Molecular Analysis of NOTCH2 in Patients with Primary Open-Angle Glaucoma

 

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Abstract

Background and Purpose: Transgenic mice overexpressing Notch2 in the uvea exhibit a hyperplastic ciliary body leading to increased IOP and glaucoma. The aim of this study was to investigate the possible presence of NOTCH2 variants in patients with primary open-angle glaucoma (POAG). Methods: We screened DNA samples from 130 patients with POAG for NOTCH2 variants by denaturing high-performance liquid chromatography after PCR amplification and validated our data by direct Sanger sequencing. Results: No mutations were observed in the coding regions of NOTCH2 or in the splice sites. 19 known SNPs (single nucleotide polymorphisms) were detected. An SNP located in intron 24, c.[4005 + 45A>G], was seen in 28.5 % of the patients (37/130 patients). As this SNP is reported to have a minor allele frequency of 7 % in the 1000 genomes database, it could be associated with POAG. However, we evaluated its frequency in an ethnic-matched control group of 96 subjects unaffected by POAG and observed a frequency of 29 %, indicating that it was not related to POAG. Conclusion: NOTCH2 seemed to be a good candidate for POAG as it is expressed in the anterior segment in the human eye. However, mutational analysis did not show any causative mutation. This study also shows that proper ethnic-matched control groups are essential in association studies and that values given in databases are sometimes misleading.

Zusammenfassung

Hintergrund: In transgenen Mäusen, welche Notch2 in der Uvea überexprimieren, wurde ein hyperplastischer Ziliarkörper beobachtet, der zu einem Anstieg des intraokularen Drucks und Glaukom führte. Ziel dieser Arbeit war es, das mögliche Auftreten von NOTCH2-Varianten bei Patienten mit primärem Offenwinkelglaukom (POAG) zu untersuchen. Methoden: Wir haben 130 DNS-Proben von Patienten mit POAG auf NOTCH2-Varianten untersucht. Nach PCR-Amplifikation wurde die denaturierte DNS per Hochleistungsflüssigkeitschromatografie analysiert und die Resultate durch Sanger-Sequenzierung bestätigt. Ergebnisse: Weder in kodierenden Regionen noch an Splice Stellen von NOTCH2 wurden Varianten festgestellt. Es wurden 19 bekannte SNPs (single nucleotide polymorphisms) entdeckt. Ein SNP in Intron 24 [c.4005 + 45A>G] wurde bei 28,5 % der Patienten beobachtet (37/130 Patienten). Da diese SNP eine geringe Allelfrequenz von 7 % in der 1000-Genom-Datenbank aufweist, könnte es mit POAG assoziiert sein. Jedoch haben wir seine Häufigkeit in einer ethnisch homogenen Kontrollgruppe von 96 nicht von POAG betroffenen Individuen analysiert und eine Allelfrequenz von 29 % beobachtet, was eine Assoziation mit POAG unwahrscheinlich macht. Schlussfolgerungen: NOTCH2 könnte mit POAG assoziert sein, da es im vorderen Segment des menschlichen Auges exprimiert wird. Jedoch konnten bisher Molekularanalysen keine ursächliche Mutation nachweisen. Diese Arbeit zeigt die Relevanz von ethnisch homogenen Kontrollgruppen in Assoziationsstudien und dass das Betrachten von reinen Zahlen aus Datenbanken manchmal irreführend sein kann.

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