Functional characterization of natural telomerase mutations found in patients with hematologic disorders

Abstract

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 dis- IntroductionTelomerase is a specialized reverse transcriptase (RT) that adds long, repetitive stretches of simple telomeric DNA sequence (ie, TTAGGG in the vertebrates) onto chromosomal termini. 1 This cellular RT protein (TERT) copies a short stretch of nucleotides located within the template region of an integral RNA component (TERC) into telomeric DNA repeats. 1 Vertebrate TERCs are believed to adopt a complex, folded secondary structure 2 as depicted for human TERC (hTERC) in Figure 1A. We have recently conducted extensive site-directed mutagenesis analysis of hTERC to show that much of the structure folded as predicted. 3 As is true of many biologically active RNA molecules, most of the internally base-paired regions of hTERC can be extensively mutated without loss of function, provided that the normal base-pairing pattern is preserved. 3 However, at certain locations, especially of the single-stranded template region that is copied into telomeric DNA, specific RNA base sequences have been shown to be required for biologic activity. 4 Telomerase catalytic proteins (TERTs) from evolutionary distant organisms share a conserved structural organization that can be divided into 3 functional domains ( Figure 1C). 5 The telomerasespecific domains exist at both the N and C termini of TERTs that are not present in any of the viral RTs. 6 The N-terminal region is required to participate in enzymatic function, 7,8 in assembly of the protein with its integral hTERC RNA component,7,9 and in the homodimerization of the protein (ie, hTERT protein-protein interaction), 7,9,10 whereas the C-terminal domain is required for telomerase-specific activity other than its catalytic function 11,12 as well as in the telomeric nucleotide addition processivity process. [13][14][15] The functional RT domain with the universally conserved RT motifs is almost centrally located in the protein primary sequence ( Figure 1C). The fact that mutations of key residues that are known to affect its conventional RT catalytic activity also negatively influence telomerase activity strongly argues that telomerase RT domain is the catalytic domain of the enzyme complex. 13,[15][16][17][18] Inherited mutations in both hTERC RNA and hTERT protein underlie rare bone marrow failure syndromes, autosomal dominant dyskeratosis congenita (DC) and acquired aplastic anemia (AA). [19][20][21] DC is characterized by abnormal skin pigmentation, nail dystrophy, and oral leukoplakia and is often complicated by life-threatening bone marrow failure and immunodeficiency. 22 Lymphocytes from patients show decreased hTERC expression, decreased t...

Telomerase reverse transcriptase-dependent telomere equilibration mitigates tissue dysfunction in mTert heterozygotes

“…Other autosomaldominant (AD) forms of aplastic anaemia, DKC and pulmonary lung fibrosis are linked to inactivating mutations in the telomerase or dyskerin complexes (the telomerase RNA, TERT, NHP2, NOP10) or components of the telomere-associated shelterin complex (TRF1, TRF2, TIN2) (Savage et al, 2006;Carroll and Ly, 2009;. Patients carrying mutations in telomerase appear to exhibit a true haploinsufficiency (Theimer et al, 2003;Armanios et al, 2005;Yamaguchi et al, 2005;Xin et al, 2007;Errington et al, 2008), suffering telomere erosion in haematopoietic stem cell (HSC) compartments and other organs that leads to proliferative defects, including bone marrow (BM) failure and cancer (Goldman et al, 2008;Aslan et al, 2009;Calado et al, 2009;). Patients diagnosed with DKC often succumb to the illness before 30 years of age .…”

Principles of adoptive T cell cancer therapy

“…Patients with various bone marrow failure syndromes have a number of structural and functional abnormalities of telomeres and telomerase (102). Some patients with aplastic anemia have mutations in the gene encoding TERT (103), and haploinsufficiency of TERC or TERT has been associated with human dyskeratosis congenita (104) and aplastic anemia (103).…”

Telomere length is inherited with resetting of the telomere set-point

“…DNA was extracted from total peripheral blood leukocytes using the Qiagen DNAeasy kit (Qiagen). Telomere length of peripheral blood leukocytes was measured after red cell lysis with ammonium chloride solution by flow-FISH as reported previously (30,31).…”