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.
- haematology (incl blood transfusion)
- clinical genetics
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Contributors MasB: planned and coordinated the study, performed experiments, analysed the data and wrote the manuscript. RoP: performed experiments and coordinated the study. MA-C, NS, JAC and PR: performed experiments. BR-S, FG and JD: bioinformatics data analysis. CB and TP: coordinated the study. JR-C: performed experiments, bioinformatics data analysis. ML-S: performed experiments and bioinformatics data analysis. LG-Q and PG: analysed the data. AiC, AR-L, IB, CD-H, RH, LS, BA, JMBB, FI, BA, ML, ÁF, AM, IPdS, InésH, JAM, MdRM, JudB, NS, EC, IC, JMC, AR, JMJ, AMG-M, LR, AnC, CB-B, AES, EsC, GdIM, RF-D, MCG-P, RS-V, MarB, RaP, FL, InesH, JMV, MT, JN, MarG, MacG, CV, EG, AV, MLA and PA: provided patients samples and clinical data. JuS: coordinated the study and analysed data. LAP-J: analysed data and corrected the manuscript. JuaB: coordinated the study and corrected the manuscript. JoS: planned and coordinated the study, provided funding and corrected the manuscript. All the authors read and approved the manuscript. MasB and JoS are guarantors for the overall content of the manuscript
Funding Surrallés laboratory is supported by ICREA-Academia programme, Spanish Ministry of Health (projects FANCOSTEM and FANCOLEN), Spanish Ministry of Economy and Competiveness (projects CB06/07/0023 and SAF2015-64152-R), European Commission (EUROFANCOLEN project HEALTH-F5-2012-305421 and P-SPHERE COFUND project), Fanconi Anemia Research Fund Inc and the ‘Fondo Europeo de Desarrollo Regional, una manera de hacer Europa’ (FEDER). This work was also funded by intramural actions (ACCI-U735/U745-2016 to LAPJ and JS) of CIBERER. CIBERER is an initiative of the Instituto de Salud Carlos III, Spain.
Competing interests JoS obtained financial support for research from Rocket Pharmaceuticals (New York, USA). The rest of the authors declare no competing financial interests.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.
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