In inherited marrow failure, clonal hematopoiesis (CH) are known to compensates for the intrinsic restricted cell fitness caused by a germline mutation (adaptive mechanisms) or associates with maladaptive mechanisms of clonal evolution, typically myelodysplastic syndromes or acute myeloid leukemia (MDS/AML). Here, we characterized for the first time the genomic landscape of telomere biology disorders (TBD), and report clinically significant genotype-phenotype associations that can serve as markers for patients’ diagnosis, clonal evolution, and survival.

Peripheral blood (PB) samples from 207 TBD patients collected at time of first visit were screened for CH in myeloid and telomere-related genes using an error-correcting DNA sequencing panel at minimum allele frequency (VAF) of 0.5%. Patients were seen at the National Institutes of Health and the Medical School of Ribeirao Preto from 1997 onwards. Clonal trajectories were assessed by single-cell proteogenomic sequencing (scDNA).

Most patients were symptomatic (83%), particularly having classical dyskeratosis congenita (DC) or marrow failure with lung or liver disease (BMF/PD_LD); 35 were asymptomatic. A germline mutation, found in 92% of patients, correlated with disease onset and inheritance. CH was restricted to symptomatic patients (80/173; 46%), being recurrently found in PPM1D, POT1, TERT promoter (TERT p), and U2AF1S34/Q157R; TP53 mutations were also common (8% of patients). PPM1D, POT1 and TERTp mutations were often at median low VAF (<2%) while U2AF1S34/Q157R and other MDS-related mutations were at VAF>10%. Abnormal karyotype was seen in 14% patients, mostly with chromosomal 1q abnormalities (Chr1q; 9/25). CH frequency increased with aging and associated with patients’ phenotypes and the underlying germline mutation: CH in TERTp, POT1¸ and MDS-associated genes were significantly enriched in TERT, TINF2, and TERC patients, respectively, rarely co-occurring with other germline mutations. CH in TERTp, TP53 and Chr1q associated with poor overall survival (OS) independently from age (p < 0.001). Presence of MDS-related mutations dominated by U2AF1S34/Q157R, TP53 mutations, and Chr1q highly associated with development of MDS/AML, regardless of VAF. The cumulative MDS/AML incidence in the cohort was 8% by age of 40, being higher in DKC1, TERC, and TERT patients with CH. Presence of TP53 mutations in PB and age were independent markers of solid cancer development, with head and neck squamous cell carcinoma predominating (43%). scDNA evidenced distinct clonal trajectories between adaptive (POT1, PPM1D, and TERTp) and maladaptive CH. U2AF1S34/Q157R and Chr1q were key drivers of malignancy due to successive acquisition of other MDS-related mutations in single cells; adaptive CH was only found in independent clones (linear vs. branched trajectories, respectively). In all, CH emerged at the HSC level and associated with a myeloid bias, being also present in up to 11% of lymphoid cells.

The clonal signatures of TBD, not found in immune mediated BMF, are molecular markers of the underlying telomere deficiency that can guide diagnosis, particularly for cases with an unknown germline mutation. CH is also important for surveillance as it identifies high-risk TBD patients with lower OS who could benefit of early intervention. Somatic and karyotypic testing is critical as detection of Chr1q and U2AF1S34/Q157R and TP53 mutations, regardless of VAFs, may warrant earlier therapeutic intervention as it is highly associated with malignancies.