Functional variations modulating PRKCA expression and alternative splicing predispose to multiple sclerosis

Abstract

The protein kinase C alpha (PRKCA) gene, encoding a Th17-cell-selective kinase, was repeatedly associated with multiple sclerosis (MS), but the underlying pathogenic mechanism remains unknown. We replicated the association in Italians (409 cases, 723 controls), identifying a protective signal in the PRKCA promoter (P = 0.033), and a risk haplotype in intron 3 (P = 7.7 × 10(-4); meta-analysis with previously published data: P = 4.01 × 10(-8)). Expression experiments demonstrated that the protective signal is associated with alleles conferring higher PRKCA expression levels, well fitting our observation that MS patients have significantly lower PRKCA mRNA levels in blood. The risk haplotype was shown to be driven by a GGTG ins/del polymorphism influencing the heterogeneous nuclear ribonucleoprotein H-dependent inclusion/skipping of a PRKCA alternative exon 3*. Indeed, exon 3* can be present in two different versions in PRKCA mRNAs (out-of-frame 61 bp or in-frame 66 bp long), and is preferentially included in transcripts generated through a premature polyadenylation event. The GGTG insertion downregulates 3* inclusion and shifts splicing towards the 66 bp isoform. Both events reduce the nonsense-mediated mRNA-decay-induced degradation of exon 3*-containing mRNAs. Since we demonstrated that the protein isoform produced through premature polyadenylation aberrantly localizes to the plasma membrane and/or in cytoplasmic clusters, dysregulated PRKCA 3* inclusion may represent an additional mechanism relevant to MS susceptibility.

The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis

“…Conversely, a significant dysregulation of GSDMB AS profile was evident in RR-MS cases, whose levels of exon 6 and exons 5–8 skippings were unrelated to the rs11078928 polymorphism. This same dysregulation of AS profiles in MS cases, characterized by the loss of genotype-dependent splicing in the presence of a polymorphism directly affecting a splice site, has been already described for at least two other genes ( PRKCA and NFAT5 ) [11,12], thus possibly representing a specific “signature” of the disease status. The loss of genotype-dependent regulatory mechanisms might be explained by the extensive general dysregulation of proteins involved in the splicing process observed by us and other groups in MS [12,35,36,37].…”

“…After 8 h of treatment, total RNA was extracted and GSDMB expression levels were evaluated by semi-quantitative real-time RT–PCRs, using primers anchored to the 3′ terminus region of the transcript, common to all splicing variants (Figure 2). Two different transcripts of the PRKCA gene (respectively sensitive and insensitive to NMD) were used as positive and negative controls of the treatment [11]. We observed a significant increase of the total level of GSDMB after cycloheximide treatment in both cell lines with respect to the untreated sample (HEK293: 2.64-fold, p = 0.0019; HepG2: 2.8-fold, p = 0.00090) (Figure 2), suggesting that, in physiologic conditions, GSDMB mRNA levels are downregulated by NMD.…”

“…It has been also suggested that AS may increase the immunogenicity of autoantigens by creating novel epitopes that might break the immune tolerance [8]. Therefore, it is not surprising that several AIDs have already been associated with an altered expression of AS isoforms, including rheumatoid arthritis, multiple sclerosis (MS), autoimmune thyroid diseases, and systemic sclerosis [9,10,11,12,13,14]. …”

LRCH1 interferes with DOCK8-Cdc42–induced T cell migration and ameliorates experimental autoimmune encephalomyelitis

“…There are conflicting reports about whether a C/T transition SNP (rs912428) in LRCH1 is a risk factor for human osteoarthritis (Snelling et al, 2007; Jiang et al, 2008). Considering that SNP polymorphisms of PKCα have been identified in MS patients, and PKCα-deficient mice are resistant to EAE (Barton et al, 2004; Meisel et al, 2013; Paraboschi et al, 2014), this makes us even more interesting to further investigate whether SNP polymorphisms in DOCK8 or LRCH1 are associated with MS using a large population of MS samples.…”

Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites

“…For both mutations, computer-assisted splice-site prediction analysis recognized a significant difference between the wild-type and the mutant sequences, suggesting that they might impact SLC45A2 splicing. These predictions were tested by a well-validated in vitro hybrid minigene approach, 26,29,30 which we have recently applied to the study of two novel splicing mutations in the OCA2 gene. 31 Functional analysis demonstrated that both mutations inactivate the relevant physiologic donor splice site and, at the same time, may lead to the activation of cryptic sites.…”