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

Richa Sharma,Charlotte M Niemeyer,Maria Spies,Miriam Erlacher,Charnise Goodings,Jill A Rosenfeld,Marc S Wold,Melanie Boerries,Dmitri Churikov,Caroline Kannengiesser,Patrick Revy,Alfonso G Fernandez,Fabian Beier,Shondra M Pruett-Miller,Vincent Géli,Sophia Polychronopoulou,Ti-Cheng Chang,Masayoshi Honda,Marcin W Wlodarski,Sandrine Hirschi,Stéphane Coulon,Hauke Busch,Louis Sanchez,Carole Saintomé,Melchior Lauten,Axel Künstner,Megan A Cooper,Marcus B Valentine,Víctor Pastor ,Sophie L Granger ,Sarah K Nicholas ,Sushree Sangita Sahoo ,Charikleia Kelaïdi

doi:10.1182/blood.2021011980

Abstract

AbstractHuman 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.

Highlights

Telomeres are complex structures made up of repetitive DNA sequences associated with specialized proteins found at natural ends of linear chromosomes in all mammals
The RPA1 c.680T.C, p.V227A variant found in patient 2 (P2) and Patient 3 (P3) occurs at ultra-low frequency in control populations
RPA1 is located on chromosome 17p13.3 and has 17 exons coding for a 70 kDa protein that is expressed in all tissues (Figure 1B, top panel)

Summary

Introduction

Telomeres are complex structures made up of repetitive DNA sequences associated with specialized proteins found at natural ends of linear chromosomes in all mammals. The hallmark function of telomeres is to protect chromosomal ends from degradation and inappropriate recombination, and activation of the DNA damage response.[1] In the absence of telomere-associated proteins, chromosomal ends undergo premature attrition with pathologic consequence of telomere biology disorders (TBD), referred to as short telomere syndromes (STS), dyskeratosis congenita or telomeropathies. TBD/STS–associated features include bone marrow failure (BMF), pulmonary and liver fibrosis, mucocutaneous fragility, and predisposition to myelodysplastic syndromes (MDS) and cancer.[2,3,4,5] Monoallelic or biallelic inactivation in 14 genes that code for telomerase holoenzyme, shelterin complex, telomere capping machinery, and accessory telomere processes have been identified as causing TBD/STS.[2,6] an estimated 30% of individuals with TBD/STS do not have a genetic resolve, which obscures timely diagnosis and clinical management.[6]

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