Mutations located in the introns of mismatch repair genes can interfere with splicing and cause aberrant spliced mRNA transcripts leading to non-functional mismatch repair proteins. Several
cis-acting elements, including the donor splice site, the acceptor splice site, the branch point, the polypyrimidine tract, and exonic/intronic splicing enhancers and silencers, are crucial for the splicing mechanism. The donor splice site consists of the conserved dinucleotides GT, whereas the acceptor splice site consists of three regions: the conserved dinucleotides AG, the polypyrimidine tract, and the branch point [
11]. Mutations in splicing motifs can lead to partial or complete skipping of the neighboring exon or inclusion of intronic sequence. Moreover, a mutation can create an ectopic splice site or activate a cryptic splice site, both of which are usually weak and only used when a mutation disrupts the normal splice site.
Ideally RNA from a patient should be examined by RT-PCR analysis to establish if a mutation has an effect on splicing. However, in many cases, RNA is not available from the patient. Alternatively, the mutation can be examined by mini-gene analysis [
12]. In fact, a high concordance between RT-PCR analysis and mini-gene assay has previously been observed [
9,
13‐
15]. As an indicative examination prior to the mini-gene assay, several
in silico prediction tools can be used to indicate which variants require further analysis.
In this study, we examined the effect on splicing of nine intronic variants identified in Danish colorectal cancer families by
in silico analysis and
in vitro using a mini-gene assay. The
in silico analysis predicted altered splicing for
MLH1 c.588 + 5G > A,
MLH1 c.677 + 3A > T,
MLH1 c.1039-8 T > A,
MLH1 c.1732-2A > T,
MSH2 c.1276 + 1G > T,
MSH2 c.1662-2A > C, and
MSH2 c.2459-18delT, whereas
MLH1 c.117-34A > T and
MSH6 c.3439-16C > T were predicted to have no effect on splicing. It should be noted that three mutations in our study (
MLH1 c.1732-2A > T,
MSH2 c.1276 + 1G > T, and
MSH2 c.1662-2A > C) are located in the highly conserved donor and acceptor splice sites and hence they are easily predicted by
in silico programs. However, mini-gene analysis revealed that the two mutations
MLH1 c.1039-8 T > A and
MSH2 c.2459-18delT had no effect on splicing, suggesting that the employed criterion (>10% difference between wild type and mutant scores in at least two programs) results in false-positive predictions as previously shown [
9].
Mini-gene analysis revealed that the
MLH1 c.117-34A > T and
MLH1 c.1039-8 T > A variants had no effect on splicing. The
MLH1 c.117-34A > T variant has not been described before, whereas our results regarding
MLH1 c.1039-8 T > A confirm previous data analyzing patient RNA [
16]. Moreover, in one Amsterdam positive family (H13), the
MLH1 c.1039-8 T > A mutation was identified together with a disease-causing
MLH1 mutation (c.1276C > T, p.Gln426X). In conclusion we classify both variants as neutral (Table
2). In contrast, the
MLH1 c.588 + 5G > A and
MLH1 c.677 + 3A > T mutations were found to lead to exclusion of exon 7 and exon 8, respectively. Ultimately, this leads to premature stop codons and, therefore, both mutations are classified as disease-causing. These findings confirm previous results showing that the
MLH1 c.588 + 5G > A mutation causes either partial skipping/deletion of exon 7 examining patient RNA [
17] or skipping of exon 7 as well as both exons 7 and 8 assesed using mini-gene assay [
15], and by results from
MLH1 c.677 + 3A > C and
MLH1 c.677 + 3A > G mutations showing skipping of exon 8 [
18,
19]. Moreover, our analysis found that
MLH1 c.1732-2A > T, which is a Danish founder mutation identified in two Amsterdam positive families (See Additional file
1), results in an in-frame deletion of exon 16, which contains the PMS2 interaction domain. In the 2 families the mutation co-segregates with the disease and has a lod score of 1.2 and 2.7, respectively, and in agreement with previous reports [
5,
7,
20] we therefore classify
MLH1 c.1732-2A > T as pathogenic. The
MSH2 c.1276 + 1G > T mutation was found to result in the activation of a cryptic splice donor site 48 bp within exon 7, leading to an in-frame deletion of 16 amino acids in the MSH6/MSH3 interaction domain. As the mutation was found to co-segregate in the affected family with a lod score of 1.5, it is regarded as pathogenic. This mutation has previously been described in microsatellite instability-high colorectal cancers, with immunohistochemical analysis of these tumors revealing the absence of nucleic MSH2 expression [
21]. Similar results and conclusions have been reported for the
MSH2 c.1276 + 1G > A mutation [
16]. The
MSH2 c.1662-2A > C mutation has not previously been described. We found that this mutation leads to skipping of exon 11 and consequently introduce a premature stop codon. Therefore, this mutation is classified as pathogenic. Finally, the
MSH2 c.2459-18delT and
MSH6 c.3439-16C > T variants were found to have no effect on splicing and are, therefore, classified as neutral. The
MSH2 c.2459-18delT variant has not been described before, whereas the
MSH6 c.3439-16C > T variant has previously been shown not to co-segregate with the disease and to be observed in healthy control individuals [
22‐
24] and in the exome sequencing project (ESP) database (0.43%), thereby supporting the notion that this variant is neutral.
Table 2
The effect on splicing determined by mini-gene assays and an overview of the mutations listed in the literature
MLH1
| 1 | c.117-34A > T | NI | No effect on splicing | Novel | Neutral |
| 7 | c.588 + 5G > A | NI | Out-of-frame skipping of exon 7 | Pagenstecher; partial deletion of exon 7 [ 17] | Pathogenic |
Tournier; deletion of exon 7 and exons 7–8 [ 15] |
8 | c.677 + 3A > T | NI | Out-of-frame skipping of exon 8 | Novel | Pathogenic |
11 | c.1039-8 T > A | NI | No effect on splicing | Betz; No effect on splicing [ 16] | Neutral |
15 | c.1732-2A > T | NI | In-frame skipping of exon 16 | | Pathogenic |
|
|
MSH2
| 7 | c.1276 + 1G > T | NI | In-frame exclusion of 48 bp of exon 7 | | Pathogenic |
10 | c.1662-2A > C | NI | Out-of-frame skipping of exon 11 | Novel | Pathogenic |
14 | c.2459-18delT | NI | No effect on splicing | Novel | Neutral |
MSH6
| 5 | c.3439-16C > T | 0.43% | No effect on splicing | | Neutral |
|
|
Overall, in all Amsterdam positive families - except one (H229) - a pathogenic mutation was identified. The index individual in family H229 had rectum cancer at age 58 and transverse colon cancer at age 66. His sister and two maternal cousins all had adenomas, while his mother has caecum cancer at age 48. Moreover his maternal aunt had transverse colon cancer at age 69 and his maternal grandmother had ascending colon cancer. The lack of a pathogenic mutation in this family could be due to an unidentified mutation in regions not previously examined, including the promoter region, the untranslated regions (UTRs) or deep intron sequences in the MLH1, MSH2 or MSH6 genes, or due to a mutation in other genes like PMS2. Future studies using exome sequencing might help identifying a putative pathogenic mutation in this family.