Table 1.
Characteristics of patients with a diagnostic biopsy.
Fig 1.
Organ telomere length per subject.
Biopsy telomere length measurements by MMqPCR in lung, kidney, thyroid, liver and bladder tissue in 2 controls (Ctrls), 2 spradic IPF patients and one pulmonary fibrosis case with a TERT mutation. In patients with pulmonary fibrosis the shortest telomere length was found in lung tissue.
Fig 2.
(a) Telomere length measurements in lungs of 32 subjects with IPF by MMqPCR. No correlation was found (Spearman correlation; r = 0.015, p = 0.935). (b) Comparison of telomere length between 8 diagnostic biopsy and 8 explant lung specimens. Samples belonging to the same patient are connected. Wilcoxon matched-pairs signed rank tests showed no differences in telomere length between samples (p = 0.25).
Fig 3.
Telomere shortening is not associated with apical or basal localisation in whole explant lung.
(a) HRCT on the left and formalin fixed explant images of IPF lung with an apicobasal fibrotic gradient on the right. The top and bottom figures represent apical and basal locations respectively, corresponding to (b) the schematic picture. (c, d) Apical and basal lung telomere length comparison of (c) 8 lungs with an apicobasal gradient and (d) 7 lungs without apicobasal gradient measured by MMqPCR. Samples belonging to the same person are connected. Wilcoxon matched-pairs signed rank tests showed no differences in telomere length between lung sections.
Fig 4.
AT2 cell telomere length in IPF explant lung tissue measured by FISH.
(a) Hematoxylin and Eosin (H&E) staining representing non-fibrotic (NF) and fibrotic (F) areas of a typical IPF lung biopsy. Black arrows indicate fibroblast foci. (b) Example of a combined fluorescent image of AT2 cells (white arrows) in a fibrotic area of IPF lung tissue. DNA in nucleus is displayed in blue (DAPI), proSP-C in green and telomeres in red (dots). (c) Within a tissue specimen, telomere length in AT2 cells was significantly longer in non-fibrotic areas than in fibrotic areas. (n = 3; Mann-Whitney test: ** < 0.01). (d) No difference in AT2 cell telomere length was observed between apical and basal lung tissue. Boxes represent data between 1st and 3rd quartile and whiskers extend to the highest and lowest values of expression that are not considered outliers.
Fig 5.
Blood and biopsy telomere length in IPF and TERT subjects measured by MMqPCR.
(a, b) Spearman correlation of blood and biopsy telomere length in (a) 32 IPF and (b) 17 TERT cases. A significant positive correlation was established in IPF cases (r = 0.531, p = 0.002). In TERT cases, no significant correlation was found (r = -0.157, p = 0.545). Dashed lines represents threshold for lung telomere length associated with telomere related pathology. IPFnormal = lung telomere length above threshold, IPFshort = lung telomere length below threshold. (c) Tukey boxplots of lung telomere length measured by MMqPCR in control (n = 13), IPFnormal (n = 17), IPFshort (n = 15) and TERT (n = 17) lungs. Lung telomere length in the IPFshort group was significantly shorter than telomere length in age-matched controls (p = 0.0014) and IPFnormal (p = 0.0001). Telomere length in the TERT group was also significantly shorter than telomere length in controls (p < 0.0001) and in IPFnormal (p < 0.0001). Telomere length in the IPFnormal group and controls were comparable. Asterisks indicate significant differences calculated by Kruskal-Wallis multiple comparison tests (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Boxes represent data between 1st and 3rd quartile and whiskers extend to the highest and lowest values of expression that are not considered outliers.
Fig 6.
AT2 cell telomere length measured by fluorescence in situ hybridization (FISH) in 5 control, 5 IPFnormal, 5 IPFshort and 6 TERT lungs.
(a) Tukey box-plots of telomere length measured in AT2 cells by fluorescence in situ hybridization (FISH). All patient groups showed significant shorter AT2 cell telomere length than in controls (p < 0.0001).Telomere length in the IPFshort group was significantly shorter than in controls (p < 0.0001) and in IPFnormal (p < 0.0001). Telomere length in TERT was also significantly shorter than in controls (p < 0.0001) and in IPFnormal (p < 0.0001). No difference in AT2 telomere length was present between IPFshort and TERT. Asterisks indicate significant differences calculated by Kruskal-Wallis multiple comparison tests. (b, c, d) AT2 cell telomere length in non-fibrotic and fibrotic lung areas in (b) IPFnormal, (c) IPFshort and (d) TERT lungs. In IPFnormal no difference was found between areas. AT2 cell telomere length in non-fibrotic areas was significantly longer than fibrotic areas in IPFshort (p = 0.0237) and TERT (p = 0.0001) lungs. Asterisks indicate significant differences calculated by Mann-Whitney analyses (ns = not significant, * = p < 0.05, *** = p < 0.001, **** = p < 0.0001). Boxes represent data between 1st and 3rd quartile and whiskers extend to the highest and lowest values of expression that are not considered outliers.
Table 2.
Telomere-related genetic variants discovered in IPF cases by WES.