Telomere Gene Complex Mutations Are Frequently Found with Shortened Telomeres in Bone Marrow Failure Syndromes and Idiopathic Pulmonary Fibrosis; Correlation with Haematological and Clinical Features

Shortened telomeres are seen in approximately a quarter of patients with idiopathic pulmonary fibrosis (IPF) and aplastic anemia (AA) and may have a role in disease pathogenesis. Mutations in the telomere gene complex (TGC) are less frequently seen in IPF (1%) and 5-10% in AA. Novel mutations affecting telomere function are increasingly being reported and involve the telomerase genes, the shelterin and helicase complexes, but their prognostic significance remains undefined. We report results on telomere length (TL) using multiplex quantitative real-time PCR (qPCR) and mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of 69 patients with AA and IPF and their effects on clinical variables and response rates.

69 patients (49 with AA, 14 with IPF and 6 with a bone marrow failure syndrome (BMFS) and pulmonary fibrosis; M/F =43/26) with a median age of 49.7 years (range 9-81) had TL (T/S ratio) tested using qPCR on peripheral blood mononuclear cells at diagnosis or pre-treatment. Massively parallel targeted sequencing of the TGC was performed in all cases and constitutional mutations identified using the exome variant server. Clinical variables studied included hemoglobin (Hb) level, neutrophil count (ANC), platelet count (PC), LDH and albumin levels, and response rates to immunosuppressive treatment (IST). In patients with AA and other BMFS, red cell and platelet transfusion dependency, bone marrow cellularity (<30), cytogenetics and p53 (Modified Quick score >1) on bone marrow (BM) staining were correlated with TL and presence of any TGC mutations. In patients with IPF, corrected transfer factor (Kco) was correlated with TL and mutation analysis.

44/69 (64%) had TL below the 1^st centile and 20 (29%) of them had TL very significantly below the 1^st centile compared to a normal cohort (n=180) of age-matched individuals. Median Hb was (11g/dl), ANC was 1.69 and PC was 96x10⁹/l. There was no statistical difference between patients whose TL was below or above the 1^st centile for Hb levels, reticulocyte count or PC. 24 patients (34.7%) were red cell transfusion dependent and 15(21.7%) needed platelet transfusions. 42/55 (76.3%) had BM cellularity < 30% and was not statistically significant between patients with TL above or below the 1^st centile. Similarly there was no statistical difference in LDH and albumin levels between the two groups. Equal number of patients were treated with IST between the two groups and response rates were similar. Median levels of Kco (0.95) were identical in IPF patients. 62 (89.8%) of patients were alive at a median follow up of 2.15 years (range 0.3-26.8).

TGC mutations were seen 16 patients (23%) in the entire cohort with 13/44 (30%) were seen where TL was below the 1^st centile as compared to 3/25 (12%) where TL was above the 1^st centile (p<0.14). 11 patients had TERT (all missense), 4 TERC (non-coding transcript) and 1 RTEL mutations were identified. TERT A279T was the commonest mutation seen, in 4 patients.

In our cohort, about two-thirds of patients had TL below the 1^st centile. TL, however, does not affect blood counts, BM cellularity and p53 staining, red cell or platelet transfusion dependency or Kco in patients with IPF. A higher proportion of patients with TL below the 1^st centile were found to have TGC mutations, although statistical significance was not achieved. Missense mutations in TERT were the most frequent followed by mutations in TERC. Patients with TL <1^st centile were diagnosed at a younger age, and where TGC mutations were present, were also likely to show other features of telomereopathy.