Figure 1.
TPP1 OB-fold variants cause short telomeres. (A) Family trees showing the segregation of ACD variants. −/−, homozygous variant; +/−, heterozygous; +/+, wild-type (WT). Arrows indicate index cases that underwent whole-exome sequencing. Gray indicates the presence of some somatic features (short stature and limbal stem cell deficiency [LSCD]) but no hematopoietic abnormality. N/A, DNA not available. (B) TPP1 topology showing 3 functional domains: telomerase interacting OB-fold, POT1 recruitment domain (RD), and TIN2 interacting domain (TID) separated by serine threonine (S/T) motif. (C) Whole-blood telomere lengths of index cases and family members are indicated (family 1 in green and family 2 in red). Telomere lengths are reduced in index cases (squares) when compared with heterozygous (diamond) and WT (triangle) family members and controls (n = 218). DAPI, 4′,6-diamidino-2-phenylindole. (D) Telomere fluorescence in situ hybridization of Epstein-Barr virus–transformed lymphoblasts derived from index case in family 2 and a healthy control. (E) Telomere chromatin immunoprecipitation of myc- POT1, FLAG- TPP1 WT, and OB-fold variants expressed in HEK293 cells. Dot blot shows input chromatin used in each immunoprecipitation (top). Immunoprecipitation with an anti-POT1 antibody, anti-FLAG antibody, anti-rabbit (Rb) immunoglobulin G (IgG) control, and protein G beads alone. Expression of TPP1 WT and variants is demonstrated by immunoblotting in the bottom panel. IB, immunoblot. TPP1 p.K170Δ variant was used as positive control, as it has been previously shown to bind to telomeres.9,10 (F) Telomere lengths of S pombe strains harboring Tpz1 (ortholog of human ACD/TPP1) OB-fold variants. Tpz1 K75A yeast strains with previously confirmed short telomeres were used as a positive control.15 Arrow indicates extremely faint telomeric band due to presence of very low telomeric DNA in the L5Q strain, and LC refers to loading control of genomic DNA stained by ethidium bromide.