期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 60, 期 8, 页码 3084-3090出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.19-26930
关键词
congenital hereditary endothelial dystrophy; SLC4A11; corneal endothelial-like cells model; induced pluripotent stem cells
资金
- National Center for Medical Genomics [LM2015091, CZ.02.1.01/0.0/0.0/16_013/0001634]
- GACR [17-12355S]
- Charles University [UNCE 204064, PROGRES Q26]
- Fight for Sight
- Moorfields Eye Charity
- National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology
- Wellcome Trust, London
- [GAUK 250361/2017]
- [SVV 260367/2017]
PURPOSE. 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. METHODS. 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. RESULTS. 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. CONCLUSIONS. This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据