September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
LOXL1 intronic variants influence LOXL1 expression through differential transcription factor binding in pseudoexfoliation syndrome/glaucoma
Author Affiliations & Notes
  • Ursula Schlötzer-Schrehardt
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Matthias Zenkel
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Ursula Hoja
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Mineo Ozaki
    Ozaki Eye Hospital, Miyazaki, Japan
  • Tin Aung
    Singapore Eye Research Institute, Singapore, Singapore
  • Chiea-Chuen Khor
    Singapore Eye Research Institute, Singapore, Singapore
  • Friedrich E Kruse
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Andre Reis
    Institute of Human Genetics, University of Erlangen-Nürnberg, Germany
  • Francesca Pasutto
    Institute of Human Genetics, University of Erlangen-Nürnberg, Germany
  • Footnotes
    Commercial Relationships   Ursula Schlötzer-Schrehardt, None; Matthias Zenkel, None; Ursula Hoja, None; Mineo Ozaki, None; Tin Aung, None; Chiea-Chuen Khor, None; Friedrich Kruse, None; Andre Reis, None; Francesca Pasutto, None
  • Footnotes
    Support  DFG Grant SCHL 366/8-1
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 789. doi:
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      Ursula Schlötzer-Schrehardt, Matthias Zenkel, Ursula Hoja, Mineo Ozaki, Tin Aung, Chiea-Chuen Khor, Friedrich E Kruse, Andre Reis, Francesca Pasutto; LOXL1 intronic variants influence LOXL1 expression through differential transcription factor binding in pseudoexfoliation syndrome/glaucoma. Invest. Ophthalmol. Vis. Sci. 2016;57(12):789.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To analyze the mechanisms by which a previously identified haplotype harboring 4 intronic regulatory variants (rs12905253, rs11638944, rs12441130, rs11631579) of LOXL1 (lysyl oxidase-like 1) may influence risk for pseudoexfoliation (PEX) syndrome/glaucoma.

Methods : Tissues obtained from donor eyes with PEX syndrome/glaucoma (n=52) and age-matched control eyes (n=51) were used for genotype-correlated expression analysis of LOXL1 by real time PCR and Western blotting. Reporter gene assays were performed in human Tenon’s capsule fibroblasts, trabecular meshwork cells, ciliary epithelial cells, limbal epithelial cells, optic nerve head astrocytes, aortic smooth muscle cells, HEK293T and 3T3 cells to determine a cell type-specific effect of SNPs on LOXL1 promoter activity. Allele-specific transcription factor binding was analyzed by electrophoretic mobility (super)shift (EMSA) assays using nuclear extracts from all cell types and antibodies against transcription factors predicted to bind by rSNP-MAPPER. Taqman primer assays were used to compare relative allelic pre-mRNA LOXL1 expression levels in heterozygous cell lines.

Results : Real time PCR showed tissue-specific dysregulation of LOXL1 expression, which was reduced by 30-60% in all anterior segment tissues, lamina cribrosa, and aortic wall of PEX patients compared to controls (p<0.001). Luciferase reporter assays showed that the risk haplotype reduced LOXL1 promoter activity by 35-45% (p<0.0001) compared to the non-risk haplotype in disease-related cell types, but not in unrelated cell models confirming a cell-type specificity of gene regulation. Allele-specific differences in DNA-protein binding efficiency were demonstrated by EMSA using DNA fragments spanning the individual SNPs or their haplotypes and nuclear extracts from disease-relevant cell types. Supershift assays provided evidence for an allele-specific binding pattern of specific transcription factors. Pre-mRNA analysis from heterozygous cells showed allelic imbalance by Taqman-based SNP analysis confirming that reduced LOXL1 tissue expression arises from the intronic risk alleles.

Conclusions : These findings provide the first functional link between noncoding LOXL1 variants and PEX pathophysiology, which can explain the mechanisms by which LOXL1 risk variants predispose to connective tissue weakness and clinical complications in PEX patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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