Expression Changes Confirm Genomic Variants Predicted to Result in Allele-Specific, Alternative mRNA Splicing

Abstract

Splice isoform structure and abundance can be affected by either noncoding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these nonconstitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays and custom designed q-RT-PCR or TaqMan assays. The majority of these SNPs (15 of 22) as well as an independent set of 24 variants were then subjected to RNAseq analysis using the ValidSpliceMut web beacon (http:// validsplicemut.cytognomix.com), which is based on data from the Cancer Genome Atlas and International Cancer Genome Consortium. SNPs from different genes analyzed with gene expression microarray and q-RT-PCR exhibited significant changes in affected splice site use. Thirteen SNPs directly affected exon inclusion and 10 altered cryptic site use. Homozygous SNP genotypes resulting in stronger splice sites exhibited higher levels of processed mRNA than alleles associated with weaker sites. Four SNPs exhibited variable expression among individuals with the same genotypes, masking statistically significant expression differences between alleles. Genome-wide information theory and expression analyses (RNAseq) in tumor exomes and genomes confirmed splicing effects for 7 of the HapMap SNP and 14 SNPs identified from tumor genomes. q-RT-PCR resolved rare splice isoforms with read abundance too low for statistical significance in ValidSpliceMut. Nevertheless, the web-beacon provides evidence of unanticipated splicing outcomes, for example, intron retention due to compromised recognition of constitutive splice sites. Thus, ValidSpliceMut and q-RT-PCR represent complementary resources for identification of allele-specific, alternative splicing.

PROM1, CXCL8, RUNX1, NAV1 and TP73 genes as independent markers predictive of prognosis or response to treatment in two cohorts of high-grade serous ovarian cancer patients

“…Splice site SNPs may contribute to the above-mentioned physiological changes by affecting the activity of cryptic sites, leading to the altered frequency of exon inclusion/exclusion, and promoting the alternate exon usage. As a result, the amounts of particular splice forms in the cell may either increase or diminish, as demonstrated for, e.g., EMID1 and IL19, respectively, by Mucaki et al [75]. Such a mechanism may also explain the correlation between the occurrence of splice site mutations and overexpression, reported herein for the PROM1 gene.…”

Late Onset Subacute Profound Biotinidase Deficiency Caused by a Novel Homozygous Variant c.466-3T>G in the BTD Gene

“…Splicing defects in the consensus sequence surrounding the canonical splice sites reduce strength of natural splice sites (weakened splice sites). Weakened splice sites can exhibit normal mRNA as well as lower levels of altered mRNA [ 9 ].…”

The Association Between Single-Nucleotide Polymorphisms of Co-Stimulatory Genes Within Non-HLA Region and the Prognosis of Leukemia Patients With Hematopoietic Stem Cell Transplantation

“…Whether intronic SNP has any effect on PDCD1 expression is not clear. Nevertheless, aberrant splicing has been linked to the intronic SNP and causes protein mutation ( 45 ).…”