RPA1 Gain of Function Causes Human Short Telomere Syndrome with Revertant Somatic Mosaicism

Dyskeratosis congenita (DC) is a short telomere syndrome with bone marrow failure (BMF), mucocutaneous fragility and predisposition to malignancy due to inherited mutations in telomere associated genes. The genetic cause remains unknown in 10-20% of DC patients. Here, we describe a new DC candidate gene, RPA1, which encodes the largest subunit of the Replication Protein A (RPA) complex, a single stranded DNA (ssDNA) binding protein essential for DNA replication, damage repair and telomere maintenance. The patient presented at the age of 5 years with pancytopenia and hypocellular marrow with morphology resembling refractory cytopenia of childhood, mucocutaneous triad and a congenital retinal anomaly. Further workup revealed telomeres in blood below 1^st percentile, normal female karyotype and a negative chromosomal breakage test. After excluding known genetic causes for DC, family whole exome sequencing identified a de novo RPA1 c.718G>A (p.E240K) mutation with a predicted pathogenic CADD score of 23 and absent in >140,000 gnomAD population controls. The mutation had 50% allelic frequency in fibroblasts in comparison to a reduced mutation burden of 27% in bone marrow (BM) cells. Using bulk and single cell sequencing of BM, we identified 2 somatic compensatory events that likely explain patient’s stable hematologic phenotype over a period of 20 years: i) an acquired RPA1 p.E579X mutation (in cis with p.E240K) was found at 10% frequency, which resulted in RNA degradation of the mutated allele as confirmed by RNA sequencing; ii) a founder clone with uniparental isodisomy on chromosome 17p encompassing RPA1 locus causing loss of germline E240K mutation. Analysis of serial BM samples using SNP arrays and deep sequencing demonstrated UPD17p clonal expansion concurrent with a decline of germline p.E240K and increase of acquired p.E579X mutation. This is consistent with a toxic, likely gain-of-function (GOF) effect of germline p.E240K mutation leading to acquired loss-of-function (LOF) escape mechanisms.

RPA1 is essential to all DNA metabolism transactions that encounter ssDNA, including binding to telomeric 3’ overhangs during late S-phase to unfold G-quadruplexes and facilitate telomerase activity. Several RPA1 genetic variants impart telomere shortening and an S-phase defect in yeast and human cell lines. However, the molecular mechanisms are not well understood and thus far, RPA1 gene has not been linked to human disease. In an iPSC-based disease model with homozygous RPA1 p.E240K knock-in, we discovered severe telomere shortening in RPA1 mutant iPSCs and iPSC derived hematopoietic progenitors (HPs) compared to WT counterparts. Furthermore, decreased erythroid differentiation capacity was noted in RPA1-mutant HPs. Because the germline p.E240K mutation is located in DNA binding domain A of RPA1, we performed electromobility shift assay to assess the DNA binding affinity of mutant RPA1 protein. The p.E240K heterotrimeric RPA complex shows increased ssDNA binding compared to wild type RPA.

In summary, we describe the first human disease manifesting as short telomere syndrome secondary to a de novo RPA1 mutation with likely GOF effect, which forces the development of multiple revertant mosaic events, similar to the newly described SAMD9/SAMD9L disorders. Our studies also have implications for the mechanistic understanding of the role of RPA1 in telomere maintenance and hematopoiesis.