Risk-associated coding synonymous SNPs in type 2 diabetes and neurodegenerative diseases: Genetic silence and the underrated association with splicing regulation and epigenetics

Highlights

• Analysis of synonymous SNPs in ND and T2D genes for their epigenetic impact.

• Several synonymous SNPs in linkage disequilibrium with T2D/ND.

• Further analysis of the SNP silence/synonymity vs. splicing/epigenetic impact is needed.

Abstract

Single nucleotide polymorphisms (SNPs) are tentatively critical with regard to disease predisposition, but coding synonymous SNPs (sSNPs) are generally considered “neutral”. Nevertheless, sSNPs in serine/arginine-rich (SR) and splice-site (SS) exonic splicing enhancers (ESEs) or in exonic CpG methylation targets, could be decisive for splicing, particularly in aging-related conditions, where mis-splicing is frequently observed.

We presently identified 33 genes T2D-related and 28 related to neurodegenerative diseases, by investigating the impact of the corresponding coding sSNPs on splicing and using gene ontology data and computational tools. Potentially critical (prominent) sSNPs comply with the following criteria: changing the splicing potential of prominent SR-ESEs or of significant SS-ESEs by >1.5 units (Δscore), or formation/deletion of ESEs with maximum splicing score. We also noted the formation/disruption of CpGs (tentative methylation sites of epigenetic sSNPs). All disease association studies involving sSNPs are also reported. Only 21/670 coding SNPs, mostly epigenetic, reported in 33 T2D-related genes, were found to be prominent coding synonymous. No prominent sSNPs have been recorded in three key T2D-related genes (GCGR, PPARGC1A, IGF1). Similarly, 20/366 coding synonymous were identified in ND related genes, mostly epigenetic. Meta-analysis showed that 17 of the above prominent sSNPs were previously investigated in association with various pathological conditions. Three out of four sSNPs (all epigenetic) were associated with T2D and one with NDs (branch site sSNP). Five were associated with other or related pathological conditions. None of the four sSNPs introducing new ESEs was found to be disease-associated. sSNPs introducing smaller Δscore changes (<1.5) in key proteins (INSR, IRS1, DISC1) were also correlated to pathological conditions.

This data reveals that genetic variation in splicing-regulatory and particularly CpG sites might be related to disease predisposition and that in-silico analysis is useful for identifying sSNPs, which might be falsely identified as silent or synonymous.

Introduction

The limited understanding of epigenetic inheritance is related to the fact that there is little experimental evidence with regard to the mechanisms influenced by epigenetics. One of the recent developments, which could shed light into the principle mechanisms involved in this process, is related to the finding that epigenetic parameters are involved in the process of alternative splicing. This discovery, which is expected to rebut several misconceptions involving the splicing process, might also be critical for evaluating the impact of genetic variations and their association to environmental parameters in disease.

The impact of environmental factors on the epigenetic profile and particularly on the DNA methylation process is presently documented through studies on identical twins [1], [2], [3] and the Agouti mice [4], [5]. This process is dynamic [6], [7], partially environmentally configured [8] and aging dependent [9], [10], [11]. On the other hand, splicing is also a process, which is affected by aging [12], [13], modified under degenerative conditions [14], [15], [16] and dependent on exposure to different environmental factors [17], [18], [19].

Although the role of epigenetic alterations in cancer is widely acknowledged, the relevance of epigenetics to common metabolic (e.g. type 2 diabetes – T2D) and neurodegenerative (e.g. Alzheimer's disease – AD) diseases (NDs), both conditions associated with aging [12], [20] and frequent global methylation modifications [21], [22], [23], [24], [25], [26], remains conspicuous to date. These processes share common pathogenic mechanisms in animal models [27]. However, more research is needed to clarify the exact role of epigenetic regulation in the proposed pathogenic mechanism shared by these two major disorders.

Coding single nucleotide polymorphisms (SNPs) are considered key to the individual's genetic predisposition. Based on the genetic code, coding SNPs which do not introduce modification of individual amino acids are classified as “synonymous” SNPs (sSNPs) and considered neutral with respect to their genetic impact. However, such a classification disregards two principle issues in functional genomics: the presence of sequence motifs which strongly affect splicing [28], [29], [30], [31], probably via nucleosomal binding [32], [33], [34], [35], [36] and the presence/absence of epigenetic modification sites which is also tentatively critical for splicing [37], [38], [39]. Recent studies have demonstrated that sSNPs can affect mRNA splicing, stability and structure as well as protein folding, indicating the role of synonymous mutations in the biosynthesis of proteins [40], [41], [42], [43], [44]. These changes can have a significant effect in the function of proteins and as a result sSNPs can be implicated in disease [45], [46], [47]. Bioinformatic analysis could provide useful tools for re-examining sSNPs with respect to these principle mechanisms of gene expression, and further our understanding on issues of epigenetic inheritance in critical pathological conditions.

In the present study we performed, using bioinformatic tools, an analysis of all sSNPs recorded in T2D and in ND disease-related genes. sSNPs introducing changes in splicing-affecting sequence elements and CpG sites were identified. Furthermore, we investigated via meta-analysis, data from clinical studies involving sSNPs from these genes. Our findings reveal high variability in epigenetic modification sites in genes related to both pathological conditions and disease-association in a large percentage of splicing-affecting sSNPs.