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Original research
Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies
  1. Massimo Bogliolo1,
  2. Roser Pujol1,
  3. Miriam Aza-Carmona2,
  4. Núria Muñoz-Subirana1,
  5. Benjamin Rodriguez-Santiago3,
  6. José Antonio Casado4,
  7. Paula Rio4,
  8. Christopher Bauser5,
  9. Judith Reina-Castillón6,
  10. Marcos Lopez-Sanchez6,
  11. Lidia Gonzalez-Quereda7,
  12. Pia Gallano7,
  13. Albert Catalá8,
  14. Ana Ruiz-Llobet9,
  15. Isabel Badell10,
  16. Cristina Diaz-Heredia11,
  17. Raquel Hladun12,
  18. Leonort Senent13,
  19. Bienvenida Argiles14,
  20. Juan Miguel Bergua Burgues15,
  21. Fatima Bañez15,
  22. Beatriz Arrizabalaga16,
  23. Ricardo López Almaraz16,
  24. Monica Lopez17,
  25. Ángela Figuera18,
  26. Antonio Molinés19,
  27. Inmaculada Pérez de Soto20,
  28. Inés Hernando21,
  29. Juan Antonio Muñoz22,
  30. Maria del Rosario Marin22,
  31. Judith Balmaña23,
  32. Neda Stjepanovic24,25,
  33. Estela Carrasco26,
  34. Isabel Cuesta27,
  35. José Miguel Cosuelo28,
  36. Alexandra Regueiro28,
  37. José Moraleda Jimenez29,
  38. Ana Maria Galera-Miñarro30,
  39. Laura Rosiñol31,
  40. Anna Carrió31,
  41. Cristina Beléndez-Bieler32,
  42. Antonio Escudero Soto32,
  43. Elena Cela32,
  44. Gregorio de la Mata33,
  45. Rafael Fernández-Delgado34,
  46. Maria Carmen Garcia-Pardos35,
  47. Raquel Sáez-Villaverde36,
  48. Marta Barragaño37,
  49. Raquel Portugal33,
  50. Francisco Lendinez38,
  51. Ines Hernadez39,
  52. José Manue Vagace40,
  53. Maria Tapia41,
  54. José Nieto42,
  55. Marta Garcia43,
  56. Macarena Gonzalez44,
  57. Cristina Vicho44,
  58. Eva Galvez45,
  59. Alberto Valiente46,
  60. Maria Luisa Antelo46,
  61. Phil Ancliff47,
  62. Francisco Garcia48,
  63. Joaquin Dopazo49,
  64. Julian Sevilla45,
  65. Tobias Paprotka5,
  66. Luis Alberto Pérez-Jurado50,
  67. Juan Bueren4,
  68. Jordi Surralles7
  1. 1 Department of Genetics and Microbiology, Universitat Autonoma de Barcelona, Barcelona, Spain
  2. 2 Hospital Universitario La Paz, Madrid, Spain
  3. 3 Hospital de la Santa Creu i Sant Pau Institut de Recerca, Barcelona, Spain
  4. 4 CIEMAT, Madrid, Spain
  5. 5 Eurofins GATC Biotech GmbH, Konstanz, Germany
  6. 6 Hospital del Mar Research Institute (IMIM), Universitat Pompeu Fabra, Barcelona, Spain
  7. 7 Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona (UAB), Barcelona, Spain
  8. 8 Department of Hematology, Hospital Sant Joan de Déu, Barcelona, Spain
  9. 9 Pediatric Hematology and Oncology Department, Hospital Sant Joan de Deu, Barcelona, Spain
  10. 10 Pediatric Hematology and HSCT Unit, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
  11. 11 Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Research Institute (VHIR), Barcelona, Spain
  12. 12 Hospital Vall d'Hebron, Barcelona, Catalunya, Spain
  13. 13 Hospital La Fe, Valencia, Valenciana, Spain
  14. 14 Pediatric Haematology Unit, Hospital de la Fe, Valencia, Spain
  15. 15 Hospital San Pedro de Alcántara, Caceres, Spain
  16. 16 Hospital de Cruces, Barakaldo, País Vasco, Spain
  17. 17 Hospital Universitario Marques de Valdecilla, Santander, Spain
  18. 18 Hospital Universitario de la Princesa, Madrid, Spain
  19. 19 C.H.U. Insular-Materno Infantil, Las Palmas de Gran Canaria, Spain
  20. 20 Hospital Universitario Virgen del Rocío, Sevilla, Spain
  21. 21 Hospital Universitario Central de Asturias, Oviedo, Spain
  22. 22 Hospital Universitario Puerta del Mar, Cadiz, Andalucía, Spain
  23. 23 Medical Oncology Department, University Hospital Vall d’Hebron, Barcelona, Spain
  24. 24 Medical Oncology Department, Hospital Vall d'Hebron, Barcelona, Spain
  25. 25 High Risk and Cancer Prevention Group, Vall d'Hebron Institut d'Oncologia, Barcelona, Spain
  26. 26 Hereditary Cancer Unit, Vall d'Hebron Institut d'Oncologia, Barcelona, Spain
  27. 27 Hospital General de Teruel Obispo Polanco, Teruel, Aragón, Spain
  28. 28 Hospital Clinico Universitario de Santiago de Compostela, Santiago de Compostela, Spain
  29. 29 Hospital Virgen de la Arrixaca, El Palmar, Murcia, Spain
  30. 30 Hospital Clinico Universitario Virgen de la Arrixaca, El Palmar, Spain
  31. 31 Hospital Clínic de Barcelona, Barcelona, Catalunya, Spain
  32. 32 Pediatric Oncology and Hematology Section, General University Hospital Gregorio Marañón, Madrid, Spain
  33. 33 Hospital Universitario de Burgos, Burgos, Spain
  34. 34 Hospital Clínico Universitario, Valencia, Spain
  35. 35 Hospital of Donostia, San Sebastian, Spain
  36. 36 Department of Genetics, Hospital Universitario Donostia, Gipuzkoa, Spain
  37. 37 San Rafael Hospital, Madrid, Spain
  38. 38 Hospital Universitario Torrecárdenas, Almeria, Spain
  39. 39 Hospital Germans Trias i Pujol, Badalona, Spain
  40. 40 Hospital Materno Infantil de Badajoz, Badajoz, Spain
  41. 41 Hospital General de la Palma, Brena Alta, Spain
  42. 42 Hospital General Universitario José M Morales Meseguer, Murcia, Spain
  43. 43 Hospital Mutua de Terrassa, Terrassa, Spain
  44. 44 Hospital Universitario de Canarias, La Laguna, Spain
  45. 45 Hospital Infantil Universitario Nino Jesus, Madrid, Spain
  46. 46 Complejo Hospitalario de Navarra, Pamplona, Spain
  47. 47 Great Ormond Street Hospital for Children, London, UK
  48. 48 Computational Genomics Department, Centro de Investigación Príncipe Felipe, Valencia, Spain
  49. 49 Fundación Pública Andaluza Progreso y Salud, Sevilla, Spain
  50. 50 Unitat de Genètica, Universitat Pompeu Fabra, Barcelona, Spain
  1. Correspondence to Dr Massimo Bogliolo, Genetics and Microbiology, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain; massimo.bogliolo{at}


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.

  • genetics
  • 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.