Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

Abstract

Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Figure 1. An SVA retrotransposal insertion induces abnormal splicing in FCMD

a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin. Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts (d) and model mice (e). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH (LDLRAP1, left), NLSDM (PNPLA2, middle), and human (AB627340, right).

Figure 2. Abnormal fukutin protein in FCMD

a-c, Immunoprecipitation analysis of fukutin protein in human lymphoblasts (a), both skeletal muscle and brain tissues from Hp/Hp mice (b), and brain tissue from FCMD patients (c). Closed arrow, abnormal fukutin. N, normal sample; F, FCMD patient sample. Hn, Hn/Hn mice; Hp, Hp/Hp mice; PI, preimmune serum. D, Duchenne muscular dystrophy (DMD) patient. d, The subcellular localization of fukutin. Top, normal fukutin; middle, mis-spliced fukutin; bottom, truncated fukutin. Stained with anti-FLAG (Left, to detect fukutin), anti-GM130 (middle, Golgi marker, top) and anti-KDEL (ER marker, middle and bottom), and merge (right, with DAPI stain). Scale bar, 10 μm.

Figure 3. AON cocktail rescues normal fukutin mRNA

a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p < 0.01 by Student’s t-test). Error bars, s.e.m.

Figure 4. AON cocktail treatment rescues normal fukutin protein and functional α-DG

a, d, Immunoprecipitation analysis of fukutin protein after local treatment with VMO (AED) in FCMD model mice (a) and human FCMD lymphoblasts (d). Arrow, normal fukutin protein. L, left TA; R, right TA. Dys, negative control. b, c, e, TA muscle after local (b) or systemic (c) AED treatment and human FCMD lymphoblasts treated with the AED (e) were analyzed by Western blot using antibodies against α-DG core protein (top panel) and glycosylated α-DG (second), and by a laminin overlay assay (third). Bottom, β-DG (internal control). f, Laminin clustering assay. Left, anti-laminin; middle, anti-glycosylated α-DG; right, merged images. Upper, normal myotubes treated with control VMO; middle, FCMD patient myotubes treated with control VMO; bottom, FCMD patient myotubes treated with AED.