The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

Abstract

A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.

A single base mutation in the 5′ splice site of intron 7 of the lck gene is responsible for the deletion of exon 7 in lck mRNA of the JCaM1 cell line

“…Thus in the JCaM1 DNA, the inability to initiate the correct excision of intron 7 at the 5' splice site could explain the absence of complete lck transcript: the exon 7 is no longer recognized as such and is thereby excluded from the mature mRNA. In agreement with Krawczak et al (1992) who observed that it is always the upstream exon immediately preceding the defect that was removed from the mature mRNA, we reported here that exon 7 is skipped as a consequence of a point mutation in intron 7. By means of COS cells transfected with minigenes from the Jurkat or JCaM1 cells, we could indeed demonstrate the critical role played by this mutation in the splicing mechanism.…”

“…Numerous studies have reported splicing defects leading to either absence or reduction of the mature mRNA. In particular the study by Krawczak et al (1992) reports more than 100 point mutations in the vicinity of exon/intron splices junctions which result in such splicing defects. Among these mutations, 60% take place at the 5' splice site and more than half lead to exon skipping.…”

Aberrant HS1 molecule in a patient with systemic lupus erythematosus

“…[33][34][35] To detect a genomic DNA mutation responsible for the skipping of HS1 exon A, we synthesized primers for HS1 exon Aspecific PCR, and conducted PCR/SSCP analysis for the patient and normal controls. However, no DNA fragment showed variations on electrophoresis (data not shown).…”

“…Aberrant splicing is mostly caused by the mutation at the splicing consensus sequence of exon-intron boundary, and, to a lesser extent, by a point mutation within the skipped exon. [33][34][35][52][53][54][55] However, there are several reports of exon skipping without any mutations in the sequence around the skipped exon. [56][57][58][59][60] Besides, a mutation in an intron away from an exon-intron boundary results in a defect in splicing.…”

Analysis of Alternatively Spliced Rice Transcripts Using Microarray Data

“…For example, relative to each other, different splice forms may have altered mRNA stability or translational efficiency or encode proteins with modified localization or function [6,19,36]. Significantly, around 15% of disease-causing mutations in human genes are point mutations in the vicinity of mRNA splice junctions, supporting the hypothesis that alternative splicing can have major effects on gene function [10,18]. Understanding the function of all rice genes, including alternative splice forms, will be invaluable toward improving rice and other grass species to meet human needs.…”