Disease-associated mutations located in the core domain or the box H/ACA and CR7 domains abolish telomerase activity. (A) Telomerase enzymatic activities as determined in VA13+hTERT cells for naturally occurring hTERC mutations and their derivatives involving the core, box H/ACA, and CR7 domains. When the natural sequence variants are located within a paired region (eg, C116T), the nucleotides that are predicted to base-pair with them are also mutated to the complementary bases (eg, C116T(op)). The compensatory mutations (eg, C116T(comp)) are created in order to restore the helical structures. The sequence changes are indicated in bold. Telomerase activity of each mutant is expressed in comparison to that of the wild-type (wt) (+++, 20%-100%; ++, 2%-20%; +, 1%-2%; −, undetectable), based on 2 or 3 independent determinations. (B, C) Representative TRAP gels showing the relative telomerase enzymatic activities obtained from the representative substitution or deletion mutations and compensatory mutations. Serial 5-fold dilutions of the transfected cell lysates (indicated by triangles) were assayed for each sample to ensure linearity of the assay. Lane 54 shows a negative control composed of wild-type (WT) cell lysate denatured at 95°C for 5 minutes prior to assay. Lane 55 shows PCR products amplified from the non-hTERC control TSR8 DNA template supplied in the TRAP kit. Lane 56 shows cells transfected with the pcDNA3.1 vector control lacking the hTERC coding sequence. “IC” indicates PCR products amplified from an unrelated DNA template, which is included as an internal control for PCR amplification efficiency in each reaction. (D) Northern blot analysis of selected naturally occurring hTERC sequence variants expressed in transfected VA13+hTERT cells (top). Lane 10 shows RNA prepared from cells that were transfected with the pcDNA3.1 vector lacking the hTERC coding sequence. Cellular β-actin mRNA (bottom) was assayed in parallel.