1. Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies
- Author
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Rafael Fernández-Delgado, Massimo Bogliolo, Jordi Surrallés, Inmaculada Pérez de Soto, Fatima Bañez, Christopher Bauser, Cristina Beléndez-Bieler, Joaquín Dopazo, Eva M. Galvez, Raquel Sáez-Villaverde, Laura Rosiñol, Antonio Molinés, José Moraleda Jimenez, Miriam Aza-Carmona, Neda Stjepanovic, Gregorio de la Mata, Núria Muñoz-Subirana, Albert Català, Juan Miguel Bergua Burgues, Maria Marín, Leonort Senent, Ines Hernadez, Cristina Diaz-Heredia, Bienvenida Argilés, A. Figuera, Judith Reina-Castillón, Estela Carrasco, Macarena Gonzalez, Marta García, José A. Casado, José Nieto, Julián Sevilla, Luis A. Pérez-Jurado, Elena Cela, Ricardo López Almaraz, Isabel Cuesta, Antonio Escudero Soto, Raquel Portugal, José Manue Vagace, Benjamín Rodríguez-Santiago, Tobias Paprotka, Isabel Badell, Inés Hernando, Raquel Hladun, Cristina Vicho, Marta Barragaño, Anna Carrió, Pia Gallano, Francisco Lendínez, José Miguel Cosuelo, Roser Pujol, Marcos López-Sánchez, Ana Ruiz-Llobet, María Tapia, Phil Ancliff, Juan Antonio Muñoz, Monica Lopez, María Luisa Antelo, Alexandra Regueiro, Alberto Valiente, F.M. Garcia, Juan A. Bueren, Paula Río, Beatriz Arrizabalaga, Ana Maria Galera-Miñarro, Maria Carmen Garcia-Pardos, Judith Balmaña, and Lidia Gonzalez-Quereda
- Subjects
Male ,0301 basic medicine ,haematology (incl blood transfusion) ,DNA Copy Number Variations ,DNA Repair ,DNA repair ,Mutation, Missense ,Single-nucleotide polymorphism ,Biology ,Polymorphism, Single Nucleotide ,Cell Line ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,FANCE ,hemic and lymphatic diseases ,Exome Sequencing ,FANCD2 ,Genetics ,Humans ,genetics ,Genetic Predisposition to Disease ,Copy-number variation ,Genetics (clinical) ,Exome sequencing ,Fanconi Anemia Complementation Group A Protein ,Point mutation ,clinical genetics, genetics, haematology (incl blood transfusion) ,FANCA ,DNA-Binding Proteins ,Fanconi Anemia ,030104 developmental biology ,030220 oncology & carcinogenesis ,Female ,clinical genetics - Abstract
PurposePatients 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.Methods68 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.ResultsWe 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)ConclusionWES 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.
- Published
- 2019