Genetic variants and haplotypes in fibulin-5 (FBLN5) are associated with pseudoexfoliation glaucoma but not with pseudoexfoliation syndrome

Abstract

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.

Keywords: fibulin-5; functional variant; pseudoexfoliation glaucoma; tag genotyping.

Figure 1. Position of Tag SNPs

Gene structure of FBLN5 showing position of the 13 tag SNPs (obtained from UCSC genome browser June 5, 2022).

Figure 2. Linkage disequilibrium pattern and haplotype association analysis

(A) LD pattern across all the SNPs. The LD blocks are defined using the confidence interval algorithm. (B) LD block pattern with rs72705342-rs17732466.

Figure 3. Putative regulatory effect of FBLN5 variants

(A) Relative luciferase activity of constructs containing rs17732466 with either ‘A’ or ‘G’ alleles cloned upstream of the minimal promoter. No difference in reporter activities was observed with either ‘A’ (84.8 ± 14.3) or ‘G’ (68.2 ± 12.9) alleles compared with empty vector (100.0 ± 15.8) with P-values 0.50 and 0.17, respectively. Also, no difference in the activity with change in alleles was observed (P=0.42). Data represent mean ± SEM of four independent experiments. (B) Relative luciferase activity of constructs containing rs72705342 with either allele ‘T’ or allele ‘C’ cloned upstream of the minimal promoter. Significant differences in reporter activities between the rs72705342 element with ‘T’ allele (203.8 ± 20.8) and ‘C’ allele (141.3 ± 14.8) or empty vector (100.0 ± 13.5) were observed with P-values of 0.03 and 0.001, respectively. No significant difference was observed between empty vector and construct with ‘C’ allele (P=0.06). Data represent mean ± SEM of four independent experiments. (C) Relative luciferase activity of constructs containing rs72705342 with either allele ‘C’ or allele ‘T’ upstream of FBLN5 core promoter. Change from allele ‘C’ to ‘T’ showed a significant increase (P=0.02) in luciferase activity. No significant difference was observed between the empty vector and the construct with allele ‘T’ (P=0.15) or allele ‘C’ (P=0.27). Data represent mean ± SEM of three independent experiments; **P< 0.01, *P<0.05.

Figure 4. Binding of specific transcription factors to rs72705342

(A) EMSA using 29bp biotinylated DNA probes with rs72705342 ‘C’ or ‘T’ allele and nuclear extract from HLE B-3 cells showed specific DNA–protein complexes which vanished on competitive inhibition with respective unlabelled excess probes. (B) Quantitative analysis of the shifted bands relative to the unshifted bands showed allele-specific differences with significantly stronger binding of the ‘C’ allele with the nuclear protein compared with the ‘T’ allele set at 100%. Data represent mean ± SEM of four independent experiments; *P<0.05. (C) Competitive EMSA on rs72705342 ‘C’ robe with increasing concentrations of unlabelled excess (50-, 100-, 200- and 400-fold) showed gradual decrease in the intensity of the shift. (D) Allele (encircled) specific transcription factor binding at rs72705342 as predicted on PROMO software. The ‘C’ allele at rs72705342 is predicted to bind to GR-α and TFII I transcription factors, the binding of which is predicted to be lost on change in allele from ‘C’ to ‘T’. (E) The EMSA shows that in the presence of TFII I specific antibody, the intensity of the shift reduced which did not happen in the presence of non-specific antibody (HSF1) taken as control. (F) The EMSA shows that in the presence of GR-α-specific antibody, the intensity of the shift reduced which did not happen in the presence of non-specific antibody taken as control.