whether there was a correlation between the expression of the B and P isoforms and clinical data (age, clinical severity, cardiac symptoms), immunohistochemical data (percentage of revertant fibers) and molecular data (vimentin expression as a marker for tissue regeneration). Skeletal muscle from four polymyositis patients was analyzed for the presence of the two non-muscle isoforms.

Section snippets

Patients

DMD and BMD patients were selected according to the following obligate criteria: male sex, clinical evidence of myopathy and hyperCKemia associated with either absent or markedly decreased dystrophin immunoreactivity at the immunohistochemical and Western blot examination using monoclonal antibodies directed against the NH₂, mid-rod and COOH domain.

BMD patients were classified as mild, moderate or severe as suggested by Beggs et al. [11]. DMD patients were defined typical or severe depending on

BMD patients

Clinical and molecular data on the 11 selected BMD patients are summarized in Table 1.

Mean age at biopsy was 26.5 (range 8–60). Patients were heterogeneous with respect to their clinical phenotype: three patients were classified as mild, five as moderate and three as severe. Only three patients manifested cardiomyopathy. All patients showed hotspot deletions of varying extension.

All of them presented dystrophin immunoreactive bands at Western blot analysis. Dystrophin levels were very variable

Discussion

The dystrophin gene is the largest human gene and has at least eight different promoters [1], [20], [21], [22]. Differential promoter utilization at the 5′ end of the gene is associated with differences in the dystrophin N-terminus. It remains unclear whether expression of the three full-length isoforms (the muscle, the brain and the Purkinje-cell type) is merely a consequence of tissue-specific promoter activation or meets the need for different N-terminal extremities with a given functional

Acknowledgements

The authors are grateful to the patients and their families for their collaboration.

We wish to thank Dr. A. Fassati for useful discussion and comments on the manuscript; A. Bordoni and F. Fortunato for technical assistance; Dr. B. Alberti for language revision.

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Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients
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Theoretically, 13% of patients with Duchenne muscular dystrophy may benefit from antisense-mediated skipping of exon 51 to restore the reading frame, which results in the production of a shortened dystrophin protein. We give a detailed description with longitudinal follow up of three patients with Becker muscular dystrophy with in-frame deletions in the DMD gene encompassing exon 51. Their internally deleted, but essentially functional, dystrophins are identical to those that are expected as end products in DMD patients treated with the exon 51 skipping therapy. The mild phenotype encourages further development of exon 51 skipping therapy.
A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro
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The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518 + 5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient’s lymphocytes, one with a 98 bp deletion of the 3′ end of exon-32 (dys32 − 98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518 + 5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3′ end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518 + 5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphism. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence.
Dystrophin and mutations: One gene, several proteins, multiple phenotypes
2003, Lancet Neurology
A large and complex gene on the X chromosome encodes dystrophin. Many mutations have been described in this gene, most of which affect the expression of the muscle isoform, the best-known protein product of this locus. These mutations result in the Duchenne and Becker muscular dystrophies (DMD and BMD). However, there are several other tissue specific isoforms of dystrophin, some exclusively or predominantly expressed in the brain or the retina. Mutations affecting the correct expression of these tissue-specific isoforms have been associated with the CNS involvement common in DMD. Rare mutations also account for the allelic disorder X-linked dilated cardiomyopathy, in which dystrophin expression or function is affected mostly or exclusively in the heart. Genotype definition of the dystrophin gene in patients with dystrophinopathies has taught us much about functionally important domains of the protein itself and has provided insights into several regulatory mechanisms governing the gene expression profile. Here, we focus on current understanding of the genotype–phenotype relation for mutations in the dystrophin gene and their implications for gene functions.
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Duchenne muscular dystrophy is an X-linked recessive muscle wasting disease caused by the absence of the muscle cytoskeletal protein, dystrophin. Dystrophin is a member of the spectrin superfamily of proteins and is closely related in sequence similarity and functional motifs to three proteins that constitute the dystrophin related protein family, including the autosomal homologue, utrophin. An alternative strategy circumventing many problems associated with somatic gene therapies for Duchenne muscular dystrophy has arisen from the demonstration that utrophin can functionally substitute for dystrophin and its over-expression in muscles of dystrophin-null transgenic mice completely prevents the phenotype arising from dystrophin deficiency. One potential approach to increase utrophin levels in muscle for possible therapeutic purpose in humans is to increase expression of the utrophin gene at a transcriptional level via promoter activation. This has lead to an interest in the identification and manipulation of important regulatory regions and/or molecules that increase the expression of utrophin and their delivery to dystrophin-deficient tissue. As pre-existing cellular mechanisms are utilized, this approach would avoid many problems associated with conventional gene therapies.
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Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle

Abstract

Introduction

Section snippets

Patients

BMD patients

Discussion

Acknowledgements

Am. J. Hum. Genet.

Neuromusc. Disord.

J. Neurol. Sci.

Neuromusc. Disord.

Genomics

Cell

Cell

J. Neurol. Sci.

Neuromusc. Disord.

Cell

The structural and functional diversity of dystrophin

Nat. Genet.

Molecular and functional analysis of the muscle-specific promoter region of the Duchenne muscular dystrophy gene

Mol. Cell Biol.

Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart and brain development

Nucleic Acids Res.

Dystrophin gene transcribed from different promoters in neuronal and glial cells

Nature

Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus

Nature

Duchenne Muscular Dystrophy gene product is not identical in muscle and brain

Nature

Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters

Hum. Mol. Genet.

Expression and regulation of the dystrophin Purkinje promoter in human skeletal muscle, heart, and brain

Hum. Genet.

Transcription of the dystrophin gene in normal tissues and in skeletal muscle of a family with X-linked dilated cardiomyopathy

Am. J. Hum. Genet.

Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies

Am. J. Hum. Genet.