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Case Reports
. 2016 Oct 20;128(16):2089-2092.
doi: 10.1182/blood-2016-04-710160. Epub 2016 Sep 1.

A novel TERC CR4/CR5 domain mutation causes telomere disease via decreased TERT binding

Baris Boyraz  1   2   3   4   5 , Courtney M Bellomo  6 , Mark D Fleming  7 , Corey S Cutler  8 , Suneet Agarwal  1   2   3   4
Affiliations
Case Reports

A novel TERC CR4/CR5 domain mutation causes telomere disease via decreased TERT binding

Baris Boyraz et al. Blood. .

Erratum in

No abstract available

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
The TERC G319A mutation impairs TERC:TERT interaction. (A) Telomere restriction fragment length (TRF) analysis of normal (WT) iPSCs, 3 independent TERC-mutant iPSC clones (cl), and TERC-mutant fibroblasts. (B) Top left, Representative Northern blot of TERC RNA from WT, TERC-mutant iPSCs, and TERC-mutant fibroblasts. Top right, Relative TERC levels are shown in the graph. Bottom, Sequencing of TERC cDNA from TERC-mutant iPSCs. (C) Left, Schematic of TERC RNA in complex with TERT and dyskerin. Right, Sequencing of TERC RT-PCR amplicons isolated from TERT RNP (top) and dyskerin RNP (bottom) isolated by immunoprecipitation (IP) from TERC-mutant iPSCs. (D) Restriction fragment length polymorphism (RFLP) of TERC amplicons from dyskerin or TERT IP of patient iPSCs (lanes 5-8). The HincII site created by the G319A mutation is depicted on the right. Lanes 1-4 show digest patterns of amplicons from WT (319G) and mutant (319A) plasmid DNA. Products were separated by agarose gel electrophoresis and band intensity was quantified using ImageLab. n.s., not significant.
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References

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