Somatic TERT promoter Mutations in a Broad Spectrum of Telomeropathies

Telomerase reactivation by acquisition of mutations in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. The most common TERTp mutations are located in positions -146, -124, and -57 upstream the initiation codon. In non-malignant diseases, TERTp mutations only have been reported in patients with idiopathic pulmonary fibrosis (IPF) caused by germline mutations in telomere biology genes, that are also etiologic in a broader spectrum of diseases collectively named telomeropathies (such as IPF, aplastic anemia [AA], dyskeratosis congenita [DC], and cirrhosis).

We screened blood from 136 patients with telomeropathies (median age=29 years; range, 1-76), 52 relatives (median age=40 years; range, 8-72), and 195 controls using a customized low-cost amplicon-based next-generation sequencing (NGS) assay for identification and quantification TERTp mutations. Patients had DC (n=21), AA (n=86), IPF with or without another telomeropathy-related phenotype (n=18), or other phenotypes (n=11). Inclusion criteria were telomere length (TL) below the 10th percentile of age-matched controls or a germline mutation in a telomere-related gene classified as pathogenic/likely pathogenic or of uncertain significance by the ACMG criteria. Patients' relatives were only studied if they carried the same germline mutation as the proband or had short telomeres, regardless of symptoms or evidence of disease. Patients with acquired AA (n=70), IPF (n=12), other inherited bone marrow failure (n=7), and acute myeloid leukemia (AML; n=106) were controls. All TERTp mutations identified by NGS were confirmed and tracked over time by droplet digital PCR.

We identified the -124 or -146 mutations in leukocytes from 12 unrelated patients diagnosed with IPF (n=6), DC (n=2), or moderate AA (n=4). Five relatives also had the -146 (n=1), and -124 (n=4) mutations, all carriers of a germline mutation in telomere biology gene. The frequency of TERTp mutations was much higher in IPF patients compared to AA cases (33% vs. 4.6%; Fisher's exact test, P=0.0016). However, no difference in frequency of TERTp mutations among patients with IPF vs. marrow failure was observed (41% vs. 58%; Fisher's exact test, P>0.05), suggesting TERTp mutations occurred in both clinical presentations. MutTERTp clones positively correlated with age, as they were only present in individuals older than 18 years old and more frequent in those 60 to 80 years old. Also, TERTp mutations more frequently co-ocurred with germline TERT mutations (n=13) compared to mutations in TERC (n=2), RTEL1 (n=1), or DKC1 (n=2) (76% vs. 23%; Fisher's exact test, P=0.002). All germline variants were pathogenic or had some evidence of pathogenicity. MutTERTp clones size varied from 1.2% to 50% in total leukocytes and was at higher allele frequencies (VAF) in the granulocytic fraction from four patients. In serial samples (available for five patients), the mutTERTp clone size expanded over time, suggesting a selective growth advantage in comparison to unmutated hematopoietic cells. Despite that, mutTERTp clones did not associate with blood counts or telomere elongation; most subjects carrying a TERTp mutation, which is known to upregulate TERT expression, nevertheless had short or very short telomeres (15 out of 17 individuals). Six patients with mutTERTp clones (VAF ranging from 3-33% in myeloid cells) were treated with danazol for two years; four were responders and two were off-study after 3-6 months. In serial samples (available for two patients), the mutTERTp clone sizes decreased during danazol treatment while blood counts improved. After treatment, mutTERTp clones VAF increased. TERTp mutations were found in telomeropathy patients who had a germline variant in telomere biology genes but not in controls or patients with very short telomeres without germline variant in telomere biology genes.

We have expanded the spectrum of non-malignant diseases associated with somatic TERTp mutations to DC, AA, and cirrhosis. Our data indicate that mutTERTp clones are specifically and randomly selected with aging in a marrow environment deficient in telomerase function, and mutTERTp selection did not associate with patients' peripheral blood counts, TL, and response to danazol treatment. TERTp emergence may be a useful clonal indicator for telomere dysfunction and may help to assess the pathogenicity of unclear constitutional variants in telomeropathies.