Missense Mutations in RPGRIP1L Are Associated With Early-Onset Retinal Degeneration and Alter Its Interaction With RPGR and Selected NPHP and BBS Ciliary Proteins

Abstract

Purpose: : Mutations in the cilia-centrosomal protein RPGR and its interacting proteins are a major cause of retinal degeneration and syndromic ciliopathies. In this study, we describe mutations and dysfunction of a RPGR-interacting protein, RPGRIP1L (mutated in Joubert Syndrome (JBTS) and Meckel-Gruber Syndrome) in Leber congenital amaurosis and RP.

Methods: : RPGR-interacting proteins were identified by yeast two-hybrid screening of a retina prey cDNA library using RPGR1-ORF15 bait. This interaction was validated by co-transfection and co-IP experiments. Over 200 LCA and RP patients were screened by dHPLC and direct sequencing of the RPGRIP1L gene.

Results: : We identified RPGRIP1L as an RPGR-interacting protein in ciliated cells as well as in bovine retinal extracts. In addition to Nephroretinin (NPHP4), RPGRIP1L interacts with RPGRIP1 and with selected Bardet-Biedl Syndrome and Nephronophthisis (NPHP) proteins. We found 11 heterozygous missense variants in RPGRIP1L in 13 LCA patients; of these, 8 also harbored significant mutations in other LCA genes: i.e LCA5 (Q279X), RPGRIP1 (R890X), CRB1 (C948Y) and RPGR (I1148V). We identified the JBTS mutation T677I in RPGRIP1L in LCA patients and excluded it from 250 normal controls; however, this mutation did not co-segregate in the pedigree. Importantly, our results show that disease-associated RPGRIP1L variants may differentially alter its interactions with RPGR, NPHP, and BBS proteins.

Conclusions: : Absence of co-segregation and a second mutation precludes RPGRIP1L as a cause of LCA. However, our data suggest that the identified missense RPGRIP1L variants may play a role in the pathogenesis of LCA, by probably acting as a modifier. We propose that disruption of the RPGR-RPGRIP1L interactome is a likely cause of associated RP and related ciliopathies.

Keywords: photoreceptors • retinal degenerations: cell biology • proteins encoded by disease genes