Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient

Kane Greer,Emily Rice,Sue Fletcher,Roberto A Barrero,Aileen Reghan Foley,Eoin O Rathallaigh,Matthew I Bellgard,Bryan J Lynch,Steve D Wilton,Kayla Mizzi,Lukas Kuster

doi:10.1002/mgg3.144

Abstract

We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45–47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

Highlights

The most common mutations in the dystrophin (DMD) gene are deletions of one or more exons (Den Dunnen et al 1989) and those that disrupt the open reading frame and ablate or dramatically reduce muscle dystrophin cause the fatal disease, Duchenne muscular dystrophy (DMD) (MIM #310200)
Patients typically present with the milder condition, Becker muscular dystrophy (BMD) (MIM #300376), and remain ambulant beyond 15 years of age, whereas DMD patients lose the ability to walk by age 12
In this study we report on a patient, identified as having a deletion of exons 45–47 and a genetic diagnosis of BMD, who presented with an intermediate/ severe BMD clinical phenotype and lost ambulation by the age of 15 years

Summary

Introduction

The most common mutations in the dystrophin (DMD) gene are deletions of one or more exons (Den Dunnen et al 1989) and those that disrupt the open reading frame and ablate or dramatically reduce muscle dystrophin cause the fatal disease, Duchenne muscular dystrophy (DMD) (MIM #310200). Dystrophin Pseudoexon Activation in Severe BMD the patient described here revealed that a pseudoexon of 82 nucleotides, derived from intron 44, (pseudoexon 44a) was included in the DMD transcript disrupting the open reading frame to yield a truncated, nonfunctional dystrophin. Since the sequence of the pseudoexon and flanking canonical splice sites are identical to the reference sequence, we concluded that the genomic deletion brought together elements promoting recognition and selection of exon 44a into the mature mRNA, causing a more severe dystrophic phenotype than would be expected from the loss of exons 45–47. Sequencing of the amplicons revealed that the junction of introns 44 and 47 in the patient lay one (or two) bases downstream of the cryptic donor splice site of the pseudoexon that adjoins ~26 kb into intron 47 (Fig. 2C).

B Exon 44

Findings

B M 1 2 3 4 5 6 7 8 -ve M M 9 10 11 12 13 14 15 16 -ve M