Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.
Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.
Invest Ophthalmol Vis Sci. 2019 Jul 1;60(8):3084-3090. doi: 10.1167/iovs.19-26930.
To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing.
Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts.
In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele.
This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
报道六名先天性遗传性内皮营养不良（CHED）患者的分子遗传学发现，这些患者的疾病表现各不相同，部分伴有听力损失（也称为 Harboyan 综合征）。此外，我们构建了一个细胞模型来确定疾病相关变异是否会诱导异常的 SLC4A11 前体 mRNA 剪接。
对五名先证者的整个 SLC4A11 编码区进行直接测序。在一名个体中进行了全基因组测序。通过暴露于 B27、PDGF-BB 和 DKK-2 的神经嵴细胞，在表达 SLC4A11 的角膜内皮样（CE 样）细胞模型中评估 c.2240+5G>A 对前体 mRNA 剪接的影响。CE 样细胞是从自体诱导多能干细胞（iPSC）衍生而来的。提取总 RNA，进行 RT-PCR，然后采用 Sanger 和靶向下一代测序（NGS）方法来鉴定和定量可变剪接转录本的相对丰度。
总共鉴定出 11 种不同的致病性 SLC4A11 突变，其中 c.1237G>A、c.2003T>C、c.1216+1G>A 和 c.2240+5G>A 为新发现的突变。c.2240+5G>A 变异被证明会导致异常的前体 mRNA 剪接。靶向 NGS 方法证实该变异会引入一个无义的隐蔽剪接供体位点，导致产生一个包含 6 个碱基插入的转录本，随后引入一个提前终止密码子（p.Thr747*）。此外，还观察到一组包含完整保留内含子 16 的转录本，导致相同的功能无效等位基因。
本概念验证研究强调了使用 CE 样细胞来研究 SLC4A11 疾病相关变异的致病后果的潜力。
