Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
ProQR Therapeutics, Leiden, The Netherlands.
J Transl Med. 2023 Aug 16;21(1):546. doi: 10.1186/s12967-023-04406-x.
ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping.
A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids.
Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids.
Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.
与斯塔加特病（STGD1）相关的 ABCA4 基因包含 50 个外显子，其中 17 个外显子包含多个三个核苷酸。框内外显子跳跃的影响尚未确定。反义寡核苷酸（AONs）已在与 Usher 综合征相关的基因中进行了研究，以诱导携带严重变异的框内外显子跳跃，并减轻其与疾病相关的影响。在确定了一名携带新型外显子 17 经典剪接位点变异的 STGD1 先证者后，研究了缺失外显子 17 编码的 22 个氨基酸的 ABCA4 的活性，随后设计了能够诱导外显子 17 跳跃的 AONs。
STGD1 先证者复合杂合了剪接变异 c.2653+1G>A，该变异预计会导致外显子 17 框内跳跃，并伴有一个无效变异[c.735T>G，p.（Tyr245*）]。使用多模态成像和全面眼科检查研究了该先证者的临床特征。使用野生型和突变中间基因在 HEK293T 细胞中体外研究了 c.2653+1G>A 的异常剪接。用全反式视黄醛激活的 ATP 酶活性测定法以及亚细胞定位分析了缺失外显子 17 编码的 Asp864-Gly885 的突变 ABCA4 蛋白的残留活性。为了诱导外显子 17 跳跃，在 WT WERI-Rb-1 细胞和 3D 人视网膜类器官中体外研究了 40 种 AON 的效果。
先证者的迟发性 STGD1 表明 c.2653+1G>A 没有完全的有害影响。体外剪接试验证实该变异导致 ABCA4 转录物没有外显子 17。与野生型 ABCA4 相比，ABC4Asp864_Gly863del 稳定并保留了 58%的全反式视黄醛激活的 ATP 酶活性。该序列位于 ABCA4 的跨膜域 6 和核苷酸结合域-1 之间的无结构连接区。设计了 AONs 以可能降低外显子 17 中严重变异的致病性。最佳 AON 在视网膜类器官中实现了 59%的外显子 17 跳跃。
当与无效等位基因在反式时，ABCA4 中外显子 17 的缺失不会导致蛋白活性缺失，也不会导致严重的 STGD1 表型。通过应用 AONs，外显子 17 中严重变异的影响可以通过外显子跳跃得到改善，从而在 STGD1 患者中产生部分 ABCA4 活性。
