• Germline RPA1 gain-of-function missense mutations result in a telomere biology disorder phenotype

  • Somatic rescue events arise in hematopoiesis secondary to germline RPA1 mutation

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 three germline heterozygous missense variants in RPA1 gene in four 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 while RPA1T270A has binding properties similar to wild type protein. To study the mutational effect in a cellular system, we used CRISPR/Cas9 to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patient with RPA1E240K, we discovered somatic genetic rescue (SGR) in hematopoietic cells due to an acquired truncating cisRPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the two SGR 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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