Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United states of America.
Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United states of America.
PLoS One. 2021 Dec 2;16(12):e0260837. doi: 10.1371/journal.pone.0260837. eCollection 2021.
Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.
CTG 三核苷酸重复（TNR）在转录因子 4（TCF4）基因中的扩增与 Fuchs 内皮角膜营养不良（FECD）高度相关。由于病变角膜内皮细胞中 DNA 的可用性有限，通常使用从外周血白细胞中分离的 DNA 样本来确定 FECD 患者的 CTG 重复次数。然而，根据目前的技术，从手术分离的 FECD 角膜内皮组织中提取足够的 DNA 来确定重复长度是不可行的。为了解决这个问题，从 FECD 角膜内皮中分离出总 RNA 并使用长读测序进行测序。还评估了从这些相同受影响个体的角膜内皮原代培养物中分离的 DNA 样本的 Southern 印迹分析。长读测序和 Southern 印迹分析均显示，FECD 患者的角膜内皮中的 CTG TNR 扩增明显更长（>1000 个重复），而同一个体的白细胞中的 CTG TNR 扩增则较短（<90 个重复）。我们的研究结果表明，FECD 角膜内皮中的 TCF4 CTG 重复扩增比白细胞中的要长得多。
