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. 2011 Apr;19(4):445-51.
doi: 10.1038/ejhg.2010.217. Epub 2011 Jan 12.

Evidence for RPGRIP1 Gene as Risk Factor for Primary Open Angle Glaucoma

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Free PMC article

Evidence for RPGRIP1 Gene as Risk Factor for Primary Open Angle Glaucoma

Lorena Fernández-Martínez et al. Eur J Hum Genet. .
Free PMC article

Abstract

Glaucoma is a genetically heterogeneous disorder and is the second cause of blindness worldwide owing to the progressive degeneration of retinal ganglion neurons. Very few genes causing glaucoma were identified to this date. In this study, we screened 10 candidate genes of glaucoma between the D14S261 and D14S121 markers of chromosome 14q11, a critical region previously linked to primary open-angle glaucoma (POAG). Mutation analyses of two large cohorts of patients with POAG, normal tension glaucoma (NTG) and juvenile open-angle glaucoma (JOAG), and control subjects, found only association of non-synonymous heterozygous variants of the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) with POAG, NTG and JOAG. The 20 non-synonymous variants identified in RPGRIP1 were all distinct from variants causing photoreceptor dystrophies and were found throughout all but one domain (RPGR-interacting domain) of RPGRIP1. Among them, 14 missense variants clustered within or around the C2 domains of RPGRIP1. Yeast two-hybrid analyses of a subset of the missense mutations within the C2 domains of RPGRIP1 shows that five of them (p.R598Q, p.A635G, p.T806I, p.A837G and p.I838V) decrease the association of the C2 domains with nephrocystin-4 (NPHPH). When considering only these five confirmed C2-domain mutations, the association remains statistically significant (P=0.001). Altogether, the data support that heterozygous non-synonymous variants of RPGRIP1 may cause or increase the susceptibility to various forms of glaucoma and that among other factors, physical impairment of the interaction of RPGRIP1with different proteins may contribute to the pathogenesis of forms of glaucoma.

Figures

Figure 1
Figure 1
RPGRIP1 protein structure and location of amino acids changes identified in this study. Schematic representation of the protein domains (modified from Roepman et al; GeneBank NP_065099) highlighting the location of the C2-domains region. All amino acids changes identified in the discovery group are listed upward; changes identified in the replication group are listed below the structure. Green bars represent coiled-coil domains, orange bar the RPGR-interacting domain, light yellow and green bar the C2 domains, white bars leucine zipper domains and dark bar the bipartite nuclear localization signal.
Figure 2
Figure 2
Effect of RPGRIP1 mutations on interaction with nephrocystin-4 (NPHP4). (a) Wild-type (wt) and mutated human RPGRIP1C2−N+C2−C proteins (fused to GAL4 transcription activation domain) were assessed for interaction with NPHP4, fused to the GAL4-binding domain. As a negative control, the known loss of function mutation, p.R890X-RPGRIP1, was used. Media lacking the amino acids Leu and Trp was used to select for cotransformants (−LW panel). Additional omission of His and Ade from the media selected for activation of associated HIS3 and ADE2 reporter genes (−LWHA panel). Blue staining indicates β-galactosidase activity by activation of the LacZ reporter gene. (b) Results of liquid β-galactosidase assays. Black bars indicate the average enzymatic activity of each construct. The error bars show standard deviation. Both approaches show that binding with NPHP4 was severely disrupted when the RPGRIP1C2−N+C2−C fragment contained the p.R598Q mutation. Although milder, an impaired interaction between the two proteins was also revealed by RPGRIP1 variants p.A635G, p.T806I, p.A837G and p.I838V. In contrast, RPGRIP1s p.Q589H, p.A764V and p.R812H did not cause any decrease in the interaction between RPGRIP1 and NPHP4, presenting similar or higher β-galactosidase activity to that of the wild-type protein.
Figure 3
Figure 3
Coimmunoprecipitation of nephrocystin-4 (NPHP4) and RPGRIP1 p.R598Q mutation. The top immunoblot indicates specific coimmunoprecipitation of full-length NPHP4 and RPGRIP1 fragments containing both C2 domains of the protein (lane 2). The RPGRIP1 construct containing the p.R598Q alteration was not found to coimmunoprecipitate with NPHP4FL (lane 1), suggesting that the interaction between both proteins was severely disrupted. As a negative control, LRRK2LRR did not coimmunoprecipitate with NPHP4FL (lane 3). Protein inputs (10%) are shown in the middle blot. The bottom immunoblot shows immunoprecipitation of HA-tagged NPHP4FL with anti-HA beads. The sizes (kDa) of the proteins corresponding with the specific antibody signals are indicated.

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