Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease

Abstract

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

Keywords: ABCA4; Stargardt disease; antisense oligonucleotides; iPSC; intronic mutations; organoids; photoreceptors; retina; splicing; stem cells.

Graphical abstract

Figure 1

Fundus Autofluorescence Images of ABCA4-Associated Retinopathy Cases Harboring c.5196+1137G>A FAF images of patients harboring c.5196+1137G>A in trans with different groups of alleles. Classification of in transABCA4 alleles was performed previously based on electrophysiology. (A) Note the increasing retinal degeneration (no flecks, fleck outside the arcades, absorbed flecks, chorioretinal atrophy) in patients harboring c.5196+1137G>A in trans with null, when arranged by age. (B and D) Relatively milder phenotypes can be observed in patients harboring c.5196+1137G>A in trans with intermediate alleles c.5714+5G>A, p.L2027F and p.[G863A, G863del] (B) and in the homozygous state (D), even at older ages. (C) Patients harboring c.5196+1137G>A in trans with alleles of unknown severity arranged by age exhibiting various FAF patterns.

Figure 2

Electroretinography of ABCA4-Associated Retinopathy Cases Harboring c.5196+1137G>A (A and B) The dark-adapted (DA) 10.0 a-wave amplitudes (A) and the light-adapted (LA) 3.0 30-Hz amplitudes (B) of “double null” patients and patients harboring c.5196+1137G>A with different mutations in trans, plotted by age. (C and D) The DA 10.0 a-wave amplitudes (C) and the LA 3.0 30-Hz amplitudes (D) of patients harboring c.5196+1137G>A in trans with null alleles and patients harboring 15 other alleles of previously determined severity, plotted by age. Data from a single recording performed at the same center (MEH in Table 2) was used for each patient. One patient (ID 17) had non-recordable 30-Hz amplitudes due to noise. The gray area represents the 95% confidence interval (CI) of the healthy volunteers. On each chart, the “double null” patients are indicated as a baseline reference with their regression line and their CIs. Note the significantly better retinal function of patients harboring c.5196+1137G>A in comparison to the “double null” patients.

Figure 3

Pseudoexon Insertion Caused by Deep-Intronic Variants in Intron 36 (A) Schematic representation of intron 36 with the location of all the mutations and the pseudoexon (PE) insertion caused by each variant (M). The number of the different PEs indicates the size in nucleotides. (B) Identification of PEs using a midigene-based approach in HEK293T cells. RHO amplification was used as transfection and loading control. (C) Identification of a 73-nt PE in patient-derived photoreceptor precursor cells (PPCs). This PE is subjected to NMD, as its detection is clearly increased upon cycloheximide (CHX) treatment. ACTB was used as a loading control. MQ, the negative control of the PCR. Semiquantitative analysis of the ratio of aberrant and correct transcripts is indicated in the graphs below each electrophoresis gel. In all graphs, results are presented as average ± SD.

Figure 4

Assessment of Splicing Redirection by AONs Using a Midigene-Based System in HEK293T Cells (A) Schematic representation of the pseudoexons (PEs) introduced by each variant (M) and the relative position of the different AONs within the region. (B and B’) Assessment by RT-PCR of AON1 to AON4 (A1 to A4) in HEK293T cells transfected with either the WT midigene or the midigene containing the c.5196+1013A>G (M4) variant (B) and its corresponding semi-quantification expressed in the ratio of correct to aberrant transcript (B’). (C and C’) Splicing redirection by AON1 to AON4 for the c.5196+1056A>G (M6) variant in HEK293T cells (C) and its semi-quantification expressed in the ratio of correct to aberrant transcript (C’). (D and D’) Effect of AON5 to AON8 (A5 to A8) in redirecting the splicing defect introduced by c.5196+1216C>A (M11) in HEK293T cells (D) and its semi-quantification expressed in the ratio of correct to aberrant transcript (D’). In all cases, a scrambled oligonucleotide (SON) was used as negative control. MQ, the negative control of the PCR. In all graphs, results are presented as average ± SD.

Figure 5

AON-Based Splicing Correction for the c.5196+1137G>A, M9, Variant Using Patient-Derived Retina-like Cells (A) RT-PCR analysis of control and STGD1 individual PPCs treated with AON5 to AON8 (A5 to A8) or scrambled oligonucleotide (SON). Pseudoexon detection was enhanced by cycloheximide (+CHX) treatment. ACTB amplification was used as loading control. (B) Representation of the ratio of correct to aberrant transcript upon semi-quantification. (C) RT-PCR analysis of STGD1 individual-derived, 185-day-old retinal organoids treated with AON6 at the indicated concentrations or SON. ACTB amplification was used as loading control (of note, primers were different from those used in B). (D) Graphical representation of the ratio of correct to aberrant transcript upon semi-quantification. MQ, the negative control of the PCR. In all graphs, results are presented as average ± SD.