Approaches to Characterize novel pathogenic missense Variants in PRPF31

Gabrielle Wheway, Liliya Nazlamova, Nervine Meshad, Samantha Hunt, Nicola Jackson, Amanda Churchill |"A combined in silico, in vitro and clinical approach to Characterize Novel Pathogenic Missense Variants in PRPF31 in Retinitis Pigmentosa"


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.


Retinitis pigmentosa (RP) is a progressive retinal degeneration characterized by night blindness and restriction of peripheral vision. Later in the course of the disease, central and color vision can be lost. Many patients experience the first signs of RP between 20 and 40 years but there is much phenotypic variability from age of onset and speed of deterioration to severity of visual impairment (Hartong et al., 2006).

Retinitis pigmentosa, whilst classified as a rare disease, is the most common cause of inherited blindness worldwide. It affects between 1:3500 and 1:2000 people (Golovleva et al., 2010; Sharon and Banin, 2015), and can be inherited in an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked (xlRP) manner. It may occur in isolation (non-syndromic RP) (Verbakel et al., 2018), or with other features (syndromic RP) as in Bardet–Biedl syndrome, Joubert syndrome and Usher syndrome (Mockel et al., 2011). The condition is extremely heterogeneous, with 64 genes identified as causes of non-syndromic RP, and more than 50 genes associated with syndromic RP (RetNet1). Even with current genetic knowledge, diagnostic detection rate in adRP cohorts remains between 40% (Mockel et al., 2011) and 66% (Zhang et al., 2016), suggesting that many disease genes remain to be identified, and many mutations within known genes require characterization to ascribe pathogenic status. Detection rates are as low as 14% in cohorts of simplex cases (single affected individuals) and multiplex cases (several affected individuals in one family but unclear pattern of inheritance) (Jin et al., 2008). Such cases account for up to 50% of RP cases, so this presents a significant challenge to diagnosis (Greenberg et al., 1993; Haim, 1993; Najera et al., 1995).

The second most common genetic cause of adRP is PRPF31, accounting for 6% of United States cases (Sullivan et al., 2013) 8% of Spanish cases (Martin-Merida et al., 2018), 8% of French Canadian cases (Coussa et al., 2015), 8% of French cases (Audo et al., 2010), 8.9% of cases in North America (Daiger et al., 2014), 11.1% in small Chinese cohort (Lim et al., 2009), 10% in a larger Chinese cohort (Xu et al., 2012) and 10.5% of Belgian cases (Van Cauwenbergh et al., 2017). However, this is likely to be an underestimate due to variable penetrance of this form of RP, complicating attempts to co-segregate the variant with clinical disease, making genetic diagnosis difficult.

Whilst the majority of reported variants in PRPF31 are indels, splice site variants and nonsense variants, large-scale deletions or copy number variations (Martin-Merida et al., 2018), which are easily ascribed pathogenic status, at least eleven missense variants in PRPF31 have been reported in the literature (Table 1). Missense variants are more difficult to characterize functionally than nonsense or splicing mutations (Cooper and Shendure, 2011) and it is likely that there are false negative diagnoses in patients carrying missense mutations due to lack of confidence in prediction of pathogenicity of such variants. This is reflected in the enrichment of PRPF31 missense variants labeled ‘uncertain significance’ in ClinVar, a public repository for clinically relevant genetic variants (Landrum et al., 2014, 2016). Furthermore, work has shown that some variants annotated as missense PRPF31 variants may in fact be affecting splicing of PRPF31, introducing premature stop codons leading to nonsense mediated decay (NMD), a common disease mechanism in RP11 (Rio Frio et al., 2008). One example is c.319C > G, which, whilst originally annotated as p.Leu107Val, actually affects splicing rather than an amino acid substitution (Rio Frio et al., 2008). The presence of exonic splice enhancers is often overlooked by genetics researchers.

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