Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies

Massimo Bogliolo, Roser Pujol, Miriam Aza-Carmona, Núria Muñoz-Subirana, Benjamin Rodriguez-Santiago, José Antonio Casado, Paula Rio, Christopher Bauser, Judith Reina-Castillón, Marcos Lopez-Sanchez, Lidia Gonzalez-Quereda, Pia Gallano, Albert Catalá, Ana Ruiz-Llobet, Isabel Badell, Cristina Diaz-Heredia, Raquel Hladun, Leonort Senent, Bienvenida Argiles, Juan Miguel Bergua BurguesFatima Bañez, Beatriz Arrizabalaga, Ricardo López Almaraz, Monica Lopez, Ángela Figuera, Antonio Molinés, Inmaculada Pérez De Soto, Inés Hernando, Juan Antonio Muñoz, Maria Del Rosario Marin, Judith Balmaña, Neda Stjepanovic, Estela Carrasco, Isabel Cuesta, José Miguel Cosuelo, Alexandra Regueiro, José Moraleda Jimenez, Ana Maria Galera-Miñarro, Laura Rosiñol, Anna Carrió, Cristina Beléndez-Bieler, Antonio Escudero Soto, Elena Cela, Gregorio De La Mata, Rafael Fernández-Delgado, Maria Carmen Garcia-Pardos, Raquel Sáez-Villaverde, Marta Barragaño, Raquel Portugal, Francisco Lendinez, Ines Hernadez, José Manue Vagace, Maria Tapia, José Nieto, Marta Garcia, Macarena Gonzalez, Cristina Vicho, Eva Galvez, Alberto Valiente, Maria Luisa Antelo, Phil Ancliff, Francisco Garcia, Joaquin Dopazo, Julian Sevilla, Tobias Paprotka, Luis Alberto Pérez-Jurado, Juan Bueren, Jordi Surralles

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3 Citations (Scopus)

Abstract

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Purpose: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. Methods: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. Results: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) Conclusion: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
Original languageEnglish
JournalJournal of Medical Genetics
DOIs
Publication statusPublished - 1 Jan 2019

Keywords

  • clinical genetics
  • genetics
  • haematology (incl blood transfusion)

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    Bogliolo, M., Pujol, R., Aza-Carmona, M., Muñoz-Subirana, N., Rodriguez-Santiago, B., Casado, J. A., Rio, P., Bauser, C., Reina-Castillón, J., Lopez-Sanchez, M., Gonzalez-Quereda, L., Gallano, P., Catalá, A., Ruiz-Llobet, A., Badell, I., Diaz-Heredia, C., Hladun, R., Senent, L., Argiles, B., ... Surralles, J. (2019). Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies. Journal of Medical Genetics. https://doi.org/10.1136/jmedgenet-2019-106249