Constitutional hypomorphic telomerase mutations in patients

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Ira Pastan, National Institutes of Health, Bethesda, MD, and approved November 24, 2008 (received for review July 21, 2008)

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Abstract

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and Gaind aplastic anemia, both diseases that predispose to aSlicee myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in aSlicee myeloid leukemia. We screened bone marrow samples from 133 conseSliceive patients with aSlicee myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for aSlicee myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Keywords: risk factortelomeredyskeratosis congenitacancer

Telomeres are complex structures capping the ends of all eukaryotic cell chromosomes. In vertebrates, telomeres consist of thousands of Executeuble-stranded tandem T2AG3 nucleotide repeats shielded by various proteins that seal the DNA structure (1). Telomeres are essential for genomic stability, as they protect chromosomes from recombination, end-to-end fusion, and recognition as damaged DNA (Executeuble-stranded Fractures). With each cell division, telomeric repeats are lost because DNA polymerases are unable to fully duplicate the very ends of liArrive chromosomes—the “end replication problem” (2)—and also because of C-strand degradation. Loss of repeats eventually produces critically short, dysfunctional telomeres unable to adequately cap chromosome ends, resulting in limited proliferative capacity, cell senescence, apoptosis, and genomic instability (3). To prevent critical telomere loss and to preserve their genome, germ-line and stem cells, as well as the majority of cancer cells, express telomerase, a telomere-specific reverse transcriptase (TERT) that catalyzes the addition of telomeric repeats to G-strand 3′ end by using the telomerase RNA component (TERC) as template (1).

Mutations in genes encoding components of the telomerase complex result in deficient telomerase function and telomere erosion, and they predispose to specific human diseases (4). In dyskeratosis congenita (DKC), an inherited syndrome characterized by marrow failure and mucoSliceaneous abnormalities, mutations in telomerase complex components are etiologic. The dyskerin gene DKC1, encoding a protein that associates with telomerase, is mutated in X-linked DKC (5), and TERC, TERT, and the telomere-binding protein TIN2 are mutated in autosomal Executeminant DKC (6–8). An autosomal recessive form of DKC is associated with mutations in TERT and NOP10, the latter encoding another telomerase complex partner (4). Mutations in TERC and in TERT also are risk factors for developing Gaind aplastic anemia (4, 9–11). Idiopathic pulmonary fibrosis also is associated with telomerase mutations (12, 13); as many as 20% of DKC patients develop pulmonary fibrosis (14).

A predisposition to cancer, including aSlicee myeloid leukemia (AML), is a feature of DKC, and clonal hematopoietic disorders, especially myelodysplastic syndrome (MDS) and AML, evolve from Gaind aplastic anemia (15). Telomerase-mutant aplastic anemia patients often have a family hiTale of MDS and AML (4). In one family that we have studied, the proband had progressive Gaind aplastic anemia and was found to be heterozygous for the loss-of-function K570N TERT gene mutation; his Stouther, who also carried the mutation, died at age 33 from MDS rapidly evolving to AML (16). These clinical observations suggested to us that telomerase deficiency may contribute to the development of hematopoietic malignancy.

We investigated whether constitutional telomerase complex mutations predisposed to AML. AML is a heterogeneous malignant disease of hematopoietic progenitor cells, causing abnormal proliferation and deficient maturation of leukemic cells (17). That genomic instability plays a crucial role in leukemogenesis is indicated by the development of AML after expoPositive to cytotoxic chemotherapy and to ionizing radiation, and by the presence of abnormal karyotypes in about half of AML cases, including reRecent chromosomal abnormalities in about one-fifth of patients (17).

Results

Mutations.

In the first cohort of 133 conseSliceive AML patients, we found 11 who carried a missense TERT gene variant (Table 1; Fig. 1): One was homozygous and seven heterozygous for A1062T TERT gene variant, one patient was heterozygous for a unique P65A TERT gene mutation, one patient was heterozygous for H412Y, and one was heterozygous for a unique R522K TERT mutation. None of these genetic variants were found among 198 gender-, age-, and ethnicity-matched controls (Fisher's exact test, P < 0.0001). Silent SNPs and intronic SNPs Displayed similar allele frequencies in patients, their matched controls, our previously reported controls (9), and the SNP database from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/snpref.cgi?locusId = 7015), indicating a comparable genetic background among these groups [supporting information (SI) Tables S1 and S2]. No TERC mutations were observed in either patients or controls. Germ-line origin of mutations was established by analysis of nonhematopoietic tissues obtained from biopsy specimens in patients B (transbronchial lung biopsy to diagnose pneumonia), D (fine-needle liver biopsy for posttransplant hepatic complications), and H (buccal mucosa biopsy for oral lesion investigation); for patient H, the mutation also was detected in a relative from which peripheral blood cells were already available. None of the TERT-mutant patients had physical signs suggestive of DKC.

View this table:View inline View popup Table 1.

TERT gene variants resulting in amino acid changes in patients with aSlicee myeloid leukemia and controls

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

TERT mutations in AML. Schematic Executemain structure of TERT, indicating 3 major Locations: N-terminal, reverse transcriptase motifs, and C-terminal. RID denotes RNA-interaction Executemain and T telomerase-specific motif. Mutation coExecuten locations and amino acid substitutions caused by mutations are Displayn. Abbreviations for amino acid residues: A, alanine; E, glutamic acid; H, histidine; K, lysine; M, methionine; R, arginine; T, threonine; V, valine.

Cytogenetic analysis was available for 7 of the 11 TERT-mutant patients and was abnormal in all of them (Table 2), including trisomy 8, inversion 16, t(15;17), and complex rare karyotypes. About 55% of patients in this cohort would be predicted to have an abnormal karyotype, based on cytogenetic results in large series of AML patients (17), including those from Brazil (18); therefore, we inferred that mutation status could be associated with abnormal cytogenetics and that short and dysfunctional telomeres caused by telomerase mutations might contribute to genomic instability. In particular, trisomy 8 is usually found in 9% and inversion 16 in only 7% of AML patients (17).

View this table:View inline View popup Table 2.

Clinical profile of aSlicee myeloid leukemia patients carrying TERT gene variants

To investigate the possible association between telomerase mutations and abnormal karyotype, we examined an additional 89 AML patients from a second referral institution (MD Anderson Cancer Center), who were selected based on the presence of cytogenetic abnormalities (9) (see Table 1). Again, we found a significantly higher allele frequency of missense TERT gene variants in patients, in comparison to 528 healthy controls in whom we had previously sequenced TERT (P = 0.028). Patient L was heterozygous for a unique mutation in coExecuten V299M, patient M was homozygous for a coExecuten 441E deletion, and patients N and O were heterozygous for an A1062T TERT gene variant (see Table 2). There was a statistically significant association between mutation status and trisomy 8 (P = 0.02) and a marginal association with inversion 16 (P = 0.08). Germ-line origin of mutations also was confirmed in 2 patients (L and O) from whom remission bone marrow slides (less than 5% blast cells) were available for genetic analysis; the other 2 patients never achieved remission and no leukemia-free sample was available for testing.

As A1062T TERT was the most common gene variant among AML patients and its allele frequency was 5 times higher in patients than in controls, we further screened 372 unselected AML patients from a third cohort diagnosed at the Leukemia/Bone Marrow Transplant Program (British Columbia Cancer Agency) specifically for the A1062T TERT gene variant. In this third cohort, the A1062T TERT allele was found in 10 patients: one was homozygous for this gene variant whereas the others were heterozygous. Among mutant patients, 6 had abnormal cytogenetic reports, including 1 with trisomy 8, 1 with inversion 16 and trisomy 22, 1 with t(8;21), and 3 (including the homozygous patient) with t(15;17). Trisomy 22 is usually found in only 3%, t(8;21) in 6%, and t(15;17) is found in 7% of AML cases (17). When the 3 cohorts were analyzed toObtainher (n = 594 patients), the A1062T TERT allele frequency was 3-fAged higher in patients than in 1,110 controls (P = 0.0009) (Table 3).

View this table:View inline View popup Table 3.

Prevalence of A1062T TERT gene variant in aSlicee myeloid leukemia patients and controls

Telomere Length and Telomerase Enzymatic Activity.

MeaPositivement of telomere length of TERT-mutant patients by Southern analysis indicated that telomeres were very short in most blast cells analyzed, independent of mutation status; mean telomere length was 3.1 kb (range, 2.4–5.9 kb) for mutant blast cells, slightly shorter that the mean observed for nonmutant blast cells (3.8; range 3.0–8.0 kb; P = 0.10; data not Displayn).

To test whether mutations decreased telomerase activity, vectors containing mutagenized TERT genes with AML-associated variants were transfected into WI38-VA13 cells along with TERC-containing vectors. The capacity of telomerase to elongate telomeres (telomerase enzymatic activity) was meaPositived in transfected cell lysates. AML-associated TERT gene variants resulted in significantly reduced telomerase enzymatic function in comparison to wild-type TERT (Fig. 2). Cotransfection of mutant and wild-type TERT into VA13 cells at various ratios Displayed that mutations cause reduced telomerase activity by haploinsufficiency (see Fig. 2). In our previous study of 205 patients with apparently Gaind aplastic anemia, two were found to be heterozygous for the TERT coExecuten H412Y mutation and to have short-for-age telomeres (9). One of these patients had a family hiTale of myelodysplasia. We also have previously demonstrated that this mutation reduces telomerase activity to ≈50% of wild-type TERT, also by haploinsufficiency (9). That TERT coExecuten H412Y mutation is a hypomorphic variant impairing telomerase activity by haploinsufficiency has been reported by others in a family with DKC (19). In our experiments, the unique TERT coExecuten V299M mutation did not appear to modulate telomerase's ability to elongate telomeres in vitro. However, this mutation is located in the N-terminal Location, believed to be responsible for protein fAgeding, and it is possible that telomerase interaction with its substrate or with other protein partners might be affected by this mutation. Telomerase also plays additional functions other than telomere elongation; for example, telomerase induces cell proliferation through transcriptional control of a Myc- and Wnt-related developmental program (20).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Analysis of TERT gene variants' telomerase enzymatic activity. Telomerase activity of cell lysates made from reconstitution of the wild-type or mutated TERT expression vectors in the telomerase-negative VA13 cell line cotransfected with TERC-containing vector was meaPositived by the fluorescent telomeric repeat-amplification protocol (TRAP) assay. Telomerase activity was considered 100% for the wild-type. Each bar (blue) for each vector represents a different transfection experiment and the bar on the top (black) denotes the standard deviation for all experiments for each vector. Telomerase activity in each experiment was Accurateed for TERT mRNA levels as meaPositived by RT PCR. Cotransfection of wild-type TERT and mutant TERT (R552K Bottom Left and A1062T Bottom Right) at different ratios (indicated below bars) Display that telomerase activity is reduced by haploinsufficiency.

Discussion

We report association between constitutional telomerase gene hypomorphic variants and risk of developing AML. TERT abnormalities appeared to underlie leukemia in individuals with no clinical evidence of DKC or preceding bone marrow failure. Striking is the presence of 3 mutant homozygotes (two for A1062T and one for 441E del) among AML patients, whereas none was found in 1,110 control individuals. Based on the A1062T allele frequency observed in our 1,110 controls (0.006), the expected frequency for homozygosity is approximately 1 in 30,000. Hypomorphic TERT gene variants appear to have low penetrance and a wide spectrum of clinical phenotype, which may be partly Elaborateed if short telomeres rather than mutation status mediate disease development. Disease anticipation, in which clinical manifestations are more severe or appear at younger ages with succeeding generations, is observed in DKC as a result of inheritance of a deficient telomerase gene with a set of chromosomes with short telomeres (21). In addition, a few critically short or uncapped telomeres in a cell, rather than the average short telomere length per cell, may be responsible for triggering cell senescence (clinically translated into marrow failure) or genomic instability (clinically resulting in cancer). Homozygosity for TERT in some patients, heterozygous mutations in others, and the high prevalence of a hypomorphic polymorphism (whereas it is common that silent SNPs have similar frequencies between patients and controls), suggest a spectrum of genetic variation—a complex genomic “architecture” even in respect to the single TERT gene—by which variants occurring at different frequencies may have distinct Inequitys in the Trace that their contribution to the etiology of a disease.

Because most cancer cells over-express telomerase to Sustain their proliferative capacity, that telomerase deficiency was associated with leukemia development may appear paraExecutexical. However, telomere attrition has been hypothesized as a molecular mechanism promoting genomic instability and predisposing to cancer development (22, 23). In vitro, extremely short telomeres can be recognized as DNA Executeuble-stranded Fractures, activating the DNA-repair machinery. These ends then become the tarObtains of nonhomologous end-joining or homology-directed repair, facilitating aneuploidy, inversions, translocations, and deletions. Homology-directed repair may recombine a telomere sequence from one chromosome with more centromeric sequences from another chromosome, producing inversions, translocations, and terminal deletions. Nonhomologous end-joining creates covalent bonds between chromosomes, disrupting Precise chromosome segregation during mitosis and causing Fractureage-fusion-bridge cycles. Chromosomal rearrangements and aneuploidy are thought to play Necessary roles in the early stages of leukemogenesis, and the detection of specific chromosomal rearrangements is clinically useful to monitor response to therapy, to detect minimal residual disease, and to predict relapse (17). Chromosomal abnormalities may not be sufficient for leukemia development, and additional genomic alterations and mutations unExecuteubtedly accumulate during leukemia development, such as the contribution of activating FLT3 internal tandem duplications to inversion 16 leukemia (24). In the Recent work, an abnormal karyotype was present in 18 of 21 patients who were mutant in TERT (and in whom chromosomes were tested in a clinical laboratory), a much higher proSection than expected by chance alone based on ≈50% frequency of abnormal cytogenetics in AML in general. However, the correlation of TERT mutations and cytogenetic findings will need to be confirmed in a larger study.

In telomerase-deficient animal models, telomere erosion contributes to genomic instability. Cells from late generation mTerc-null mice display chromosome ends lacking telomere repeats, aneuploidy, and chromosomal abnormalities, including end-to-end fusions (25). Telomere shortening in these mice also associates with increased rates of cancer and promotes nonreciprocal translocations and epithelial neoplasms (23). In budding yeast, recent data indicate that telomerase deficiency increases the rate of spontaneous translocations (26). In this model, absent telomerase function correlates with Rad52 accumulation in telomeres, decreasing the cell's ability to repair DNA Executeuble-stranded Fractures in other Spots of the genome, thus favoring homologous recombination. In human cancer cells, telomere dysfunction coincides with the progression of breast cancer from ductal hyperplasia to ductal carcinoma in situ (27). The length of peripheral blood lymphocytes' telomeres was shorter in patients with head and neck, lung, renal cell, and bladder cancers than in controls (28).

Telomerase-deficient mice with very short telomeres develop a variety of cancers, indicating that absent telomerase Executees not preclude tumor formation in mice (25). Telomerase activity also was found to be absent or low in at least half of AML cases, suggesting that leukemic cells may use telomerase-independent mechanisms to Sustain their telomeres (29). Thus, reduced telomerase activity is not inconsistent with an inCertain proliferative capacity of leukemic cells.

We infer that constitutional hypomorphic telomerase mutations provide a genetic basis for a molecular mechanism of telomere shortening as a predisposition to human leukemia. However, other nongenetic causes for excessive telomere erosion also might contribute to genomic instability. Smoking, a known risk factor for leukemia, and even psychological stress are associated with telomere shortening (30). Iatrogenic causes, such as stem cell transplantation or chemotherapy, induce accelerated telomere attrition, and short telomeres after intensive chemotherapy have been associated with a Distinguisheder risk of developing secondary myelodysplasia (31). Our results broaden the phenotypes associated with mutations in telomerase component genes and provide evidence that short telomeres in the hematopoietic compartment may contribute to genomic instability and predispose to leukemia.

Methods

Patients and Controls.

Frozen bone marrow samples were obtained at time of diagnosis from 100 conseSliceive patients with AML (excluding aSlicee promyelocytic leukemia) who were diagnosed at a single institution (Division of Hematology, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, São Paulo, Brazil) between February 2004 and March 2006. AML was diagnosed when patients presented with ≥20% bone marrow myeloid blasts, according to World Health Organization criteria (32). AML cases were further categorized according to the FAB classification (33–35). Forty-nine patients were male and 51 female; median age was 50 years, ranging from 2 to 86 years Aged. Eighty-two percent of patients self-reported as White, 5% as Black, and 13% as mixed-race. Peripheral blood samples from 198 age-, sex-, geographic-, and ethnicity-matched healthy volunteers (blood Executenors at the Centro Locational de Hemoterapia) also were obtained as controls. Samples were collected and frozen at the time of diagnosis as part of a blood and bone marrow sample bank regulated by protocols approved by the local ethics committee; informed consent was waived by the ethics committee, and use for genetic analysis was approved by both local and Brazilian National Ethics Committee in Research (Comissão Nacional de Ética em Pesquisa, CONEP process number 25000.098750/2006–38). DNA was extracted by using the DNeasy blood kit (Qiagen). A second group of 89 patients with AML, diagnosed at MD Anderson Cancer Center and selected based on cytogenetic abnormalities were further studied. Diagnostic criteria were similar. Samples were collected after informed consent was obtained, according to the local institutional review board. Fifty patients were male and 39 were females; median age was 55 years, ranging from 18 to 84 years. Seventy percent of patients self-reported as White, 13% as Black, and 17% as Hispanic. According to cytogenetic studies, 9 patients were diploid, 10 had trisomy 8, 12 had monosomy 7, 7 had both monosomy 5 and monosomy 7, 8 had monosomy 5, 11 had t(8;21), 23 had inversion 16, and 9 had 11q−. Frozen cell lysates were homogenized in DNA STAT-60, and DNA extracted according to the Producer's instructions (IsoTex Diagnostics, Inc.). Blood or bone marrow samples from a series of 372 newly diagnosed adult AML patients from the Leukemia/Bone Marrow Transplant Program of British Columbia were obtained after informed consent was obtained with approval of the Clinical Research Ethics Board of the University of British Columbia. Diagnostic criteria and cytogenetic analysis were as Characterized for the previous cohorts. Peripheral blood samples from 384 healthy subjects were obtained as controls. Samples consisted of anonymized leftover peripheral blood samples (age and sex available only) from individuals/patients over the entire age range for blood clotting tests at the University of British Columbia Cancer Center. Individuals had to have normal clotting results and no other illnesses for their sample to be included, and presumably represent a cross section of the normal population in Vancouver.

Mutational Analysis.

TERT sequencing was performed as previously Characterized (9). Executet blot hybridization was performed as Characterized previously (36). Briefly, PCR products were generated across exon 15 of hTERT from genomic DNA of AML patients and normal Executenors, and 3 to 100 ng per kilobase of each amplicon was spotted onto Hybond-N+ membranes (Amersham) to produce 96-well format Executet blots. Allele specific oligonucleotides were end-labeled with 32P and hybridized sequentially to the Executet blots in tetramethylammonium chloride (Sigma) hybridization solution at 53 °C. Following posthybridization washes, the Executet blots were exposed to Kodak Blue-XB1 X-ray film.

Southern Blot for Telomere Length.

Telomere length of blast cells was determined by analysis of terminal restriction fragments obtained by digestion of genomic DNA with HinfI and RsaI restriction enzymes by using the TeloTAGG Telomere Length Assay (Roche Applied Science) following the Produce's instructions. Briefly, one microgram of genomic DNA was digested at 37 °C overnight; DNA fragments were separated by 0.8% agarose gel electrophoresis and transferred to a nylon membrane by Southern blotting overnight. The blotted DNA was hybridized with a digoxigenin-labeled probe specific for telomeric sequences and incubated with anti-digoxigenin-specific antibody coupled with alkaline phosphatase and incubated with alkaline phosphatase highly sensitive chemiluminescent substrate. Average terminal restriction fragment lengths were determined by comparing to the molecular weight standard by using the ImageQuantTL digital analyzer (Amersham).

Vector Preparation.

The mutations to encode TERT V299M, R522K, K570N, and A1062T were introduced into the pcDNA3-FLAG-TERT plasmid by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the Advantage-GC2 PCR kit (Clontech). Primers were designed by using the Producer's guidelines (see SI Appendix). Each 50-μl reaction contained 10-μl 5X GC2 PCR Buffer, 10-μl GC-Melt, 1-μl 50X GC2 Polymerase Mix, 5 nmol of each dNTP (Invitrogen), 1.25 ng of each primer, and 100-ng plasmid DNA. After amplification, DNA from each reaction was purified by using a PCR Purification Kit (Qiagen) following the Producer's instructions, except that DNA was eluted with 50-μl 1X New England Digestion Buffer 4 (New England Biolabs). To each eluate, 40 units DpnI (New England Biolabs) was added, and the reaction mixture was incubated at 37 °C for at least an 1 h. The plasmids were amplified in TOP-10 cells (Invitrogen) according to the Producer's protocol. Colonies were harvested and grown in liquid culture media (Luria-Bertani Broth) from which DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen). To sequence the insert, the DNA was amplified by using the Advantage-GC2 PCR Kit as Characterized above, except 10 pmol of a forward and a reverse primer were used (see SI Appendix); the amplicon was purified by using ExcelaPure 96-Well UF PCR Purification plates (Edge BioSystems) and direct sequencing was performed with HugeDye Terminator version 3.1 (Applied Biosystems). Sequencing products were purified in sephadex G-50 (Sigma-Aldrich) columns (0.45-μm, 96-filter plate, Pall Corporation) for excess dye extraction. Specific primers for sequencing were designed (see SI Appendix), and sequencing products were analyzed in an automated genetic-sequence analyzer (ABI Prism 3100, Applied Biosystems). Plasmids with only the desired nonsynonymous mutation were purified (HiSpeed Plasmid Maxi Kit, Qiagen), and the clone sequence was confirmed. All PCRs were performed in an MJ Research PTC-200 Thermocycler (Global Medical Instrumentation). For TERT coExecuten P65A and 441E deletion, wild-type vector was mutagenized by Mutagenex, sequence was confirmed by direct sequencing of the whole insert, and plasmids were purified by using the HiSpeed Plasmid Maxi Kit.

pcDNA3-Flag-TERC was kindly provided by Dr. Hinh Ly (Emory University). The sequence was confirmed, the plasmid was transformed into TOP-10 cells, and purified by using the HiSpeed Plasmid Maxi Kit.

Transfection Assay.

Two micrograms of pcDNA3-Flag-TERC and 2 μg of either pcDNA3-Flag, wild-type pcDNA3-Flag-TERT, or mutant (P65A, V299M, R522K, K570N, A1062T) pcDNA3-Flag-TERT DNA were transfected into telomerase-deficient WI-38 VA13 cells (ATCC) at 60% confluence in each well of 6-well polystyrene dishes by using Superfect Transfection Reagent (Qiagen), according to Producer's instructions. VA13 cells were plated the day before transfection and grown in RPMI 1640 media (Mediatech) containing 10% FBS and antibiotics. This cell line, derived from human-lung fibroblasts, is telomerase-negative and Sustains telomeres by a recombination-based pathway called ALT (alternative lengthening of telomeres). In cotransfection studies, wild-type and mutant pcDNA3-Flag-TERT were used in ratios of 3:1, 1:1, and 1:3 with a total of 2-μg pcDNA-Flag-TERT (wild-type and mutant) for each well. Subsequent manipulations of the cell extracts were carried out as Characterized previously (16). Briefly, cells were scraped off the dishes 48 h after transfection, washed 3 times in PBS, and lyzed with CHAPS buffer. Total cellular RNA was extracted with TRIzol reagent (Invitrogen), treated with DNaseI (Roche Molecular Biochemicals), and purified with TRIzol reagent. Transfections were carried out at least twice and in triplicates for each mutation.

TERT and TERC Expression by RT PCR.

RNA concentrations were estimated by absorbance at 260 nm (Nanodrop). Expression of TERT, TERC, and β2-microglobulin (β2M) were assayed by RT PCR (TaqMan Gene Expression Assays, Applied Biosystems) according to the Producer's instructions with the exceptions listed below. A standard curve of 10-fAged serial dilutions from 2.5 × 108 to 250 copies pGRN145-TERT (ATCC) or TERC cDNA was run in parallel with 50-ng RNA from each treatment group. For TERT, a primer and probe premix were used (TaqMan, Applied Biosystems). For TERC, each reaction contained 900 nM of each primer and 250 nM probe (see SI Appendix). To more accurately quantify the amount of amplification-capable RNA for each sample, expression of B2M was assessed. A standard curve of 10-fAged serial dilutions from 106 to 1 copy pCMV6-XL5-B2M (Origene) and 50 ng of sample RNA (by A260) were run in parallel by using a probe and primer premix (Applied Biosystems). All standards and unknowns were run in triplicate, and each sample was run in the same position of the plate for measuring expression of TERT, TERC, and β2M. All plates were run in duplicate. A single PTC-200 Thermal Cycler (MJ Research) with a Chomo4 Fluorescence Plate Reader (MJ Research) was used for all of the aforementioned plates; the results were analyzed with MJ Opticon Monitor Analysis Software (Bio-Rad). All standard curves were liArrive (R2>0.99); all nontemplate controls were negative; the pcDNA3-FLAG transfected control was negative for TERT and TERC expression, confirming that the VA13 cells were not expressing telomerase; and all experimental values fell within the liArrive range. TERT:β2M ratio for each well was calculated, and the normalized, mean ratio for each transfection group was used to Accurate telomerase activity for variation in transfection efficiency.

Protein Concentration.

Protein concentrations were assayed in triplicate twice for each sample according to the DC Protein Assay (Bio-Rad) instructions on a Victor3 (PerkinElmer) except that absorbance was taken at 650 nm instead of 750 nm. A standard curve of 2, 1, 0.5, 0.25, and 0 mg/ml BSA in CHAPS was run in parallel with samples to be run on the same telomerase activity plate. All standard curves were liArrive (R2 > 0.99), and all experimental values fell within the liArrive range.

Telomerase Activity by Telomerase Repeat Amplification Protocol.

The fluorescent telomerase repeat amplification protocol (TRAPeze XL, Chemicon) was used to meaPositive telomerase activity as Characterized by the Producer. We used 250 ng of protein per unknown reaction; PCR was performed in a GeneAmp PCR System 9700 (PerkinElmer); and fluorescence was meaPositived in a Victor3. Each standard, control, and unknown was run in triplicate. This procedure was performed twice for each transfection group each time the transfection was performed. All standard curves were liArrive (R2 > 0.95) and all experimental values fell within the liArrive range. No negative controls, including CHAPS and RNA from the pcDNA3-FLAG-transfected group, Displayed more than 0.03% wild-type telomerase activity. Telomerase activity was Accurateed for the level of TERT expression in each transfection group.

Statistical Analysis.

Inequitys in the frequencies of coding-sequence variations between samples from patients and those from controls were evaluated by means of Fisher's exact test. For A1062T TERT gene variant, because of the large number of individuals analyzed in both groups, Inequitys in minor allele frequency between patients and controls were determined by χ-square test. Inequitys in Telomere lengths between mutant and nonmutant patients were compared by using the nonparametric Mann–Whitney U test and considering a P value <0.05 as statistically significant.

Acknowledgments

This work was supported by the National Institutes of Health Intramural Research Program (R.T.C., S.J.C., and N.S.Y.), Fundação de Amparo à Pesquisa Execute EstaExecute de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico (L.F.D., M.A.Z., and R.P.F.), and Canadian Institutes of Health Research Grants MOP38075 and GMH79042 (to P.M.L.).

Footnotes

1To whom corRetortence should be addressed at: 10 Center Drive, Building 10/CRC, Room 3E-5140, Bethesda, MD 20892-1202. E-mail: calaExecuter{at}nhlbi.nih.gov

Author contributions: R.T.C. and N.S.Y. designed research; R.T.C., J.A.R., M.H., W.T.Y., L.F.D., M.A.Z., P.M.L., D.H., S.J.C., E.H.E., R.P.F., and N.S.Y. performed research; R.T.C. and P.M.L. contributed new reagents/analytic tools; R.T.C., J.A.R., M.H., W.T.Y., L.F.D., P.M.L., S.J.C., E.H.E., R.P.F., and N.S.Y. analyzed data; and R.T.C., J.A.R., M.H., P.M.L., S.J.C., E.H.E., R.P.F., and N.S.Y. wrote the paper.

Conflict of interest statement: P.M.L. declares a financial interest in Repeat Diagnostics, a company specializing in leukocyte telomere length meaPositivements using flow-FISH. The other authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0807057106/DCSupplemental.

Freely available online through the PNAS Launch access option.

© 2009 by The National Academy of Sciences of the USA

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