UCLA Stein Eye Institute and Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA.
Transl Vis Sci Technol. 2022 Mar 2;11(3):33. doi: 10.1167/tvst.11.3.33.
Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease.
Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system.
Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease.
Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue.
This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.
现代分子遗传学彻底改变了基因发现、基因诊断和精准医学，但仍有许多患者无法从中受益，因为多达一半的孟德尔遗传疾病的致病变体仍然难以捉摸。本研究采用患者来源的诱导多能干细胞（iPS）和转录组学来确定尚未解决的斯塔加特病患者 ABCA4 等位基因的命运。
从 3 名携带单一已识别致病性 ABCA4 变体的斯塔加特病患者的皮肤活检中生成了多个独立的 iPS 系。从正常对照和患者细胞中衍生的视网膜色素上皮细胞（dRPE）在 Novaseq6000 平台上进行 RNA-Seq 分析，使用 DESeq2 进行分析，并从致病性或已知连锁变体计算等位基因特异性失衡。使用自动化 Simple Western 系统进行蛋白质分析。
生成了 9 个 dRPE 样本，其中 8 个进行了转录组分析。等位基因特异性表达表明，尽管水平较低，但剪接变体表达的正常转录本，错义转录本表达水平接近正常。相应的蛋白质不易检测到。患者表型相关性表明，高水平表达的错义变体具有更具破坏性的后果。转录组分析表明，线粒体膜动力学和未折叠蛋白反应途径可能与斯塔加特病有关。
患者特异性 iPS 衍生的 RPE 细胞为使用容易活检的组织评估难以检测的基因组区域中不表达的变体奠定了基础。
许培扬
