Identification of a robust DNA methylation signature for Fanconi anemia

Daria Pagliara; Andrea Ciolfi; Lucia Pedace; Sadegheh Haghshenas; Marco Ferilli; Michael A Levy; Evelina Miele; Claudia Nardini; Camilla Cappelletti; Raissa Relator; Angela Pitisci; Rita De Vito; Simone Pizzi; Jennifer Kerkhof; Haley McConkey; Francesca Nazio; Sarina G Kant; Maddalena Di Donato; Emanuele Agolini; Marta Matraxia; Barbara Pasini; Alessandra Pelle; Tiziana Galluccio; Antonio Novelli; Tahsin Stefan Barakat; Marco Andreani; Francesca Rossi; Cristina Mecucci; Anna Savoia; Bekim Sadikovic; Franco Locatelli; Marco Tartaglia

doi:10.1016/j.ajhg.2023.09.014

Identification of a robust DNA methylation signature for Fanconi anemia

Am J Hum Genet. 2023 Nov 2;110(11):1938-1949. doi: 10.1016/j.ajhg.2023.09.014. Epub 2023 Oct 20.

Authors

Daria Pagliara¹, Andrea Ciolfi², Lucia Pedace¹, Sadegheh Haghshenas³, Marco Ferilli², Michael A Levy³, Evelina Miele¹, Claudia Nardini¹, Camilla Cappelletti², Raissa Relator³, Angela Pitisci¹, Rita De Vito⁴, Simone Pizzi², Jennifer Kerkhof³, Haley McConkey⁵, Francesca Nazio¹, Sarina G Kant⁶, Maddalena Di Donato⁷, Emanuele Agolini⁷, Marta Matraxia⁷, Barbara Pasini⁸, Alessandra Pelle⁸, Tiziana Galluccio⁹, Antonio Novelli⁷, Tahsin Stefan Barakat¹⁰, Marco Andreani⁹, Francesca Rossi¹¹, Cristina Mecucci¹², Anna Savoia¹³, Bekim Sadikovic⁵, Franco Locatelli¹⁴, Marco Tartaglia¹⁵

Affiliations

¹ Department of Hematology/Oncology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.
² Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.
³ Verspeeten Clinical Genome Centre, London Health Sciences Centre, London, ON N6A 5W9, Canada.
⁴ Department of Laboratories, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.
⁵ Verspeeten Clinical Genome Centre, London Health Sciences Centre, London, ON N6A 5W9, Canada; Department of Pathology and Laboratory Medicine, Western University, London, ON N6A 3K7, Canada.
⁶ Department of Clinical Genetics, Erasmus MC University Medical Center, 3015 Rotterdam, the Netherlands.
⁷ Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Gesù Children Hospital, IRCCS, 00146 Rome, Italy.
⁸ AOU Città della salute e della scienza di Torino, Molinette's Hospital, 10126 Torino, Italy.
⁹ Laboratory of Transplant Immunogenetics, Department of Hematology/Oncology, Cell and Gene Therapy, IRCCS Bambino Gesù Children's Hospital, 00146 Rome, Italy.
¹⁰ Department of Clinical Genetics, Erasmus MC University Medical Center, 3015 Rotterdam, the Netherlands; ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC University Medical Center, 3015 Rotterdam, the Netherlands.
¹¹ Department of Woman, Child and of General and Specialist Surgery, University of Campania "Luigi Vanvitelli," 80138 Naples, Italy.
¹² Institute of Hematology and Center for Hemato-Oncology Research, University and Hospital of Perugia, 06123 Perugia, Italy.
¹³ Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, 37134 Verona, Italy.
¹⁴ Department of Hematology/Oncology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy; Department of Pediatrics, Catholic University of the Sacred Hearth, 00168 Rome, Italy. Electronic address: [email protected].
¹⁵ Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy. Electronic address: [email protected].

Abstract

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.

Keywords: DNA methylation profiling; Fanconi anemia; classifier; diagnostic tool; episignature; gene conversion; hematological disorders; machine learning; mosaicism; variant classification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA / metabolism
DNA Methylation / genetics
Fanconi Anemia Complementation Group Proteins / genetics
Fanconi Anemia Complementation Group Proteins / metabolism
Fanconi Anemia* / diagnosis
Fanconi Anemia* / genetics
Fanconi Anemia* / metabolism
Humans
Proteins / genetics

Substances

Fanconi Anemia Complementation Group Proteins
Proteins
DNA