A Combined in silico, in vitro and Clinical Approach to Characterize Novel Pathogenic Missense Variants in PRPF31 in Retinitis Pigmentosa.

Gabrielle Wheway,Amanda Churchill,Liliya Nazlamova,Samantha Hunt,Nicola Jackson,Nervine Meshad

doi:10.3389/fgene.2019.00248

Abstract

At least six different proteins of the spliceosome, including PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200, are mutated in autosomal dominant retinitis pigmentosa (adRP). These proteins have recently been shown to localize to the base of the connecting cilium of the retinal photoreceptor cells, elucidating this form of RP as a retinal ciliopathy. In the case of loss-of-function variants in these genes, pathogenicity can easily be ascribed. In the case of missense variants, this is more challenging. Furthermore, the exact molecular mechanism of disease in this form of RP remains poorly understood. In this paper we take advantage of the recently published cryo EM-resolved structure of the entire human spliceosome, to predict the effect of a novel missense variant in one component of the spliceosome; PRPF31, found in a patient attending the genetics eye clinic at Bristol Eye Hospital. Monoallelic variants in PRPF31 are a common cause of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance. We use in vitro studies to confirm pathogenicity of this novel variant PRPF31 c.341T > A, p.Ile114Asn. This work demonstrates how in silico modeling of structural effects of missense variants on cryo-EM resolved protein complexes can contribute to predicting pathogenicity of novel variants, in combination with in vitro and clinical studies. It is currently a considerable challenge to assign pathogenic status to missense variants in these proteins.

Highlights

Retinitis pigmentosa (RP) is a progressive retinal degeneration characterized by night blindness and restriction of peripheral vision
PRPF31 is a component of the major and minor spliceosome, the huge macromolecular ribonucleoprotein (RNP) complex which catalyzes the splicing of pre-messenger RNAs to remove introns and produce mature mRNAs
In the absence of PRPF31, U4/U6 di-snRNP accumulates in the splicing-rich Cajal bodies in the nucleus, preventing formation of the tri-snRNP, and subsequently efficient splicing (Schaffert et al, 2004)

Summary

INTRODUCTION

Retinitis pigmentosa (RP) is a progressive retinal degeneration characterized by night blindness and restriction of peripheral vision. Pre-mRNA splicing factors may have additional roles beyond splicing in the nucleus, after a study recently found that PRPF6, PRPF8, and PRPF31 are all localized to the base of the retinal photoreceptor connecting cilium and are essential for ciliogenesis, suggesting that this form of RP is a ciliopathy (Wheway et al, 2015) Missense variants in these proteins are, collectively, a common cause of adRP. We show that analysis of protein complexes in silico can complement clinical and laboratory studies in predicting pathogenicity of novel genetic variants

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION