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

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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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Availability of data and material statement

Data from the participating hereditary cancer registers, this is indeed available for researchers following direct contact with the register (thus not freely available online).

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Acknowledgements

We thank the patients and their families who participated in this work. We also want to thank Fabiana Alejandra Ferro, Pablo Germán Kalfayan and Juan Pablo Santino from Pro.Can.He for their support in Argentinean patient’s management and preparation of all the familial pedigrees, genetic variant analysis and IHC data analysis, respectively and the A.C. Camargo Biobank for sample processing. This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica [PICT-2017-3210 to C.V. and W.P.]; and the Instituto Nacional del Cáncer de Argentina [INC-Grant AFIV-2018/2020 to C.V. and W.P.]; and by Fundação de Amparo à Pesquisa do Estado de São Paulo [2014/50943-1 to DMC and GTT]. Moreover, this study was sponsored by the Groupement des Entreprise Françaises dans la Lutte contre le Cancer (Gefluc), and co-supported by the European Union and Région Normandie. Europe gets involved in Normandy with European Regional Development Fund (ERDF). This work was also supported by the Radium Hospital Foundation (Oslo, Norway), the Norwegian Cancer Society, contract 194751-2017 and Helse Sør-Øst (Norway).

Funding

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica [PICT-2017-3210 to C.V. and W.P.]; and the Instituto Nacional del Cáncer de Argentina [INC-Grant AFIV-2018/2020 to C.V. and W.P.]; and by Fundação de Amparo à Pesquisa do Estado de São Paulo [2014/50943-1 to DMC and GTT]. Moreover, this study was sponsored by the Groupement des Entreprise Françaises dans la Lutte contre le Cancer (Gefluc), and co-supported by the European Union and Région Normandie. Europe gets involved in Normandy with European Regional Development Fund (ERDF). This work was also supported by the Radium Hospital Foundation (Oslo, Norway), the Norwegian Cancer Society, contract 194751-2017 and Helse Sør-Øst (Norway).

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Author notes

  1. Tamara Alejandra Piñero and Omar Soukarieh contributed equally to this work.

Authors and Affiliations

  1. Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB) [HIBA-IUHI-CONICET], C1199ABH, Buenos Aires, Argentina

    Tamara Alejandra Piñero, Carlos Alberto Vaccaro & Walter Hernán Pavicic

  2. Programa de Cáncer Hereditario (Pro.Can.He.), Hospital Italiano de Buenos Aires, C1199ABH, Buenos Aires, Argentina

    Tamara Alejandra Piñero, Carlos Alberto Vaccaro & Walter Hernán Pavicic

  3. Inserm U1245, IRIB, UNIROUEN, Normandie Univ, Normandy Centre for Genomic and Personalized Medicine, 76183, Rouen Cedex 1, France

    Omar Soukarieh, Marion Rolain & Alexandra Martins

  4. Laboratorio de Oncología y Genética Molecular, Unidad de Coloproctología, Clínica Las Condes, 7591046, Santiago de Chile, Chile

    Karin Alvarez & Francisco López-Köstner

  5. Genomic and Molecular Biology Group, International Research Center, A. C. Camargo Cancer Center, São Paulo, 01508-010, Brazil

    Giovana Tardin Torrezan & Dirce Maria Carraro

  6. Laboratório de Imunologia e Biología Molecular, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, BA, 40110-902, Brazil

    Ivana Lucia De Oliveira Nascimento, Thaís Ferreira Bomfim, Taísa Manuela Bonfim Machado-Lopes, Juliana Côrtes Freitas, Maria Betânia Toralles & Kiyoko Abe Sandes

  7. Departamento de Ciências da Vida, Universidade do Estado da Bahia, Salvador, BA, 41150-000, Brazil

    Juliana Côrtes Freitas

  8. Hospital Sirio Libanes, São Paulo, 01308-050, Brazil

    Benedito Mauro Rossi

  9. Colorectal Tumor Nucleus of the Pelvic Surgery Department, A.C. Camargo Cancer Center, São Paulo, SP, 01509-010, Brazil

    Samuel Aguiar Junior

  10. Hospital Universitário Prof. Edgard Santos, Universidade Federal da Bahia, Salvador, 40301-155, Brazil

    Joanna Meira

  11. Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, 0369, Oslo, Norway

    Mev Dominguez-Valentin

  12. Instituto de Investigación, Universidad Católica de Trujillo, Chimbote, Peru

    Mev Dominguez-Valentin

  13. Department of Tumor Biology, The Norwegian Radium Hospital, Part of Oslo University Hospital, 0369, Oslo, Norway

    Pål Møller

Authors
  1. Tamara Alejandra Piñero
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  2. Omar Soukarieh
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  3. Marion Rolain
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  18. Pål Møller
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Contributions

TAP, OS, MDV, PM, CAV, AM and WHP contributed to conception and design of the study, and wrote the manuscript. TAP, OS, MR, KA, FLK, GTT, DMC, ILON, TFB, TMBML, JCF, MBT, KAS, BMR, SAJ, JM, MDV, CAV, AM and WHP contributed to acquisition and analysis of data. TAP, OS, GTT, AM and WHP prepared the figures. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Walter Hernán Pavicic.

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The authors declare no conflict of interest.

Consent to participate and for publication

Patients were informed about their inclusion into the registries and written informed consent was obtained from all participants during genetic counseling sessions.

Ethics approval

This study was approved by the institutional review board of the Hospital Italiano de Buenos Aires (Buenos Aires, Argentina), the A.C. Camargo Cancer Center (São Paulo, Brazil), the Federal University of Bahia (Bahia, Brazil) and the Clinica Las Condes (Santiago de Chile, Chile). All procedures followed were in accordance with the ethical standards of the Helsinki Declaration.

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Piñero, T.A., Soukarieh, O., Rolain, M. et al. MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing. Familial Cancer 19, 323–336 (2020). https://doi.org/10.1007/s10689-020-00182-5

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