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

J Med Genet. 2020 Apr;57(4):258-268. doi: 10.1136/jmedgenet-2019-106249. Epub 2019 Oct 5.


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.

Keywords: clinical genetics; genetics; haematology (incl blood transfusion).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • DNA Copy Number Variations / genetics
  • DNA Repair / genetics
  • DNA-Binding Proteins / genetics
  • Exome Sequencing*
  • Fanconi Anemia / genetics*
  • Fanconi Anemia / pathology
  • Fanconi Anemia Complementation Group A Protein / genetics*
  • Female
  • Gene Knockout Techniques
  • Genetic Predisposition to Disease*
  • Humans
  • Male
  • Mutation, Missense / genetics
  • Polymorphism, Single Nucleotide / genetics


  • DNA-Binding Proteins
  • FANCA protein, human
  • Fanconi Anemia Complementation Group A Protein