Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants (original) (raw)

Abstract

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level.

Access this article

Log in via an institution

Subscribe and save

• Get 10 units per month
• Download Article/Chapter or eBook
• 1 Unit = 1 Article or 1 Chapter
• Cancel anytime Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

• Aretz S, Uhlhaas S, Sun Y, Pagenstecher C, Mangold E, Caspari R, Moslein G, Schulmann K, Propping P, Friedl W (2004) Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene. Hum Mutat 24:370–380
  Article PubMed CAS Google Scholar
• Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3:285–298
  Article PubMed CAS Google Scholar
• Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31:3568–3571
  Article PubMed CAS Google Scholar
• Ellison AR, Lofing J, Bitter GA (2001) Functional analysis of human MLH1 and MSH2 missense variants and hybrid human–yeast MLH1 proteins in Saccharomyces cerevisiae. Hum Mol Genet 10:1889–1900
  Article PubMed CAS Google Scholar
• Gorlov IP, Gorlova OY, Frazier ML, Amos CI (2003) Missense mutations in hMLH1 and hMSH2 are associated with exonic splicing enhancers. Am J Hum Genet 73(5):1157–1161
  Article PubMed CAS Google Scholar
• Guerrette S, Acharya S, Fishel R (1999) The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer. J Biol Chem 274(10):6336–6341
  Article PubMed CAS Google Scholar
• Kohonen-Corish M, Ross VL, Doe WF, Kool DA, Edkins E, Faragher I, Wijnen J, Khan PM, Macrae F, St John DJ (1996) RNA-based mutation screening in hereditary nonpolyposis colorectal cancer. Am J Hum Genet 59:818–824
  PubMed CAS Google Scholar
• Kondo E, Suzuki H, Horii A, Fukushige S (2003) A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations. Cancer Res 63:3302–3308
  PubMed CAS Google Scholar
• Kruger S, Plaschke J, Pistorius S, Jeske B, Haas S, Kramer H, Hinterseher I, Bier A, Kreuz FR, Theissig F, Saeger HD, Schackert HK (2002) Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer. Hum Mutat 19:82
  Article PubMed CAS Google Scholar
• Lastella P, Resta N, Miccolis I, Quagliarella A, Guanti G, Stella A (2004) Site directed mutagenesis of hMLH1 exonic splicing enhancers does not correlate with splicing disruption. J Med Genet 41:e72
  Article PubMed CAS Google Scholar
• Lynch HT, de la Chapelle A (1999) Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet 36:801–818
  PubMed CAS Google Scholar
• Mangold E, Pagenstecher C, Friedl W, Mathiak M, Buettner R, Engel C, Loeffler M, Holinski-Feder E, Muller-Koch Y, Keller G, Schackert HK, Kruger S, Goecke T, Moeslein G, Kloor M, Gebert J, Kunstmann E, Schulmann K, Ruschoff J, Propping P (2005) Spectrum and frequencies of mutations in MSH2 and MLH1 identified in 1,721 German families suspected of hereditary nonpolyposis colorectal cancer. Int J Cancer 116:692–702
  Article PubMed CAS Google Scholar
• Nystrom-Lahti M, Perrera C, Raschle M, Panyushkina-Seiler E, Marra G, Curci A, Quaresima B, Costanzo F, D’Urso M, Venuta S, Jiricny J (2002) Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer. Genes Chromosomes Cancer 33(2):160–167
  Article PubMed CAS Google Scholar
• Peltomaki P (2005) Lynch syndrome genes. Fam Cancer 4(3):227–232
  Article PubMed CAS Google Scholar
• Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch E, Lonnqvist KE, Holinski-Feder E, Sutter C, McKinnon W, Duraisamy S, Gerdes AM, Peltomaki P, Kohonen-Ccorish M, Mangold E, Macrae F, Greenblatt M, de la Chapelle A, Nystrom M (2005) Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology 129(2):537–549
  Article PubMed CAS Google Scholar
• Sharp A, Pichert G, Lucassen A, Eccles D (2004) RNA analysis reveals splicing mutations and loss of expression defects in MLH1 and BRCA1. Hum Mutat 24:272
  Article PubMed CAS Google Scholar
• Stella A, Wagner A, Shito K, Lipkin SM, Watson P, Guanti G, Lynch HT, Fodde R, Liu B (2001) A nonsense mutation in MLH1 causes exon skipping in three unrelated HNPCC families. Cancer Res 61:7020–7024
  PubMed CAS Google Scholar
• Vasen HF (2005) Clinical description of the Lynch Syndrome [Hereditary Nonpolyposis Colorectal Cancer (HNPCC)]. Fam Cancer 4(3):219–225
  Article PubMed CAS Google Scholar
• Wang Y, Friedl W, Lamberti C, Junck M, Mathiak M, Pagenstecher C, Propping P, Mangold E (2003) Hereditary nonpolyposis colorectal cancer: frequent occurrence of large genomic deletions in MSH2 and MLH1 genes. Int J Cancer 2003 Feb 20 103(5):636–641
  Article CAS Google Scholar
• Wehner M, Mangold E, Sengteller M, Friedrichs N, Aretz S, Friedl W, Propping P, Pagenstecher C (2005) Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes. Eur J Hum Genet 13:983–986
  Article PubMed CAS Google Scholar

Download references

Acknowledgements

We would like to thank the patients who participated in this study and their attending doctors. The work of the consortium is supported by a multicenter grant from the Deutsche Krebshilfe, Bonn, Germany, project no. 70-3027-Ma1.

Author information

Authors and Affiliations

1. Institute of Human Genetics, University of Bonn, Wilhelmstrasse 31, 53111, Bonn, Germany
   Constanze Pagenstecher, Maria Wehner, Waltraut Friedl, Nils Rahner, Stefan Aretz, Marlies Sengteller, Peter Propping & Elisabeth Mangold
2. Institute of Pathology, University of Bonn, Bonn, Germany
   Nicolaus Friedrichs & Reinhard Buettner
3. Institute of Human Genetics, Saarland University, Homburg/Saar, Germany
   Wolfram Henn

Authors

1. Constanze Pagenstecher
   You can also search for this author inPubMed Google Scholar
2. Maria Wehner
   You can also search for this author inPubMed Google Scholar
3. Waltraut Friedl
   You can also search for this author inPubMed Google Scholar
4. Nils Rahner
   You can also search for this author inPubMed Google Scholar
5. Stefan Aretz
   You can also search for this author inPubMed Google Scholar
6. Nicolaus Friedrichs
   You can also search for this author inPubMed Google Scholar
7. Marlies Sengteller
   You can also search for this author inPubMed Google Scholar
8. Wolfram Henn
   You can also search for this author inPubMed Google Scholar
9. Reinhard Buettner
   You can also search for this author inPubMed Google Scholar
10. Peter Propping
    You can also search for this author inPubMed Google Scholar
11. Elisabeth Mangold
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toConstanze Pagenstecher.

Additional information

Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: http://www.insight-group.org/, Programs: BDGP: http://www.fruitfly.org/seq_tools/splice-instrucs.html, ESEfinder program: http://exon.cshl.edu/ESE/

Rights and permissions

About this article

Cite this article

Pagenstecher, C., Wehner, M., Friedl, W. et al. Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants.Hum Genet 119, 9–22 (2006). https://doi.org/10.1007/s00439-005-0107-8

Download citation

• Received: 04 August 2005
• Accepted: 31 October 2005
• Published: 08 December 2005
• Issue Date: March 2006
• DOI: https://doi.org/10.1007/s00439-005-0107-8

Keywords