Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements

Exonic mutations associated with exon-skipping events.

<p>On the left, semi-quantification of alternative transcripts by QF-PCR on muscle biopsy cDNA. In the centre, schematic representation of the detected transcript species and their relative ratio. On the right, mutation sequence context and predicted ESE motifs: blue bars indicate ESE finder SR protein binding sites; violet bars indicate Rescue-ESE hexamers; red bars indicate PESE octamers. The mutated nucleotide is indicated in red.</p

Factors determining the main splicing pathway in 5′ ss mutations: cryptic site activation versus exon-skipping.

<p><b>A</b>) Relative 3′ ss strength (MaxEnt score difference with next distal natural 3′ ss) of exons exhibiting mainly cryptic site activation and exon exhibiting mainly exon-skipping. Box plots indicate the lowest and highest observation, lower and upper quartile, and median. <b>B</b>) Mean density of ESE motifs predicted by different matrices. <b>C</b>) Individual exons are plotted by the relative 3′ ss strength and density of Rescue-ESE motifs. Black-filled circles represent exons showing cryptic site activation. Non-filled circles represent exons showing exon-skipping. Exon numbers are indicated beside circles.</p

Splicing pathways in splice site mutations.

<p>Natural (wt) and cryptic splice sites were scored using Human Splicing Finder (HSF) and Maximum Entropy Scan matrices (MaxEnt).</p

Representative results of dystrophin immunostaining.

<p>A healthy control and five patients presenting different types of <i>DMD</i> point mutations are shown. BMD patient #1665 shows dystrophin reduction. This patient presented a 3′ ss disrupting mutation causing mainly exon 3 in-frame skipping. Patient #1973 presents the rare combination of DMD phenotype and reduction of dystrophin expression. In this patient, a missense mutation in CH1 of ABD1 domain may cause impaired actin-binding activity. DMD patient #1775 carrying a nonsense mutation in exon 26 shows absence of dystrophin (an isolated revertant fibre can be observed in DYS2). In contrast, patient #1472 carrying a nonsense mutation in exon 28 shows reduced dystrophin expression and milder BMD phenotype. mRNA analysis in this patient revealed in-frame exon-skipping due to the disruption of an ESE motif. In the last row, BMD patient #1497 shows a very mild reduction of dystrophin expression. This patient presented a missense mutation in the ZZ domain that may compromise β-distroglycan binding.</p

Summary of <i>DMD</i> point mutations, clinical phenotype and muscle dystrophin immunostaining in 105 dystrophinopathy patients.

<p>Asterisks indicate novel mutations not previously reported in the LOVD (<a href="http://www.dmd.nl" target="_blank">www.dmd.nl</a>). Protein domains: ABD, actin-binding domain; CH1-2, calponin homology; R1-24, spectrin-like repeats; H1-4, hinge regions; CRD, cysteine-rich domain; WW, domain with a signature of two tryptophan that binds to proline-rich proteins; EF-1/2, EF-hand domains found in calcium-binding proteins; ZZ, zinc-finger domain; CTD, c-terminal domain; SBS α1-syntrophin-binding site.</p

Relative ESE density versus relative 3′ ss strength in in-frame exons presenting nonsense and frameshift mutations.

<p>On the y axis: difference between exon PESE density and mean density of all <i>DMD</i> exons. On the x axis: difference between 3′ ss MaxEnt score and next distal natural 3′ ss score. Non-filled circles represent exons with DMD/IMD-associated mutations, while grey-filled circles represent exons with BMD-associated mutations. Black bordered circles indicate exons without PESE disruptions. Blue bordered circles indicate exons with PESE disruption. Exon numbers are indicated beside circles.</p