GALC Deletions Increase the Risk of Primary Open-Angle Glaucoma: The Role of Mendelian Variants in Complex Disease

DNA copy number variants (CNVs) have been reported in many human diseases including autism and schizophrenia. Primary Open Angle Glaucoma (POAG) is a complex adult-onset disorder characterized by progressive optic neuropathy and vision loss. Previous studies have identified rare CNVs in POAG; however, their low frequencies prevented formal association testing. We present here the association between POAG risk and a heterozygous deletion in the galactosylceramidase gene (GALC). This CNV was initially identified in a dataset containing 71 Caucasian POAG cases and 478 ethnically matched controls obtained from dbGAP (study accession phs000126.v1.p1.) (p = 0.017, fisher's exact test). It was validated with array comparative genomic hybridization (arrayCGH) and realtime PCR, and replicated in an independent POAG dataset containing 959 cases and 1852 controls (p = 0.021, OR (odds ratio) = 3.5, 95% CI −1.1–12.0). Evidence for association was strengthened when the discovery and replication datasets were combined (p = 0.002; OR = 5.0, 95% CI 1.6–16.4). Several deletions with different endpoints were identified by array CGH of POAG patients. Homozygous deletions that eliminate GALC enzymatic activity cause Krabbe disease, a recessive Mendelian disorder of childhood displaying bilateral optic neuropathy and vision loss. Our findings suggest that heterozygous deletions that reduce GALC activity are a novel mechanism increasing risk of POAG. This is the first report of a statistically-significant association of a CNV with POAG risk, contributing to a growing body of evidence that CNVs play an important role in complex, inherited disorders. Our findings suggest an attractive biomarker and potential therapeutic target for patients with this form of POAG

Three-Primer PCR assay for <i>GALC</i> deletion.

<p>(A) Schematic diagram of a portion of the <i>GALC</i> gene showing the region around the known GALC deletion. Both primers Del_P1 and Del_P3 are located outside of the deletion while primer Del_P2 is inside the deletion and close to the left breakpoint. (B) DNA samples with heterozygous deletion produced two DNA bands (329 bp and 615 bp in size) while DNA samples without GALC deletion generated one DNA band of 615 bp in size. The white arrow indicates the DNA sample with heterozygous deletion.</p

Validation of <i>GALC</i> deletion with array CGH.

<p>The <i>GALC</i> deletion from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027134#pone-0027134-g001" target="_blank">Figure 1</a> was validated with high resolution chromosome 14 specific CGH array. This figure showed a 31 kb deletion in the <i>GALC</i> gene, indicated by the red line.</p

Heterogeneity of <i>GALC</i> deletions in different DNA samples.

<p>Two heterozygous <i>GALC</i> deletions with different end-points were identified by array CGH, indicated by the red line. The lower diagram shows the previously reported 31 kb <i>GALC</i> deletion while the upper diagram shows a smaller deletion of approximately 15 kb.</p

Heterozygous DNA deletion in the GALC gene.

<p>A heterozygous DNA deletion in the galactosylceramidase (<i>GALC</i>) gene present in 2 of 71 POAG cases and absent in 478 controls. The deletion was identified through six probes common to both the Illumina HumanCNV370-Duo and Human610-Quad BeadChips. The deletion indicated by the red-colored box includes five exons (exons 11–15) and abolishes enzymatic activity.</p

Summary of <i>GALC</i> deletion screening in all datasets.

<p>The CHOP dataset was obtained from the Copy Number Variation project at the Children's Hospital of Philadephia (CHOP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027134#pone.0027134-Shaikh1" target="_blank">[25]</a>; Fisher's exact p-value was based on one-tailed test, as we hypothesize that loss or decrease of GALC function results in POAG risk.</p