A novel variant in the CR4/5 region of TERC in a family with telomere disease. (A) Pedigree of the family; the proband is indicated with an arrow. Affected individuals are shown in gray and symptoms are indicated. Mutations in the individuals genotyped are shown. IPF, idiopathic pulmonary fibrosis; Skin, pigmentation abnormalities; TP, thrombocytopenia. (B) Bone marrow histology. Top, Hypercellular, fibrotic bone marrow with dyspoiesis (hematoxylin and eosin [H&E]). Bottom, CD34 staining shows increased percentage of CD34⁺ cells (scale bar, 20 µm). (C) Telomere length measurement by flow cytometry fluorescence in situ hybridization (flow-FISH) in lymphocytes in the proband and his sister. (D) Top, Schematic of telomerase RNP. The location of the variant in the TERC CR4/CR5 region is indicated with an arrow. Bottom, Sequencing of TERC in the proband shows the c.319G>A variant. (E) Telomeric repeat amplification protocol (TRAP) assay for telomerase activity in VA13 cells transfected with TERT and WT TERC, G319A TERC, or control plasmid. Threefold dilutions of input cell extract. Internal control (IC) amplification standard is indicated. Relative telomerase activities are shown in the graph. (F) Top, Immunoblot of TERT and actin protein levels. Bottom, Northern blot of TERC RNA from VA13 cells transfected with TERT plus WT TERC, G319A TERC, or control plasmid. Ethidium bromide staining of 28S ribosomal RNA (rRNA) is used as a loading control.