Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue

Blood. 2022 Feb 17;139(7):1039-1051. doi: 10.1182/blood.2021011980.

Richa Sharma ¹, Sushree S Sahoo ¹, Masayoshi Honda ², Sophie L Granger ², Charnise Goodings ¹, Louis Sanchez ³, Axel Künstner ⁴ ⁵, Hauke Busch ⁴ ⁵, Fabian Beier ⁶, Shondra M Pruett-Miller ⁷, Marcus B Valentine ⁸, Alfonso G Fernandez ¹, Ti-Cheng Chang ⁹, Vincent Géli ¹⁰, Dmitri Churikov ¹⁰, Sandrine Hirschi ¹¹, Victor B Pastor ⁹, Melanie Boerries ¹² ¹³, Melchior Lauten ¹⁴, Charikleia Kelaidi ¹⁵, Megan A Cooper ¹⁶, Sarah Nicholas ¹⁷, Jill A Rosenfeld ¹⁸, Sophia Polychronopoulou ¹⁵, Caroline Kannengiesser ¹⁹, Carole Saintomé ³ ²⁰, Charlotte M Niemeyer ¹³ ²¹, Patrick Revy ²², Marc S Wold ², Maria Spies ², Miriam Erlacher ¹³ ²¹, Stéphane Coulon ¹⁰, Marcin W Wlodarski ¹ ²¹

Affiliations

1 Department of Hematology, St Jude Children's Research Hospital, Memphis, TN.
2 Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA.
3 Structure et Instabilité des Génomes, Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 7196, INSERM Unité1154, Paris, France.
4 Lübeck Institute of Experimental Dermatology and Institute of Cardiogenetics, University of Lübeck, Lübeck, Germany.
5 University Cancer Center Schleswig-Holstein, University Hospital of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
6 Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
7 Department of Cell and Molecular Biology.
8 Cytogenetics Core Facility, and.
9 Center for Applied Bioinformatics, St Jude Children's Research Hospital, Memphis, TN.
10 Marseille Cancer Research Centre, Unité1068 INSERM, UMR 7258 CNRS, Aix-Marseille University (UM 105), Institut Paoli-Calmettes, Equipe Labellisée par la Ligue Nationale contre le Cancer, Marseille, France.
11 Department of Respiratory Medicine and Rare Pulmonary Diseases, Strasbourg University Hospital, Strasbourg, France.
12 Institute of Medical Bioinformatics and Systems Medicine, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
13 German Cancer Consortium (DKTK), Freiburg, Germany, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
14 University Hospital Schleswig-Holstein, Department of Pediatrics, University of Lübeck, Lübeck, Germany.
15 Department of Pediatric Hematology/Oncology, Aghia Sophia Children's Hospital, Athens, Greece.
16 Department of Pediatrics, Washington University School of Medicine, St Louis, MO.
17 Department of Allergy and Immunology and.
18 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
19 Department of Genetics, Bichat Hospital, Assistance Publique-Hôpitaux de Paris, Paris University, INSERM U1152, Paris, France.
20 Sorbonne Université, Education and Research Unit for Life Sciences (UFR 927), Paris, France.
21 Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Medical Center, University of Freiburg, Freiburg, Germany; and.
22 Université de Paris, Imagine Institute, Laboratory of Genome Dynamics in the Immune System, Laboratoire Labellisé Ligue, INSERM UMR 1163, Paris, France.

PMID: 34767620 DOI: 10.1182/blood.2021011980

Abstract

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

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