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

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

Author information

1
Department of Genetics and Microbiology, Universitat Autonoma de Barcelona, Barcelona, Spain massimo.bogliolo@uab.es.
2
Department of Genetics and Microbiology, Universitat Autonoma de Barcelona, Barcelona, Spain.
3
Hospital Universitario La Paz, Madrid, Spain.
4
Hospital de la Santa Creu i Sant Pau Institut de Recerca, Barcelona, Spain.
5
CIEMAT, Madrid, Spain.
6
Eurofins GATC Biotech GmbH, Konstanz, Germany.
7
Hospital del Mar Research Institute (IMIM), Universitat Pompeu Fabra, Barcelona, Spain.
8
Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona (UAB), Barcelona, Spain.
9
Department of Hematology, Hospital Sant Joan de Déu, Barcelona, Spain.
10
Pediatric Hematology and Oncology Department, Hospital Sant Joan de Deu, Barcelona, Spain.
11
Pediatric Hematology and HSCT Unit, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
12
Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Research Institute (VHIR), Barcelona, Spain.
13
Hospital Vall d'Hebron, Barcelona, Catalunya, Spain.
14
Hospital La Fe, Valencia, Valenciana, Spain.
15
Pediatric Haematology Unit, Hospital de la Fe, Valencia, Spain.
16
Hospital San Pedro de Alcántara, Caceres, Spain.
17
Hospital de Cruces, Barakaldo, País Vasco, Spain.
18
Hospital Universitario Marques de Valdecilla, Santander, Spain.
19
Hospital Universitario de la Princesa, Madrid, Spain.
20
C.H.U. Insular-Materno Infantil, Las Palmas de Gran Canaria, Spain.
21
Hospital Universitario Virgen del Rocío, Sevilla, Spain.
22
Hospital Universitario Central de Asturias, Oviedo, Spain.
23
Hospital Universitario Puerta del Mar, Cadiz, Andalucía, Spain.
24
Medical Oncology Department, University Hospital Vall d'Hebron, Barcelona, Spain.
25
Medical Oncology Department, Hospital Vall d'Hebron, Barcelona, Spain.
26
High Risk and Cancer Prevention Group, Vall d'Hebron Institut d'Oncologia, Barcelona, Spain.
27
Hereditary Cancer Unit, Vall d'Hebron Institut d'Oncologia, Barcelona, Spain.
28
Hospital General de Teruel Obispo Polanco, Teruel, Aragón, Spain.
29
Hospital Clinico Universitario de Santiago de Compostela, Santiago de Compostela, Spain.
30
Hospital Virgen de la Arrixaca, El Palmar, Murcia, Spain.
31
Hospital Clinico Universitario Virgen de la Arrixaca, El Palmar, Spain.
32
Hospital Clínic de Barcelona, Barcelona, Catalunya, Spain.
33
Pediatric Oncology and Hematology Section, General University Hospital Gregorio Marañón, Madrid, Spain.
34
Hospital Universitario de Burgos, Burgos, Spain.
35
Hospital Clínico Universitario, Valencia, Spain.
36
Hospital of Donostia, San Sebastian, Spain.
37
Department of Genetics, Hospital Universitario Donostia, Gipuzkoa, Spain.
38
San Rafael Hospital, Madrid, Spain.
39
Hospital Universitario Torrecárdenas, Almeria, Spain.
40
Hospital Germans Trias i Pujol, Badalona, Spain.
41
Hospital Materno Infantil de Badajoz, Badajoz, Spain.
42
Hospital General de la Palma, Brena Alta, Spain.
43
Hospital General Universitario José M Morales Meseguer, Murcia, Spain.
44
Hospital Mutua de Terrassa, Terrassa, Spain.
45
Hospital Universitario de Canarias, La Laguna, Spain.
46
Hospital Infantil Universitario Nino Jesus, Madrid, Spain.
47
Complejo Hospitalario de Navarra, Pamplona, Spain.
48
Great Ormond Street Hospital for Children, London, UK.
49
Computational Genomics Department, Centro de Investigación Príncipe Felipe, Valencia, Spain.
50
Fundación Pública Andaluza Progreso y Salud, Sevilla, Spain.
51
Unitat de Genètica, Universitat Pompeu Fabra, Barcelona, Spain.

Abstract

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)

Conflict of interest statement

Competing interests: JoS obtained financial support for research from Rocket Pharmaceuticals (New York, USA). The rest of the authors declare no competing financial interests.

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