School of Biological Sciences, National Institute of Science Education and Research (NISER) Bhubaneswar, P.O. Bhimpur-Padanpur, Jatni, Khurda, Odisha 752050, India.
Homi Bhabha National Institute (HBNI), Training School Complex, Anushaktinagar, Mumbai, 400094, India.
Biosci Rep. 2023 Mar 31;43(3). doi: 10.1042/BSR20221622.
Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular proteinaceous aggregates on anterior ocular tissues. The present study aims to identify functional variants in fibulin-5 (FBLN5) as risk factors for the development of PEX. Thirteen tag single-nucleotide polymorphisms (SNPs) in FBLN5 were genotyped using TaqMan SNP genotyping technology to identify association between SNPs of FBLN5 and PEX in an Indian cohort comprising 200 control and 273 PEX patients (169 PEXS and 104 PEXG). Functional analysis of risk variants was done through luciferase reporter assays and electrophoretic mobility shift assay (EMSA) using human lens epithelial cells. Genetic association and risk haplotype analysis showed a significant association of rs17732466:G>A (NC_000014.9:g.91913280G>A) and rs72705342:C>T (NC_000014.9:g.91890855C>T) within FBLN5 as risk factors with the advanced severe stage of the disease, pseudoexfoliation glaucoma (PEXG). Reporter assays showed allele-specific regulatory effect of rs72705342:C>T on gene expression, wherein, construct containing the risk allele showed a significant decrease in the reporter activity compared with the one with protective allele. EMSA further validated higher binding affinity of the risk variant to nuclear protein. In silico analysis predicted binding sites for two transcription factors, GR-α and TFII-I with risk allele at rs72705342:C>T, which were lost in the presence of protective allele. The EMSA showed probable binding of both these proteins to rs72705342. In conclusion, the present study identified the novel association of two genetic variants in FBLN5 with PEXG but not with PEXS, distinguishing between the early and the later forms of PEX. Further, rs72705342:C>T was found to be a functional variant.
假性剥脱（PEX）是一种与年龄相关的多因素疾病，涉及细胞外蛋白质聚集物在前眼部组织上的沉积。本研究旨在鉴定纤维连接蛋白 5（FBLN5）中的功能变体作为 PEX 发展的风险因素。使用 TaqMan SNP 基因分型技术对 FBLN5 中的 13 个标签单核苷酸多态性（SNP）进行基因分型，以鉴定印度队列中 SNP 与 200 名对照和 273 名 PEX 患者（169 名 PEXS 和 104 名 PEXG）之间的关联。通过荧光素酶报告基因检测和电泳迁移率变动分析（EMSA）在人晶状体上皮细胞中对风险变异进行功能分析。遗传关联和风险单倍型分析显示，rs17732466：G>A（NC_000014.9：g.91913280G>A）和 rs72705342：C>T（NC_000014.9：g.91890855C>T）在 FBLN5 内作为风险因素与疾病的晚期严重阶段，假性剥脱性青光眼（PEXG）显著相关。报告基因检测显示 rs72705342：C>T 对基因表达具有等位基因特异性调节作用，其中含有风险等位基因的构建体与含有保护等位基因的构建体相比，报告活性显著降低。EMSA 进一步验证了风险变异与核蛋白的更高结合亲和力。计算机分析预测了 rs72705342 处的两个转录因子，GR-α 和 TFII-I 的结合位点，而在保护性等位基因存在的情况下，该位点丢失。EMSA 显示这两种蛋白可能与 rs72705342 结合。总之，本研究鉴定了 FBLN5 中两个遗传变异与 PEXG 的新关联，但与 PEXS 无关，区分了 PEX 的早期和晚期形式。此外，rs72705342：C>T 被发现是一个功能性变体。
