Identification of a Novel Mucin Gene HCG22 Associated With Steroid-Induced Ocular Hypertension

Abstract

Purpose: The pathophysiology of ocular hypertension (OH) leading to primary open-angle glaucoma shares many features with a secondary form of OH caused by treatment with glucocorticoids, but also exhibits distinct differences. In this study, a pharmacogenomics approach was taken to discover candidate genes for this disorder.

Methods: A genome-wide association study was performed, followed by an independent candidate gene study, using a cohort enrolled from patients treated with off-label intravitreal triamcinolone, and handling change in IOP as a quantitative trait.

Results: An intergenic quantitative trait locus (QTL) was identified at chromosome 6p21.33 near the 5' end of HCG22 that attained the accepted statistical threshold for genome-level significance. The HCG22 transcript, encoding a novel mucin protein, was expressed in trabecular meshwork cells, and expression was stimulated by IL-1, and inhibited by triamcinolone acetate and TGF-β. Bioinformatic analysis defined the QTL as an approximately 4 kilobase (kb) linkage disequilibrium block containing 10 common single nucleotide polymorphisms (SNPs). Four of these SNPs were identified in the National Center for Biotechnology Information (NCBI) GTEx eQTL browser as modifiers of HCG22 expression. Most are predicted to disrupt or improve motifs for transcription factor binding, the most relevant being disruption of the glucocorticoid receptor binding motif. A second QTL was identified within the predicted signal peptide of the HCG22 encoded protein that could affect its secretion. Translation, O-glycosylation, and secretion of the predicted HCG22 protein was verified in cultured trabecular meshwork cells.

Conclusions: Identification of two independent QTLs that could affect expression of the HCG22 mucin gene product via two different mechanisms (transcription or secretion) is highly suggestive of a role in steroid-induced OH.

Figures

Figure 1

Manhattan plot of the GWAS for steroid-induced OH. The Manhattan plot depicts the genome-wide P values. The results for the total of 1,545,328 genotyped SNPs that passed the quality control criteria are plotted as −log10(P value) by genomic position. The red line indicates the genome-wide significance level of P = 5 × 10−8.

Figure 2

Regional association plot for the lead SNP rs2523864 (marked as purple diamond) on Chr6. All SNPs shown as circles are plotted with their respective P values against their genomic location. The solid diamond indicates the top-ranked SNP rs2523864. The colored box at the right or left corner of each plot indicates the pairwise correlation (r2) between the top SNP and the other SNPs in the region. The gray circles indicate the imputed SNPs from the CEU population of the HapMap. Each plot was created using LocusZoom (available in the public domain at http://csg.sph.umich.edu/locuszoom/) for the top-ranked SNP in each region with a 400-kb region surrounding it. The SNPs are plotted at the top of the figure. The box underneath each plot shows the gene annotations in the region, with the arrow indicated the DNA strand for transcription. The lower LD plot was created using Haploview (available in the public domain at http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview).

Figure 3

Location and expression of genes surrounding the identified QTL in chromosomal region 6p21.32-33. Top: Schematic of chromosomal region 6p21.32-33 from the NCBI Gene website depicting annotated genes surrounding the identified QTL (red arrow). Bottom: Total RNA was purified from cultured primary HCE and TM-1 cells and used for cDNA synthesis. Reverse transcription-PCR using cDNA was performed using gene-specific primers from MUC21, MUC22, and HCG22, and the products were resolved on a 1.5% agarose gel. Primers for HCG22 were designed to detect only the coding transcript. Similar results were obtained using three primary TBM cell lines (not shown). RTase, reverse transcriptase; HCE, primary corneal epithelial cells obtained from corneal rims.

Figure 4

Transcriptional analysis. Trabecular meshwork-1 cells in 12-well plates were treated with IL-1β (10 ng/mL), TGF-β2 (10 ng/mL), and TA (100 ng/mL), individually or in combination, in serum-free media for 17 hours. The HCG22 mRNA levels were measured by qRT-PCR. Relative expression of HCG22 was normalized by the expression of GAPDH in individual samples. Student's t-test was used to identify the statistical significance between treated and untreated samples: *P < 0.01 (n = 5), **P = 0.02 (n = 5), P = 0.07 (n = 4).

Figure 5

Translation, O-glycosylation and secretion of HCG22 protein. The HEK293 cells expressing HCG22-His tag protein were cultured in the presence of benzyl-GalNAc at indicated doses. After 24 hours, the conditioned media were harvested. Secreted proteins were resolved on a denaturing 10% SDS/PAGE gel, blotted, and probed with anti-His antibody.