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01-10-2020 | Original Article

MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing

Authors: Tamara Alejandra Piñero, Omar Soukarieh, Marion Rolain, Karin Alvarez, Francisco López-Köstner, Giovana Tardin Torrezan, Dirce Maria Carraro, Ivana Lucia De Oliveira Nascimento, Thaís Ferreira Bomfim, Taísa Manuela Bonfim Machado-Lopes, Juliana Côrtes Freitas, Maria Betânia Toralles, Kiyoko Abe Sandes, Benedito Mauro Rossi, Samuel Aguiar Junior, Joanna Meira, Mev Dominguez-Valentin, Pål Møller, Carlos Alberto Vaccaro, Alexandra Martins, Walter Hernán Pavicic

Published in: Familial Cancer | Issue 4/2020

Abstract

Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

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Carethers JM, Stoffel EM (2015) Lynch syndrome and Lynch syndrome mimics: the growing complex landscape of hereditary colon cancer. World J Gastroenterol 21:9253–9261. https://doi.org/10.3748/wjg.v21.i31.9253CrossRefPubMedPubMedCentral

Aarnio M, Sankila R, Pukkala E et al (1999) Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J Cancer 81:214–218CrossRefPubMed

Engel C, Loeffler M, Steinke V et al (2012) Risks of less common cancers in proven mutation carriers with lynch syndrome. J Clin Oncol 30:4409–4415. https://doi.org/10.1200/JCO.2012.43.2278CrossRefPubMed

Dominguez-Valentin M, Sampson JR, Seppälä TT et al (2020) Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database. Genet Med 22:15–25. https://doi.org/10.1038/s41436-019-0596-9CrossRefPubMed

Lynch HT, Lanspa S, Shaw T et al (2017) Phenotypic and genotypic heterogeneity of Lynch syndrome: a complex diagnostic challenge. Fam Cancer. https://doi.org/10.1007/s10689-017-0053-3CrossRefPubMedPubMedCentral

Ligtenberg MJL, Kuiper RP, Chan TL et al (2008) Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3′ exons of TACSTD1. Nat Genet 41:112CrossRefPubMed

Takahashi M, Furukawa Y, Shimodaira H et al (2012) Aberrant splicing caused by a MLH1 splice donor site mutation found in a young Japanese patient with Lynch syndrome. Fam Cancer 11:559–564. https://doi.org/10.1007/s10689-012-9547-1CrossRefPubMed

Bianchi F, Raponi M, Piva F et al (2011) An intronic mutation in MLH1 associated with familial colon and breast cancer. Fam Cancer 10:27–35. https://doi.org/10.1007/s10689-010-9371-4CrossRefPubMed

Cartegni L, Wang J, Zhu Z et al (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31:3568–3571. https://doi.org/10.1093/nar/gkg616CrossRefPubMedPubMedCentral

10.

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. https://doi.org/10.1002/humu.9267CrossRefPubMed

11.

Stella A, Shito K, Liu B et al (2001) A nonsense mutation in MLH1 causes exon skipping in three unrelated HNPCC families. Cancer Res 61:7020–7024PubMed

12.

Tournier I, Vezain M, Martins A et al (2008) A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. Hum Mutat 29:1412–1424. https://doi.org/10.1002/humu.20796CrossRefPubMed

13.

Soukarieh O, Gaildrat P, Hamieh M et al (2016) Exonic splicing mutations are more prevalent than currently estimated and can be predicted by using in silico tools. PLoS Genet 12:1–26. https://doi.org/10.1371/journal.pgen.1005756CrossRef

14.

Thompson BA, Spurdle AB, Plazzer J-P et al (2013) Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database. Nat Genet 46:107–115. https://doi.org/10.1038/ng.2854CrossRefPubMedPubMedCentral

15.

Richards S, Aziz N, Bale S et al (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405–423. https://doi.org/10.1038/gim.2015.30CrossRefPubMedPubMedCentral

16.

Dominguez-Valentin M, Nakken S, Tubeuf H et al (2018) Identification of genetic variants for clinical management of familial colorectal tumors. BMC Med Genet 19:26. https://doi.org/10.1186/s12881-018-0533-9CrossRefPubMedPubMedCentral

17.

Köger N, Paulsen L, López-Kostner F et al (2018) Evaluation of MLH1 variants of unclear significance. Genes Chromosom Cancer 57:350–358. https://doi.org/10.1002/gcc.22536CrossRefPubMed

18.

Dominguez-Valentin M, Evans DGR, Nakken S et al (2018) Genetic variants of prospectively demonstrated phenocopies in BRCA1/2 kindreds. Hered Cancer Clin Pract 16:4. https://doi.org/10.1186/s13053-018-0086-0CrossRefPubMedPubMedCentral

19.

Vaccaro CA, López-Kostner F, Adriana DV et al (2018) From colorectal cancer pattern to the characterization of individuals at risk: Picture for genetic research in Latin America. Int J Cancer. https://doi.org/10.1002/ijc.31920CrossRefPubMedPubMedCentral

20.

Dominguez-Valentin M, Nakken S, Tubeuf H et al (2018) Potentially pathogenic germline CHEK2 c.319+2T%3eA among multiple early-onset cancer families. Fam Cancer 17:141–153. https://doi.org/10.1007/s10689-017-0011-0CrossRefPubMed

21.

Baralle D, Lucassen A, Buratti E (2009) Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep 10:810–816. https://doi.org/10.1038/embor.2009.170CrossRefPubMedPubMedCentral

22.

Dominguez-Valentin M, Nilbert M, Wernhoff P et al (2013) Mutation spectrum in South American Lynch syndrome families. Hered Cancer Clin Pract 11:18. https://doi.org/10.1186/1897-4287-11-18CrossRefPubMedPubMedCentral

23.

Rossi BM, Palmero EI, López-Kostner F et al (2017) A survey of the clinicopathological and molecular characteristics of patients with suspected Lynch syndrome in Latin America. BMC Cancer 17:623. https://doi.org/10.1186/s12885-017-3599-4CrossRefPubMedPubMedCentral

24.

Carneiro da Silva F, de Ferreira JRO, Torrezan GT et al (2015) Clinical and molecular characterization of Brazilian patients suspected to have lynch syndrome. PLoS ONE 10:e0139753–e0139753. https://doi.org/10.1371/journal.pone.0139753CrossRefPubMedPubMedCentral

25.

Della Valle A, Rossi BM, Palmero EI et al (2019) A snapshot of current genetic testing practice in Lynch syndrome: the results of a representative survey of 33 Latin American existing centres/registries. Eur J Cancer 119:112–121. https://doi.org/10.1016/j.ejca.2019.07.017CrossRefPubMed

26.

den Dunnen JT, Antonarakis SE (1999) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15:7–12CrossRef

27.

Valentin MD, Da Silva FC, Dos SEMM et al (2011) Characterization of germline mutations of MLH1 and MSH2 in unrelated south American suspected Lynch syndrome individuals. Fam Cancer 10:641–647. https://doi.org/10.1007/s10689-011-9461-yCrossRefPubMed

28.

Houdayer C, Caux-Moncoutier V, Krieger S et al (2012) Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants. Hum Mutat 33:1228–1238. https://doi.org/10.1002/humu.22101CrossRefPubMed

29.

Gaildrat P, Killian A, Martins A et al (2010) Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. In: Webb M (ed) Cancer susceptibility: methods and protocols. Humana Press, Totowa, pp 249–257CrossRef

30.

Ho SN, Hunt HD, Horton RM et al (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59. https://doi.org/10.1016/0378-1119(89)90358-2CrossRefPubMed

31.

Pavicic W, Nieminen TT, Gylling A et al (2014) Promoter-specific alterations of APC are a rare cause for mutation-negative familial adenomatous polyposis. Genes Chromosom Cancer. https://doi.org/10.1002/gcc.22197CrossRefPubMed

32.

Casey G, Lindor NM, Papadopoulos N, Al E (2005) Conversion analysis for mutation detection in mlh1 and msh2 in patients with colorectal cancer. JAMA 293:799–809CrossRefPubMedPubMedCentral

33.

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

34.

Petersen SM, Dandanell M, Rasmussen LJ et al (2013) Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients. BMC Med Genet 14:103. https://doi.org/10.1186/1471-2350-14-103CrossRefPubMedPubMedCentral

35.

Lefevre JH, Colas C, Coulet F et al (2010) MYH biallelic mutation can inactivate the two genetic pathways of colorectal cancer by APC or MLH1 transversions. Fam Cancer 9:589–594. https://doi.org/10.1007/s10689-010-9367-0CrossRefPubMed

36.

Maquat LE (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1:453–465PubMedPubMedCentral

37.

Silva FCC, Torrezan GT, Ferreira JRO et al (2017) Germline mutations in MLH1 leading to isolated loss of PMS2 expression in Lynch syndrome. Am J Surg Pathol 41:861–864. https://doi.org/10.1097/PAS.0000000000000827CrossRefPubMed

38.

Thompson BA, Martins A, Spurdle AB (2015) A review of mismatch repair gene transcripts: issues for interpretation of mRNA splicing assays. Clin Genet 87:100–108. https://doi.org/10.1111/cge.12450CrossRefPubMed

39.

Goldberg Y, Porat RM, Kedar I et al (2008) Mutation spectrum in HNPCC in the Israeli population. Fam Cancer 7:309–317. https://doi.org/10.1007/s10689-008-9191-yCrossRefPubMed

40.

Wolf B, Henglmueller S, Janschek E et al (2005) Spectrum of germ-line MLH1 and MSH2 mutations in Austrian patients with hereditary nonpolyposis colorectal cancer. Wien Klin Wochenschr 117:269–277CrossRefPubMed

41.

Burn J, Gerdes AM, MacRae F et al (2011) Long-term effect of aspirin on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial. Lancet 378:2081–2087. https://doi.org/10.1016/S0140-6736(11)61049-0CrossRefPubMedPubMedCentral

42.

Sahashi K, Masuda A, Matsuura T et al (2007) In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5′ splice sites. Nucleic Acids Res 35:5995–6003. https://doi.org/10.1093/nar/gkm647CrossRefPubMedPubMedCentral

43.

van der Klift HM, Jansen AML, van der Steenstraten N et al (2015) Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses. Mol Genet Genom Med 3:327–345. https://doi.org/10.1002/mgg3.145CrossRef

44.

Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3:285–298. https://doi.org/10.1038/nrg775CrossRefPubMed

45.

Krawczak M, Thomas NST, Hundrieser B et al (2007) Single base-pair substitutions in exon–intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat 28:150–158. https://doi.org/10.1002/humu.20400CrossRefPubMed

46.

Buratti E, Chivers M, Královičová J et al (2007) Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 35:4250–4263. https://doi.org/10.1093/nar/gkm402CrossRefPubMedPubMedCentral

Title: MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing
Authors: Tamara Alejandra Piñero
Omar Soukarieh
Marion Rolain
Karin Alvarez
Francisco López-Köstner
Giovana Tardin Torrezan
Dirce Maria Carraro
Ivana Lucia De Oliveira Nascimento
Thaís Ferreira Bomfim
Taísa Manuela Bonfim Machado-Lopes
Juliana Côrtes Freitas
Maria Betânia Toralles
Kiyoko Abe Sandes
Benedito Mauro Rossi
Samuel Aguiar Junior
Joanna Meira
Mev Dominguez-Valentin
Pål Møller
Carlos Alberto Vaccaro
Alexandra Martins
Walter Hernán Pavicic
Publication date: 01-10-2020
Publisher: Springer Netherlands
Published in: Familial Cancer / Issue 4/2020
Print ISSN: 1389-9600
Electronic ISSN: 1573-7292
DOI: https://doi.org/10.1007/s10689-020-00182-5

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