A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

Isabelle Tournier; Myriam Vezain; Alexandra Martins; Francoise Charbonnier; Stéphanie Baert-Desurmont; Sylviane Olschwang; Qing Wang; Marie Pierre Buisine; Johann Soret; Jamal Tazi; Thierry Frebourg; Mario Tosi

doi:10.1002/humu.20796

Article dans une revue

A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

Isabelle Tournier ^{1, 2} Myriam Vezain ² Alexandra Martins ^{3, 2, 4} Francoise Charbonnier ^{3, 2} Stéphanie Baert-Desurmont ^{1, 2} Sylviane Olschwang ⁵ Qing Wang ⁶ Marie Pierre Buisine ⁷ Johann Soret ⁸ Jamal Tazi ⁸ Thierry Frebourg ^{1, 2} Mario Tosi ^{1, 2}

1 Génétique médicale et fonctionnelle du cancer et des maladies neuropsychiatriques

2 GPMCND - Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques

3 GMFC - Génétique du cancer et des maladies neuropsychiatriques

4 Inserm U614, IFRMP, Institute for Biomedical Research, Rouen University Medical School, Rouen, France

5 Genetique des Tumeurs

6 Chimie du solide et de l'énergie

7 Hôpital Claude Huriez

8 IGMM - Institut de Génétique Moléculaire de Montpellier

Abstract : 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 ∼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.

Domaine :

Sciences du Vivant [q-bio]

Liste complète des métadonnées

https://hal-normandie-univ.archives-ouvertes.fr/hal-02336389
Contributeur : Alexandra Martins <>
Soumis le : lundi 28 octobre 2019 - 17:57:28
Dernière modification le : vendredi 13 décembre 2019 - 11:48:50

A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

Identifiants

Collections

Citation

Exporter

Métriques

49

A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

Identifiants

Collections

Citation

Exporter

Partager

Métriques

49