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

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.

Schlüsselwörter

primäres Offenwinkelglaukom - NOTCH2 - Hochleistungsflüssigkeitschromatografie - Genetik

Key words

primary open angle glaucoma - NOTCH2 - high-performance liquid chromatography - genetics

• References

• 1 Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996; 80: 389-393
• 2 Quigley HA. Glaucoma. Lancet 2011; 377: 1367-1377
• 3 Coleman AL, Brigatti L. The glaucomas. Minerva Med 2001; 92: 365-379
• 4 Mataftsi A, Achache F, Heon E et al. MYOC mutation frequency in primary open-angle glaucoma patients from Western Switzerland. Ophthalmic Genet 2001; 22: 225-231
• 5 Stone EM, Fingert JH, Alward WL et al. Identification of a gene that causes primary open angle glaucoma. Science 1997; 275: 668-670
• 6 Rezaie T, Child A, Hitchings R et al. Adult-onset primary open angle glaucoma caused by mutations in optineurin. Science 2002; 295: 1077-1079
• 7 Monemi S, Spaeth G, DaSilva A et al. Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum Mol Genet 2005; 14: 725-733
• 8 Footz TK, Johnson JL, Dubois S et al. Glaucoma-associated WDR36 variants encode functional defects in a yeast model system. Hum Mol Genet 2009; 18: 1276-1287
• 9 Pasutto F, Matsumoto T, Mardin CY et al. Heterozygous NTF4 mutations impairing neutrophin-4 signaling in patients with primary open-angle glaucoma. Am J Hum Genet 2009; 85: 447-456
• 10 Guo Y, Chen X, Zhang H et al. Association of OPA1 polymorphisme with NTG and HTG: a meta-analysis. PloS One 2012; 7: e42387
• 11 Aung T, Ocaka L, Ebenezer ND et al. A major marker for normal tension glaucoma: association with polymorphisms in the OPA1 gene. Hum Genet 2002; 110: 52-56
• 12 Kaut K, Mandal AK, Chakrabarti S et al. Primary congenital glaucoma and the involment of CYP1B1. Middle East Afr J Ophthalmol 2011; 18: 7-16
• 13 Jelodari-Mamaghani S, Haji-Seyed-Javadi R, Suri F et al. Contribution of the latent transforming growth factor-beta binding protein 2 gene etiology of primary open angle glaucoma and pseudoexfoliation syndrome. Mol Vis 2013; 19: 333-347
• 14 Pérez-Rico C, Gutiérrez-Diaz E, Mencia-Gutiérrez E et al. Obstructive sleep apnea-hypopnea syndrome (OSAHS) and glaucomatous optic neuropathy. Graefes Arch Clin Exp Ophthalmol 2014; 252: 1345-1357
• 15 Weinreb RN, Aung T, Medeiros FA. The pathophysiology and treatment of glaucoma: a review. JAMA 2014; 14: 1901-1911
• 16 Goel M, Picciani RG, Lee RK et al. Aqueous humor dynamics: a review. Open Ophthalmol J 2010; 4: 52-59
• 17 Howell GR, Libby RT, John SW et al. Mouse genetic models: an ideal system for understanding glaucomatous neurodegeneration and neuroprotection. Prog Brain Res 2008; 173: 303-321
• 18 Sigal IA, Ethier CR. Biomechanics of the optic nerve haed. Exp Eye Res 2009; 88: 799-807
• 19 Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signalling: cell fate control and signal integration in development. Science 1999; 284: 770-776
• 20 Koch U, Radtke F. Notch and cancer: a double-edged sword. Cel Mol Life Sci 2007; 64: 2746-2762
• 21 Turnpenny PD, Ellard S. Alagille syndrome: Pathogenesis, diagnosis and management. Eur J Hum Genet 2012; 20: 251-257
• 22 Zhou Y, Tanzie C, Yan Z et al. Notch2 regulates BMP signalling and epithelial morphogenesis in the ciliary body of the mouse eye. Proc Natl Acad Sci U S A 2013; 110: 8966-8971
• 23 Andersson ER, Sandberg R, Lendahl U. Notch signalling: simplicity in design, versatility in function. Development 2011; 138: 3593-3612
• 24 Sarode B, Nowell CS, Jungeun I et al. Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation. Pigment Cell Melanoma Res 2014; 27: 580-589
• 25 Mendoza-Reinoso V, Patil TS, Guevara-Fujita ML et al. Novel and known MYOC exon 3 mutations in an admixed Peruvian primary open-angle glaucoma population. Mol Vis 2012; 18: 2067-2075
• 26 Hung CC, Su YN, Lin CY et al. Comparison of the mismatch-specific endonuclease method and denaturing high performance liquid chromatography for the identification of HBB gene mutations. BMC Biotechnol 2008; 8: 62