A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects.
Numerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild-type exons to be tested were PCR-amplified from patient genomic DNA together with approximately 150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE-dependent central exon of a three-exon splicing minigene and we showed in cell transfection experiments that the wild-type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases.
Inserm U614, Federate Institute for Multidisciplinary Research on Peptides, Faculty of Medicine, University of Rouen, Department of Genetics and Institute for Biomedical Research, Rouen University Hospital, Rouen, France.
Isabelle
I
Tournier
Myriam
M
Vezain
Alexandra
A
Martins
Françoise
F
Charbonnier
Stéphanie
S
Baert-Desurmont
Sylviane
S
Olschwang
Qing
Q
Wang
Marie Pierre
MP
Buisine
Johann
J
Soret
Jamal
J
Tazi
Thierry
T
Frébourg
Mario
M
Tosi
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D005190:Family; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D012326:RNA Splicing
D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein