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

2019; BMJ; Volume: 57; Issue: 4 Linguagem: Inglês

10.1136/jmedgenet-2019-106249

1468-6244

Massimo Bogliolo, Roser Pujol, Miriam Aza‐Carmona, Núria Muñoz-Subirana, Benjamín Rodríguez‐Santiago, José Antonio Casado, Paula Rı́o, Christopher Bauser, Judith Reina-Castillón, Marcos López‐Sánchez, Lidia González‐Quereda, P. Gallano, Albert Catalá, Ana Ruiz-Llobet, Isabel Badell, Cristina Díaz de Heredia, Raquel Hladun, Leonort Senent, Bienvenida Argilés, Juan Bergua, Fatima Bañez, Beatriz Arrizabalaga, Ricardo López Almaraz, Monica López, Á Figuera, Antonio Molinés, Inmaculada Pérez de Soto, Inés Hernando, Juan Antonio Muñoz, Maria Marín, Judith Balmañà, Neda Stjepanovic, Estela Carrasco, Isabel Cuesta, José Miguel Cosuelo, Alexandra Regueiro, José M. Moraleda, Ana Maria Galera-Miñarro, Laura Rosiñol, Ana 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 Lendínez, Ines Hernadez, José Manue Vagace, Maria C. Tapia, J. M. Nieto, Marta Garcia, M J Garcia Gonzalez, Cristina Vicho, Eva Heredero Gálvez, Alberto Valiente, María Luisa Antelo, Phil Ancliff, Francisco García‐García, Joaquı́n Dopazo, Julián Sevilla, Tobias Paprotka, Luis A. Pérez‐Jurado, Juan A. Bueren, Jordi Surrallés,

Chromosomal and Genetic Variations

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.