Clonal Hematopoiesis in Telomere Biology Disorders Associates with the Underlying Germline Defect and Somatic Mutations in POT1, PPM1D, and TERT promoter

Introduction: Telomere biology (TBD) disorders are caused by pathogenic germline variants in genes related to telomere maintenance. In TBD, clonal hematopoiesis (CH) has been hypothesized to compensate for restricted cell fitness and to lead to development of myelodysplastic syndromes and acute myeloid leukemia (MDS/AML). We sought to characterize the clonal landscape and dynamics by deep sequencing of a large cohort of TBD patients with a broad spectrum of phenotypes and ages.

Methods: We screened 120 TBD patients (median age=29) from the National Institutes of Health and the University of Sao Paulo for somatic mutations in genes related to myeloid malignancies and telomere diseases using an error-correcting DNA sequencing panel (minimum allele frequency [VAF] of 0.5%). Patients had either a pathogenic germline variant in telomere-related genes or short telomeres in blood and a strong clinical suspicion for TBD. Relatives were included if they harbored the proband's germline mutation. Single-cell DNA sequencing was performed in marrow samples from two TBD patients with MDS (TBD-MDS) to elucidate clonal trajectories

Results: Fifty-eight TBD patients (48%) had somatic mutations in peripheral blood (median age and range, 42 years; 9-57), most frequently in PPM1D (all exon 6 truncated; n=18) , TERTp (-57, -124, and -146; n=14), POT1 (n=12), U2AF1 (n=12), and other MDS-associated genes. Clinically, these patients had dyskeratosis congenita (DC; n=12/27), aplastic anemia (AA; n=11/27), isolated cytopenias (n=7/10), MDS/AML (n=7/8), pulmonary or liver fibrosis (n=4/8), and multi-organ disease (n=19/26). In this series, no relatives had somatic mutations (n=14). CH frequency increased with age and was significantly more frequently observed than in healthy controls, regardless of age (p<0.001). POT1, PPM1D, and TERTp clones size was lower than the size of MDS-associated clones (VAF of 1% vs 8%). These mutations often co-occurred, except for POT1 and TERTp mutation.

Patients' clonal profiles correlated with the underlying germline defect. Somatic P OT1 mutations strongly associated with TINF2 germline variants, and consequently DC: 5/9 TINF2 patients had one (n=2) or >2 POT1 clones (n=3). In contrast, both TERTp and PPM1D clones were mostly detected in TERT/TERC patients with multi-organ disease, especially pulmonary fibrosis and marrow failure. No telomere elongation or improved blood counts were seen in serial samples. TINF2 patients with somatically mutated POT1 clones were older despite their DC diagnosis (median age=19 vs 5 years in POT1 mutated and wild type, respectively). A single patient with a germline TINF2 R282C and somatically mutated POT1 clone at VAF=29%, which was stable for 5 years, had MAA. The median ages (range) of TERT/TERC patients with TERTp and PPM1D mutations were 41 (25-64) and 43 (12-72), respectively, whereas TERT/TERC patients without TERTp and PPM1D mutations were at a median age of 27 (8-58). Most clones were stable regardless of clinical phenotype, even after danazol treatment. PPM1D clones were stable for 2-9 years of follow-up. TERTp and POT1 clones' size decreased while on androgens but consistently increased after the drug was discontinued.

In single-cell DNA analysis of two TBD-MDS patients, the U2AF1 S34F and Q157R were driver mutations and occurred with mutations in RUNX1, ETV6, ASXL1; these clones were stable for 3-6 years. In the first case, the U2AF1 clone subsequently acquired a RUNX1 mutation; this clone was coincident with an independent clone containing PPM1D and POT1 mutations. In the second patient, a U2AF1 clone acquired successive mutations in SETBP1 and AXSL1; a second clone with U2AF1 and additional mutations in GATA2 and KRAS arose at evolution to AML.

Conclusion: In TBD, the somatic landscape differed from age-related CH, with recurrent TERTp, POT1, and truncated PPM1D mutations. Mutations' frequency increased with age but was related to the underlying germline mutation. It is uncertain whether clonal selection is a probabilistic consequence of older age or the cause of mild phenotypes and prolonged lifespan. Despite the association of POT1 and PPM1D with malignancies, no patients in our cohort had POT1-related cancers or had received chemotherapy. POT1 and PPM1D like TERTp mutations may arise to compensate for cell fitness. Clinically, this distinct clonal landscape, not found in immune BMF, could serve as a molecular marker of underlying TBD.