Introduction:
Glaucoma describes a highly heritable group of ocular conditions characterized by the progressive degeneration of the optic nerve. It is a leading cause of adult-onset vision loss worldwide and is often attributable to an increase in intraocular pressure (IOP). However, roughly one-third of cases occur in patients with a clinically measured IOP that is within normal range, which is classified as normal-pressure glaucoma (NPG). A subset of these patients fail to respond to conventional IOP-lowering therapies. Disease gene discovery projects elucidating the poorly understood molecular mechanisms of NPG pathogenesis are therefore necessary to inform new treatment options. To that end, our group has identified a 54-member pedigree with an aggressive and autosomal dominant form of early-onset NPG that has continued to progress despite successful IOP-lowering surgery.

Methods:
Logarithm of the odds (LOD) scores across the familial genome were calculated using a dominant, fully penetrant model in conjunction with an IlluminaQC SNP array. We then performed genome and exome sequencing, with resulting variants prioritized by familial/population allele frequencies, conservation scores, in silico predictors of pathogenicity, and gene expression data from publicly available RNAseq datasets. RNA was extracted from familial blood samples for Illumina RNAseq and processed through the DESeq2 pipeline to identify differentially expressed genes (DEGs). PANTHER Gene-Ontology (GO) analysis was also utilized to define dysregulated pathways. Finally, population databases such as GeneMatcher and the Undiagnosed Diseases Network were parsed to identify relevant phenotypes in other individuals with similar variants.

 

Results:
Clinical characterization and familial linkage/segregation results mapped the predominant NPG locus to chromosome 12q13 with a LOD peak of 2.6. This region contains a single plausible candidate variant that, in addition to being rare in the general population, is computationally predicted to be deleterious and occurs at a highly conserved residue: RPAP3 c.77G>T (p.R26L). The RPAP3 variant was absent in all unaffected individuals and segregated in all but two affected individuals. These two outliers likely developed optic atrophy secondary to extreme myopia, which had confounded diagnosis due to myopic degeneration.  RPAP3 is a known glaucoma GWAS hit that appears to be associated with the neurodegenerative component of the disease rather than an elevated IOP, consistent with NPG. Expression data demonstrated that this gene is highly expressed in the optic nerve head and the classes of astrocytes predicted by single-cell enrichment to produce the lamina cribrosa. Moreover, the resulting protein may indirectly modulate TGFβ signaling and extracellular matrix (ECM) protein production through its enzymatic interaction with molecular chaperone HSP90. DEGs analysis of familial RNA samples thus showed a significant degree of downregulation in TGFβ effectors and ECM components tracking with the RPAP3 variant. Specifically, GO annotation revealed some of the most affected pathways included production of ECM structural constituents (FDR 5.4E-9), as well as proteins involved in ECM, fibrinogen, fibronectin, and integrin binding (FDRs 2.3E-2, 4.3E-2, 7.5E-3, and 2E-3) and collagen metabolism (FDR 1E-2). Subsequently, population data suggested a strong association between biallelic loss of RPAP3 function and epileptic encephalopathy in pediatric patients. The great-aunt of one such proband was noted to also have aggressive, treatment-resistant NPG and carry the RPAP3 c.125T>C (p.L42P) variant. Genome sequencing of this family uncovered no additional variants in known glaucoma disease genes.

 

Conclusion:
These data support a role for RPAP3 in NPG pathogenesis and suggest ECM production/maintenance as a possible therapeutic target.