Identification and Characterization of Genetic Factors Responsible for Cavitary Optic Disc Anomalies

Abstract

Purpose: To identify and characterize the gene that causes autosomal dominant, congenital malformations of the optic nerve known as cavitary optic disc anomaly (CODA) in a multiplex family with 17 affected members. Features of the nerve disease in CODA closely resemble that of glaucoma (excavated optic nerve head appearance) that occurs in the absence of elevated IOP, suggesting that the gene that causes CODA may also contribute to the pathophysiology of glaucoma.

Methods: The gene that causes CODA was previously mapped to a 13.5Mb locus on chromosome 12q14. The proband of the CODA pedigree was tested for copy number variations (CNVs) in the chromosome 12q14 region with custom comparative genomic hybridization using the NimbleGen platform (Madison, WI). CNVs were confirmed with quantitative PCR in the proband and in the remaining 16 affected family members as well as in a panel of 78 controls. Confirmed CNVs were analyzed for their effect on downstream genes using a luciferase reporter gene construct (pGL3, Promega, Madison, WI) in HEK293T cells.

Results: CGH experiments identified a triplication of a 6kb segment of DNA upstream of matrix metalloproteinase 19 (MMP19) in the proband of the CODA pedigree. Quantitative PCR experiments confirmed that the MMP19 promoter CNV is present in the proband as well as in the additional 16 affected family members. No control subjects carried this mutation. Furthermore there were no instances of this variant in the database of genomic variants (projects.tcag.ca/variation). The luciferase reporter gene assay showed that the 6kb sequenced spanned by the CNV in CODA patients increased luciferase activity and functioned as a transcription enhancer. Moreover, a similar analysis of overlapping subdivisions of the DNA sequence in the 6kb CNV showed that a 1kb segment had a strong positive influence (8-fold higher) on downstream gene expression.

Conclusions: We have identified a copy number variation mutation in the promoter sequence of the MMP19 gene that co-segregates with CODA in our large 17 member pedigree. Moreover we have shown that the CNV spans DNA sequences that powerfully enhance downstream genes (i.e. MMP19). These functional data strongly suggest that the genetic defects in MMP19 cause CODA and that over-activity of this gene has an important role in the pathogenesis of this optic nerve disease.

Keywords: 539 genetics • 627 optic disc • 629 optic nerve