ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease

Abstract

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.

Figure 1.

Large minigene enables accurate RNA analysis of the ABCA4 noncanonical splice site variant c.5714+5G>A. (A) Minigene containing the genomic region encompassing ABCA4 exon 40 (MG_ex40, black rectangle) and flanked by rhodopsin (RHO) exons 3 and 5 (open boxes) was designed to investigate the effect of the noncanonical splice site variant c.5714+5G>A. Reverse-transcription polymerase chain reaction (RT-PCR) performed using primers (open arrowheads) targeting RHO exons 3 and 5 showed an expected 404-bp wild-type fragment and a 274-bp fragment corresponding to exon 40 skipping (Δ) in control minigene (+5G) and full exon 40 skipping in mutant minigene (+5A). (B) Larger minigene containing the genomic region encompassing ABCA4 exons 39–41 (MG_ex39–41) was designed to investigate the same variant. RT-PCR using primers (black arrowheads) targeting exons 39 and 41 showed a single 260-bp wild-type fragment in the control minigene, while in the mutant minigene fragments of 260 and 130 bp corresponding to wild-type and exon 40 skipping were found.

Figure 2.

Overview of the wild-type midigene splice constructs of ABCA4 and locations of all 47 noncanonical splice site variants. Exons are represented here as black rectangles. Employing bacterial artificial chromosome DNA and Gateway sequence-tagged primers, we amplified 29 genomic fragments and cloned these into the pCI-NEO-RHO Gateway-adapted vector (BA1–BA29). Genomic DNA from control persons (MG30 and MG31) and a patient carrying c.6729+5_6729+19del (MG30) were used to clone wild-type and mutant fragments.

Figure 3.

Overview of splice defects due to nine noncanonical splice site variants in ABCA4. All wild-type and mutant midigenes were transfected in HEK293T cells and their RNA subjected to RT-PCR. (A,B) RT-PCR for wild-type and c.160+5G>C mutant BA1 midigene showed complex splicing defects when using primers in RHO exon 3 and ABCA4 exon 4. Five defects (fragments 2 through 6) were observed next to the wild-type fragment 1. Asterisks denote fragments for which we obtained no sequence information. The RT-PCR products showed skipping of exon 2, exon 3, or exons 2 and 3. (C) Sequencing of pGEM-T-cloned fragment 4 revealed a 14-nt insertion due to the activation of a cryptic splice donor site in intron 2 and the absence of exon 3. The triangle points to the +5G>C variant. (D,E) RT-PCR products of wild-type and mutant BA1 containing NCSS variants residing at splice sites of exons 3 and 4. Primers in ABCA4 exons 2 and 4 reveal exon 3 skipping and/or exon 4 elongation. Note that the wild-type vector BA1 shows exon 3 skipping (fragment 5), which is more pronounced when using a forward RHO exon 3 primer (A) compared to using a forward ABCA4 exon 2 primer (D). (F) Sanger sequence of gel-purified fragment 3 shows a 2-nt elongation at the 3′ end of exon 4 due to the activation of a cryptic splice acceptor site in intron 3. Sanger sequencing results for all fragments without an asterisk are provided in Supplemental Figure S3. (G,H) RT-PCR and Sanger sequencing of RNA extracted from c.768G>T mutant BA4 midigene showed a 35-nt exon 6 elongation. (I) Human Splice Finder (HSF) splice site scores (blue arrowheads) for wild-type and mutant sites. (J,K) RT-PCR, gel analysis, and Sanger sequencing showed the wild-type product and a 12-nt exon 13 elongation due to c.1937+13T>G. M and WT denote mutant and wild-type splice products. (L) Details of the exon elongation defect. As represented by the HSF prediction software, the scores of the normal and intronic splice donor sites (SDSs) are not changed due to this variant. Most of the RT-PCR product consists of the exon elongation which introduces a premature stop-codon. (M–O) Variants at the penultimate position of exon 30 (c.4538A>G and c.4538A>C) resulted in a correct mRNA (fragment 1), a 30-nt exon 30 elongation due to the presence of a strong cryptic intronic splice donor site (fragment 2), a deletion of 73 nt of the 3′ end of exon 30 (fragment 3), and a 30-nt exon 30 elongation in combination with a 114-nt deletion of the 5′ end of exon 30 (fragment 4). Fragments 3 (227 bp) and 4 (216 bp) could not be resolved. The exon 30 deletions are the results of cryptic splice donor and acceptor sites that overlap at exonic positions c.4465–4468 (Supplemental Fig. S2, BA20). (P) RT-PCR of BA27 carrying the c.5898+5del variant resulted in at least two exon 42 elongation products of 717 and 780 bp, as well as intron 42 retention. (Q) The exon elongation products were due to the reduction of the strength of the exon 42 splice donor site, as shown with red numbers, in combination with the presence of nearby cryptic intronic splice donor sites with strong predicted scores. Only HSF and SpliceSiteFinder (SSF)-like scores are provided. Note that SSF-like is the only software that predicts the rare splice donor sites (positions c.5898+108 and c.5899−23) that contain the canonical GC sequence instead of GU. Intron 42 retention likely is due to the very strong splice donor site (HSF 98.0) flanking exon 43. The open arrowhead denotes the 1-bp deletion.

Figure 4.

Percentages of remaining normal ABCA4 transcripts due to noncanonical splice site variants based on capillary electrophoresis system analyses. The percentages of normal ABCA4 transcript for 45 noncanonical splice site variants are represented by black bars. Nineteen variants showed varying fractions (4.3%–100%) of correct ABCA4 mRNA. Four of them also result in missense changes as depicted, but only p.(Ser1545Asn) and p.(Lys2160Glu) are likely to have an effect on protein function, as significant amounts of ABCA4 protein will be produced. For the remaining 26 variants (in the square box), no residual RNA was observed. (#) For this variant, densitometric scanning was performed.