Association of two synonymous splicing-associated CpG single nucleotide polymorphisms in calpain 10 and solute carrier family 2 member 2 with type 2 diabetes.

Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.

Synonymous polymorphisms at splicing regulatory sites are associated with CpGs in neurodegenerative disease-related genes.

Neuronal plasticity is associated with alternative splicing and epigenetic modulation. Recent evidence reveals the association of cytosine methylation with alternative splicing and splicing regulatory mechanisms. Single nucleotide polymorphisms (SNPs) are generally less frequent in conserved coding regions and probably in splice sites, compared to non-coding regions. CpG polymorphisms in coding regions and splice sites and their association with splicing regulatory elements have not been investigated till presently. We currently analyzed the CpG variability in 28 genes (361 constitutive and 105 alternative exons and the corresponding splice sites) associated with neurodegenerative diseases (ND). CpG polymorphisms in the splice sites of these genes are particularly frequent when compared to those at AG sequences. Moreover, in both constitutive and alternative exons, polymorphisms in CpGs are more frequent than in AG, GT sequences. On the contrary, in the polypyrimidine acceptor sequence C/T conservation is prominent indicating that in this locus the sequence of cytosines and thymines is preserved. Bioinformatic analysis of the splicing-associated regulatory elements in these exons and splice sites reveals that 18 out of a total of 39 SNPs which could strongly affect splicing (>1.5 score difference) contain CpG sequences. Cytosines are considerably more frequent and variable than expected at the position preceding the GT splice donors, while sites of epigenetic modification are absent from acceptors. The high CpG frequency in polymorphic splicing-associated sites implicates the involvement of epigenetic mechanisms in splicing selection decisions regulated by these sites, and indicates the complexity of genetic studies involving these, tentatively critical, polymorphisms in ND.