05180nas a2201117 4500008004100000022001400041245011300055210006900168260001200237300001200249490000700261520174900268653001402017653003102031653001502062653002502077653001902102653005102121653001102172653002902183653003802212653001102250653000902261653002302270653003602293653002702329100002202356700001702378700002402395700002802419700003302447700002702480700001502507700002402522700002902546700002602575700002802601700001702629700002002646700002102666700001902687700002702706700001902733700002002752700002402772700003002796700001902826700002602845700002902871700001802900700002102918700002202939700003202961700002002993700002603013700002903039700002103068700002203089700002103111700001903132700002703151700002403178700002903202700003203231700002003263700001803283700003103301700002803332700001603360700002503376700003103401700003303432700002903465700002203494700002103516700002403537700001903561700002503580700001703605700001703622700001803639700002303657700002003680700001603700700002203716700002503738700001803763700002303781700002003804700002003824700002103844700003303865700001703898700002103915856012603936 2020 eng d a1468-624400aOptimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.0 aOptimised molecular genetic diagnostics of Fanconi anaemia by wh c2020 04 a258-2680 v573 a
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 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 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.
10aCell Line10aDNA Copy Number Variations10aDNA Repair10aDNA-Binding Proteins10aFanconi Anemia10aFanconi Anemia Complementation Group A Protein10aFemale10aGene Knockout Techniques10aGenetic Predisposition to Disease10aHumans10aMale10aMutation, Missense10aPolymorphism, Single Nucleotide10awhole exome sequencing1 aBogliolo, Massimo1 aPujol, Roser1 aAza-Carmona, Miriam1 aMuñoz-Subirana, Núria1 aRodriguez-Santiago, Benjamin1 aCasado, José, Antonio1 aRio, Paula1 aBauser, Christopher1 aReina-Castillón, Judith1 aLopez-Sanchez, Marcos1 aGonzalez-Quereda, Lidia1 aGallano, Pia1 aCatalá, Albert1 aRuiz-Llobet, Ana1 aBadell, Isabel1 aDiaz-Heredia, Cristina1 aHladun, Raquel1 aSenent, Leonort1 aArgiles, Bienvenida1 aBurgues, Juan, Miguel Ber1 aBañez, Fatima1 aArrizabalaga, Beatriz1 aAlmaraz, Ricardo, López1 aLopez, Monica1 aFiguera, Ángela1 aMolinés, Antonio1 ade Soto, Inmaculada, Pérez1 aHernando, Inés1 aMuñoz, Juan, Antonio1 aMarin, Maria, Del Rosari1 aBalmaña, Judith1 aStjepanovic, Neda1 aCarrasco, Estela1 aCuesta, Isabel1 aCosuelo, José, Miguel1 aRegueiro, Alexandra1 aJimenez, José, Moraleda1 aGalera-Miñarro, Ana, Maria1 aRosiñol, Laura1 aCarrió, Anna1 aBeléndez-Bieler, Cristina1 aSoto, Antonio, Escudero1 aCela, Elena1 ade la Mata, Gregorio1 aFernández-Delgado, Rafael1 aGarcia-Pardos, Maria, Carmen1 aSáez-Villaverde, Raquel1 aBarragaño, Marta1 aPortugal, Raquel1 aLendinez, Francisco1 aHernadez, Ines1 aVagace, José, Manue1 aTapia, Maria1 aNieto, José1 aGarcia, Marta1 aGonzalez, Macarena1 aVicho, Cristina1 aGalvez, Eva1 aValiente, Alberto1 aAntelo, Maria, Luisa1 aAncliff, Phil1 aGarcía, Francisco1 aDopazo, Joaquin1 aSevilla, Julian1 aPaprotka, Tobias1 aPérez-Jurado, Luis, Alberto1 aBueren, Juan1 aSurralles, Jordi uhttps://www.clinbioinfosspa.es/content/optimised-molecular-genetic-diagnostics-fanconi-anaemia-whole-exome-sequencing-and