Identification of functional single nucleotide polymorphisms in the branchpoint site

Hung-Lun Chiang,Jer-Yuarn Wu,Yuan-Tsong Chen

doi:10.1186/s40246-017-0122-6

Abstract

BackgroundThe human genome contains millions of single nucleotide polymorphisms (SNPs); many of these SNPs are intronic and have unknown functional significance. SNPs occurring within intron branchpoint sites, especially at the adenine (A), would presumably affect splicing; however, this has not been systematically studied. We employed a splicing prediction tool to identify human intron branchpoint sites and screened dbSNP for identifying SNPs located in the predicted sites to generate a genome-wide branchpoint site SNP database.ResultsWe identified 600 SNPs located within branchpoint sites; among which, 216 showed a change in A. After scoring the SNPs by counting the As in the ± 10 nucleotide region, only four SNPs were identified without additional As (rs13296170, rs12769205, rs75434223, and rs67785924). Using minigene constructs, we examined the effects of these SNPs on splicing. The three SNPs (rs13296170, rs12769205, and rs75434223) with nucleotide substitution at the A position resulted in abnormal splicing (exon skipping and/or intron inclusion). However, rs67785924, a 5-bp deletion that abolished the branchpoint A nucleotide, exhibited normal RNA splicing pattern, presumably using two of the downstream As as alternative branchpoints. The influence of additional As on splicing was further confirmed by studying rs2733532, which contains three additional As in the ± 10 nucleotide region.ConclusionsWe generated a high-confidence genome-wide branchpoint site SNP database, experimentally verified the importance of A in the branchpoint, and suggested that other nearby As can protect branchpoint A substitution from abnormal splicing.

Highlights

The human genome contains millions of single nucleotide polymorphisms (SNPs); many of these Single nucleotide polymorphism (SNP) are intronic and have unknown functional significance
Splicing depends on the spliceosome, which is a large complex of small nuclear ribonucleoproteins and non-snRNPs; these components recognize the target sequence and assemble on the pre-mRNA [4]
A previous report showed that IVS4,22A>G in the LCAT gene, which is an A to G change at the splicing branchpoint, resulted in intron inclusion and exon skipping of the mRNA and caused the Fish-eye disease [14]

Summary

Introduction

The human genome contains millions of single nucleotide polymorphisms (SNPs); many of these SNPs are intronic and have unknown functional significance. SNPs occurring within intron branchpoint sites, especially at the adenine (A), would presumably affect splicing; this has not been systematically studied. We employed a splicing prediction tool to identify human intron branchpoint sites and screened dbSNP for identifying SNPs located in the predicted sites to generate a genome-wide branchpoint site SNP database. A previous report showed that IVS4,22A>G in the LCAT gene, which is an A to G change at the splicing branchpoint, resulted in intron inclusion and exon skipping of the mRNA and caused the Fish-eye disease [14]. There is a report suggesting that mutations in the branchpoint sequence, especially the adenine (A) may result in aberrant pre-mRNA splicing and give rise to human genetic disorders [15]

Methods

Results

Conclusion