Pedigrees showing Telomerase Mutations and Disease Phenotypes.

<p><b>(A)</b> The <i>TERT</i> K570N mutation tracked with hematological disorders and severe liver disease (lower pedigree) in Family A. In the extended family (upper pedigree), several disorders are found, including autoimmune diseases, anemia, thyroid diseases, liver diseases, and multiple miscarriages; however, the mutation was only associated with liver disease and multiple miscarriages. Two consanguineous relationships are not show: Subject A-IV-17 is a grand-daughter of Subjects A-II-7 and A-II-8, and Subject A-IV-7 is a grandson of Subjects A-III-14 and A-III-15. The genetic status for the immediate family (lower pedigree) and its association with bone marrow failure have been previously reported by us <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007926#pone.0007926-Xin1" target="_blank">[15]</a>. In smaller pedigrees, <b>(B)</b><i>TERC</i> nucleotide 341-360 deletion tracked to liver disease in family B, <b>(C)</b> liver disease occurred in a family with a <i>TERC</i> nucleotide 28–34 deletion, and <b>(D)</b> in a family with <i>TERC</i> nucleotide 109–123 deletion. The following are denoted by their abbreviations: common variable immunodeficiency (CVID), aplastic anemia (AA), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), idiopathic thrombocytic purpura (ITP), and non-alcoholic steatohepatitis (NASH).</p

Hepatic profile of patients with telomerase mutations and liver disease.

<p>Hepatic profile of patients with telomerase mutations and liver disease.</p

Functional analysis of telomerase mutations.

<p><b>(A)</b> Telomere length in peripheral-blood total white blood cells (ordinate) from patients and their relatives with or without telomerase gene mutations as a function of age (abscissa) compared to healthy controls. Telomere lengths were measured by flow fluorescence <i>in situ</i> hybridization (flow-FISH). Small gray circles represent the telomere lengths for 400 healthy volunteers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007926#pone.0007926-Yamaguchi1" target="_blank">[10]</a>, and the curve marks the 50^th percentile for healthy controls as a function of age. <b>(B)</b> Telomerase activity - measured by telomeric-repeat amplification assay - of lysates of telomerase-negative WI38-VA13 cells cotransfected with mutated <i>TERC</i> and wild-type <i>TERT</i> expression vectors (2 µg per vector per transfection reaction). Enzymatic activity was normalized to <i>TERC</i> expression as measured by Real Time RT-PCR and to the telomerase activity of wild-type <i>TERC</i>, which was set at 100%. Quadruplicate measurements were performed using one microgram of cell lysate protein per reaction. “Empty vector” refers to protein from VA13 cells transfected with an empty pcDNA3-Flag vector in lieu of <i>TERC</i>.</p

Hematoxylin and Eosin Bone Marrow and Liver Sections from Probands and Relatives with Aplastic Anemia, Acute Myeloid Leukemia, and Severe Liver Disease.

<p><b>(A)</b> Family A proband's bone marrow was hypocellular with isolated regions of normal cellularity (hematoxylin and eosin [H&E] staining; low power magnification). <b>(B)</b> Proband's father's bone marrow smear (Subject A-IV-29), illustrating dysplastic changes and increased number of blasts (H&E, high power magnification). <b>(C)</b> Subject A-IV-23's liver biopsy revealing islands of liver surrounded by zones of necrosis and parenchymal collapse (H&E, low magnification). The necrosis was far enough in the past that hepatocytes have mostly disappeared. In the inset, in some of the areas where hepatocytes were preserved there was still necrosis near the central veins. Little evidence of inflammation exists. <b>(D)</b> Subject's A-IV-25's liver biopsy showing small portal areas and poorly formed veins (H&E, low power magnification). <b>(E)</b> Same liver biopsy exhibiting widened hepatocyte plates on the reticulin stain (high power magnification), but clear changes of nodular regenerative hyperplasia were not seen. <b>(F)</b> The CD34 stain by was positive in sinusoidal endothelial cells consistent with an abnormal proportion of arterial blood flow to the sinuses (immunohistochemistry, low power magnification). <b>(G)</b> Liver biopsy of Subject A-III-11 in which the hepatic architecture is distorted by bridging fibrosis (low power magnification); the inset gives a close-up of the fibrosis. The biopsy revealed moderate inflammation but not elevated levels of plasma cells relative to other inflammatory cells. Other changes included interface hepatitis and cholatestasis. <b>(H)</b> Subject B-II-3's liver biopsy demonstrating portal inflammation with interface hepatitis (H&E, low power magnification). In the inset, Masson staining shows sclerosis around central vein with perisinusoidal fibrosis. <b>(I)</b> Subject B-III-7's liver biopsy with mild, macrovesicular steatosis in a zone 3 distribution. The inset indicates that there is mild lymphocytic portal inflammation with focal interface hepatitis (H&E). <b>(J)</b> Subject C-III-3's liver biopsy shows mild hepatocellular iron accumulation in a pericanalicular pattern; the sinusoidal-lining cells show mild to moderate iron accumulation. The inset illustrates mild variation in hepatocyte nuclear size. <b>(K)</b> Subject C-III-3's reticulin staining exemplifying several zones in which the hepatocyte plates were abnormally widened, consistent with regeneration. <b>(L)</b> Subject E-II-1's liver biopsy revealing some portal areas with mild inflammation and all with poorly formed, slit-like veins (H&E). <b>(M)</b> The reticulin stain showed evidence of nodular regenerative hyperplasia, with zones of plate widening alternating with areas of compression. <b>(N)</b> CD34 stain was abnormally positive in the sinusoidal endothelial cells by immunohistochemistry, indicating abnormal proportion of arterial blood flow to the sinuses.</p

Telomerase Variant A279T Induces Telomere Dysfunction and Inhibits Non-Canonical Telomerase Activity in Esophageal Carcinomas

<div><p>Background</p><p>Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.</p><p>Methods</p><p>Sequencing techniques were used to evaluate mutational status of <i>telomerase reverse transcriptase (TERT)</i> and <i>telomerase RNA component (TERC)</i> in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.</p><p>Results</p><p>Sequencing analysis revealed one deletion involving <i>TERC (TERC del 341-360)</i>, and two non-synonymous <i>TERT</i> variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.</p><p>Conclusions</p><p>A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.</p></div

Gene expression levels of A279T-transduced cells normalized to wtTERT-transduced cells.

<p>*ND: Not detected.</p

Effects of A279T on genomic stability in normal cells.

<p>A. SKY assay demonstrating that A279T-induces genomic instability in Zeocin™-treated MEF-1 cells. Upper panel: translocations, dicentric, and rearranged chromosomes are present in cells expressing A279T compared to wtTERT. Lower left panel: a multi-centric chromosome observed in cells harboring A279T. Lower right panel: a ring chromosome is formed and every chromosome is rearranged in cells transfected with A279T. See text for additional details. B. Upper panel: representative results of SKY analysis Li Fraumeni fibroblasts constitutively expressing wtTERT or A279T-TERT. Lower panel: close-up of chromosomes 1 and 16. C. Summary of results of two independent experiments demonstrating that A279T expression increases genomic instability in Li Fraumeni cells.</p

Effects of A279T on tumorigenicity and telomere length of esophageal cancer cells in vivo.

<p>A. A279T significantly inhibits growth of subcutaneous EsC2 xenografts in athymic nude mice. B. Representative results of telomere-specific FISH analysis of ExC2 xenografts depicting shortened telomere length in xenografts from EsC2-A279T cells compared to those derived from EsC2-TERT cells; Red: telomere; green: centromere; blue: DAPI.</p

A279T inhibits proliferation of esophageal cancer cells.

<p>(*p<0.05; **p<0.01). A. MTS assay demonstrating inhibition of EsC1 (left) and EsC2 (right) proliferation by A279T relative to wtTERT. B. Immunofluorescence analysis (left panel) with corresponding summary (right panel) of Ki67 expression in esophageal cancer cells expressing wtTERT or A279T (Red: Ki67; blue: DAPI). EsC1 and EsC2 cells expressing A279T exhibit decreased Ki67 levels relative to respective cells expressing wtTERT. C. Annexin V-FITC assay demonstrating A279T-induces apoptosis in EsC2 but not EsC1 cells. Results are expressing as mean ± SD of triplicate experiments. D. Graphic summarization of immunofluorescence analysis of β-galactosidase expression in EsC1 and EsC2 following constitutive expression of wtTERT or A279T. Red: β-galactosidase; blue: DAPI.</p

A279T down-regulates β-catenin independent of telomerase activity.

<p>A. Telomerase enzymatic activity of TERT and TERC mutations in VA13 cells, measured by TRAPeze assay. Telomerase activity is defined as 100% of wtTERT. B. Quantitative PCR analysis of telomere lengths in EsC1, EsC2 and OE21 esophageal cancer cells transfected with empty vector, wtTERT, or A279T-TERT. C. Immunoblot analysis of TERT and related shelterin protein levels in EsC1 and EsC2 cells transduced with wtTERT, A279T-TERT or empty vector. Expression of A279T depletes several shelterin proteins in esophageal cancer cells. D. Upper panel: Immunoprecipitation experiments demonstrating that A279T disrupts TERT-BRG-1-β-catenin complex. This phenomenon was not seen in cells expressing G260D. Lower panel: immunoblot experiments demonstrating decreased β-catenin levels in EsC1 and EsC2 expressing A279T. E. Representative immunofluorescence analysis of β-catenin expression in EsC1 and EsC2 cells cultured in normal media in the presence or absence of proteasome inhibitors (red: β-catenin; blue: DAPI).</p