Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat

Abstract

Background: The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain) is its distant relative, alpha-dystrobrevin. The alpha-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands) in the dystrophin glycoprotein complex.

Results: We show here that, contrary to the literature, most alpha-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse alpha-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms alpha-dystrobrevin-4 and -5) are present in most mammals but specifically ablated in mouse and rat.

Conclusion: Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the alpha-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the alpha-dystrobrevins (and therefore the dystrophin glycoprotein complexes) of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.

Figure 1

Comparison of α-dystrobrevin gene structure and isoforms in humans and mice. The central genomic DNA structure and observed transcript isoforms are shown for A) most tetrapods, including humans, and B) mice and rats. In each instance, the partial genomic DNA structure (not to scale) is shown at the top, with the coarse isoforms (numbered as in Peters et al. [42]) shown below. To the right are shown the fine isoforms generated by alternative splicing of the SBS-encoding region, labelled from 'a' to 'c' according to the SBS content, and '+' or '-' according to whether tiny exon 9 is included. We suggest an extension to the existing nomenclature system, such that, for example, a transcript starting with exon 7b and ending at the polyadenylation site in intron 18, containing exon 11b spliced to exon 14 and including exon 9, would be called 'α-dystrobrevin-5b+'. Red boxes - constitutive coding exons. Orange boxes - alternatively spliced coding regions. White boxes - untranslated regions. Green boxes - encoded SBSs. Black crosses - exons disrupted in murids. Exon numbering according to Ambrose et al. [41], apart from exons 7b and 11b, which are described first here.

Figure 2

Exon 11b and exon 7b are highly conserved but disrupted in murids. A) The murid exon 11b sequence has experienced multiple catastrophic defects, including frameshifting deletions and mutation of acceptor (mouse), donor (rat) and lariat branch (both species) sites. Red boxes - probable deleterious mutations. Yellow box - exon 11b. Arrow - unusual pyrimidine at +3 position. Broad taxonomy is shown at left; E - superorder Euarchontoglires; L - order Lagomorphia; R - order Rodentia; M - family Muridae. B) Selected phylogeny of superorder Euarchontoglires (based on Huchon et al. [61]), showing species in which exon 7b and exon 11b are known to be intact (bold lines) or disrupted (faint lines). Red star indicates most parsimonious time of mutation of both elements. C) Disruption of murid exon 7b by retroviral insertion. The upper panel shows the exon structure present in most mammals (to scale), while the lower shows that found in murids. Large horizontal arrow - majority transcriptional start site observed in non-murid transcript sequences. Red cross - donor splice site mutation. Small vertical arrows - murid-specific substitutions in exon 7. A detailed annotated alignment of this mammalian genomic region appears in Additional file 3.

Figure 3

Expression of α-dystrobrevin isoforms in human and mouse tissues. The relative expression of the discernable coarse and fine isoforms in A) human and B) mouse heart, brain, skeletal muscle and colon, as determined by quantitative RT-PCR. As indicated, horizontal axes show the tissues tested and the coarse and fine isoforms, while the vertical axis shows the relative expression level in arbitrary units. For human tissues, presence and absence of exon 9 (+ and -) was also assessed; these are shown as differently coloured portions of the bars. Asterisks in mouse indicate isoforms which while present in other mammals, are non-existent in murids. For display purposes, brain α-dystrobrevin-1a was used to normalise between species. Although α-dystrobrevin-3 is not directly comparable to the other reactions, heart α-dystrobrevin-3 has been normalised to brain α-dystrobrevin-1a, an approximate equivalence that is evident in published northern blots.

Figure 4

The exon 11b encoded sequence is a functional SBS. A) Comparison of exon 11b encoded protein sequence (containing SBS1') with those of other vertebrate SBS-encoding dystrobrevin exons. Vertical dotted lines delimit the 15-residue pan-SBS consensus, and specific SBS consensus sequence logos are presented below the alignment. Arrow at top indicates the leucine mutated to proline in the yeast two-hybrid constructs. B) Schematic diagram showing observed α-dystrobrevin SBS isoform repertoires in various vertebrates. 'a*' is a one-SBS isoform seen in zebrafish, where the 'a' form is not observed. C) Yeast two-hybrid analysis of the interactions between mutant and wildtype α-dystrobrevin constructs and full-length β₁-syntrophin. Fine isoforms are shown as 'a', 'b', 'c', 'd'; mutant status of respective SBSs is shown as superscript 'w' (wild-type) or 'm' (mutant) and by green and red boxes, respectively. Growth on AHLT- plates was scored as '-' (0-10 colonies), '+' (10-100), '++' (100-1000) or '+++' (>1000).