Identification and Functional Characterization of Telomerase RNA Variant Alleles in Japanese Patients with Aplastic Anemia or Myelodysplastic Syndrome.
Authors: Takeuchi Junko , Hinh Ly , Adam Beauchamp , Inokuchi Koiti , Kosaka Fumiko , Sawaguchi Kazuhiro , Mitamura Yoshio , Watanabe Ayako , Gomi Seiji , Yamaguchi Hiroki , Dan Kazuo 
Published: April 2020

Abstract
Dyskeratosis Congenita (DKC) is an inherited bone marrow failure (BMF) characterized by abnormal skin pigmentation, nail dystrophy and mucosa leucoplakia. Mutations in the DKC1 and TERC genes occur in the X-linked and autosomal dominant DKC, respectively. Several groups have recently reported that mutations of the telomerase TERC RNA and of the telomerase catalytic TERT protein caused short telomere and developed DKC and other BMF syndromes, such as aplastic anemia (AA) and myelodysplasia (MDS). As the incidence for BMF seems to be higher in East Asia than in the West, we investigated whether mutations in TERC and TERT genes are indeed associated with BMF patients in Japan.
We examined blood or marrow cells from 73 BMF patients; 27 samples collected from patients with acquired AA (n=27) who did not respond to immunosuppressive therapy, and 46 samples from patients with MDS (RA). We also analyzed blood samples from 120 healthy donors as controls. Polymerase-chain-reaction (PCR) amplification of TERC and TERT genes was performed with DNA samples extracted from peripheral blood or bone marrow cells. PCR products were purified and direct sequencing was performed. Altered sequences were transfected into telomerase-negative cell line in order to address the effect of the mutations on telomerase enzymatic function. We found two polymorphic sequence changes (n-717A/G and n-713 deletion) in the promoter region of the hTERC gene and nine TERT polymorphic sequences in the hTERT gene. These hTERT sequence polymorphisms, which were also identified in healthy individuals, did not introduce an amino acid change in the corresponding protein. To our knowledge, the two TERC polymorphisms and three of the nine TERT polymorphisms (IVS6 −93 G/A, codon837 CTC/CTG and codon 840 CTG/CTA) have not been reported elsewhere. Among the 73 BMF patients, 1(1.4%) heterozygous TERC mutation (C323T) was identified in an MDS patient. This mutation was not observed in normal controls. The C323T mutation is located on the P5 stem of the predicted hTERC RNA secondary structure, which suggests that the mutation may disrupt basepairing interactions of this stem. We introduced this C233T mutation, as well as additional sequence changes to test for the effect of the mutation in the formation of the P5 stem, into VA13+hTERT cells. We found that the mutations located on the individual strands of the P5 stem (either the natural C323T(rt) mutation or the corresponding C323T(lt) mutation located at position 246 alone) abolished telomerase activity to about the same degree. Interestingly, the compensatory mutation (C323T(comp)), in which the natural C323T mutation was accompanied by complementary mutation on the opposite strand of the P5 stem, did not restore telomerase activity, suggesting that both the primary and secondary structure of the P5 stem are important to confer optimal telomerase enzymatic activity. Moreover, no effects were observed between cells that carried only the wild type TERC vector and those that carried both the wild type and the individual mutated TERC copy. These results suggest that the natural hTERC variant functions in a haploinsufficient manner to modulate wild type telomerase function.