Identification and functional characterization of novel telomerase variant alleles in Japanese patients with bone-marrow failure syndromes

Abstract Abstract 1028 The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes. Disclosures: No relevant conflicts of interest to declare.

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Genomic Characterization of the Inherited Bone Marrow Failure Syndromes

Seminars in Hematology ◽

10.1053/j.seminhematol.2013.09.002 ◽

2013 ◽

Vol 50 (4) ◽

pp. 333-347 ◽

Cited By ~ 49

Author(s):

Payal P. Khincha ◽

Sharon A. Savage

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Bone Marrow Failure Syndromes ◽

Genomic Characterization

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Identification and Functional Characterization of Novel Telomerase Variant Alleles in Patients with Bone-Marrow Failure Syndromes

Blood ◽

10.1182/blood.v112.11.4113.4113 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4113-4113

Author(s):

Kathryn A Carroll ◽

Rodrigo Calado ◽

Neal S Young ◽

Shamika Danzy ◽

Hinh Ly

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Functional Characterization ◽

Luciferase Reporter ◽

Cell Renewal ◽

Blood Disorders ◽

Hematological Disorders ◽

Enzymatic Function ◽

Bone Marrow Failure Syndromes ◽

The Individual

Abstract As multiple studies have shown a link between mutations in telomerase components and the disease pathogenesis of bone-marrow failure syndromes (BMFS), we screened blood or marrow cells from patients with various forms of blood disorders for novel pathogenic mutations in the telomerase hTERC and hTERT genes. We identified several heterozygous mutations in these genes, which were observed only in patient samples and not in controls. As these mutations are predicted to affect proper telomerase function, we introduced these and other mutations identified previously in patients with other forms of blood disorders into telomerase-negative cells in order to reconstitute telomerase enzymatic activity and to assess the effect of the individual mutations on modulating a wild-type telomerase function. All disease-associated mutations disrupted telomerase function to various degrees and most modulated a wildtype telomerase enzymatic function by haploinsufficiency based on results obtained from the telomeric repeat amplification protocol (TRAP). We also tested several previously reported natural sequence changes in the promoter region of the hTERC gene for their effect on gene expression and telomerase function via luciferase-reporter assay, TRAP, and Northern blot. The results obtained from these studies appear to contradict those that have recently been reported for some of the naturally occurring hTERC-promoter associated mutations. This study, therefore, offers new insights into the mechanism of natural telomerase mutations in regulating telomere lengths and marrow stem cell renewal capacity in patients with hematological disorders.

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

Blood ◽

10.1182/blood-2006-07-035089 ◽

2006 ◽

Vol 109 (2) ◽

pp. 524-532 ◽

Cited By ~ 76

Author(s):

Zhong-Tao Xin ◽

Adam D. Beauchamp ◽

Rodrigo T. Calado ◽

Jennifer W. Bradford ◽

Joshua A. Regal ◽

...

Keyword(s):

Bone Marrow Failure ◽

Functional Characterization ◽

Gene Mutations ◽

Dyskeratosis Congenita ◽

Dominant Negative ◽

Sequence Variants ◽

Hematologic Disorders ◽

Bone Marrow Failure Syndromes ◽

Human Telomerase

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 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.

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Diagnostic Significance of Haptoglobin Concentration in Patients with Bone Marrow Failure Syndromes.

Blood ◽

10.1182/blood.v108.11.3772.3772 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3772-3772

Author(s):

Kazuhiko Ikeda ◽

Naoto Takahashi ◽

Junichi Kameoka ◽

Katsushi Tajima ◽

Akiko Nakamura ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Clinical Course ◽

Bone Marrow Failure ◽

Japanese Patients ◽

Diagnostic Significance ◽

Serum Haptoglobin ◽

Bone Marrow Failure Syndromes ◽

Haptoglobin Concentration ◽

Gpi Proteins

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is one of the bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndromes (MDS). Recently, the International PNH Interest Group proposed that evidence of a population of erythrocytes and granulocytes deficient in glycosylphosphatidylinositol (GPI) proteins and assessment of hemolytic parameters, including haptoglobin concentration, are important as minimal essential diagnostic criteria of PNH and that less than 1.0% GPI-deficient erythrocytes and granulocytes identifies subclinical PNH from classic PNH (Parker C et al, Blood, 2005). To know whether haptoglobin can be a hallmark which expects the occurrence of classic PNH during the clinical course in AA and MDS patients, we examined the expressions of CD59 on erythrocytes and granulocytes by flow cytometry and relationship between proportions of negative populations of them and various clinical parameters, including haptoglobin concentrations, in Japanese patients with AA (n=23; M:F=11:12; 50.5 ± 19.1 years), PNH (n=28; M:F=14:14; 42.7 ± 16.1 years), and MDS (n=29; M:F=20:9; 66.1 ± 13.9 years). Less than 20 mg/dl of haptoglobin were judged as significant decrease. Flow cytometry showed that the proportions of CD59− erythrocytes (38.11 ± 35.49%) and granulocytes (52.57 ± 42.39%) from PNH patients were significantly higher than those from AA and MDS patients and healthy individuals (n=21; M:F=12:9; 41.3 ± 12.2 years). The values of serum asparatate aminotransferase (AST) and lactate dehydrogenase (LDH) were significantly higher in PNH patients (54.9 ± 53.1U/l and 1035 ± 1052 U/l, respectively) than AA (20.8 ± 9.1 U/l and 205.8 ± 45.0 U/l, respectively) and MDS (22.4 ± 16.9 U/l and 217.7 ± 64.0 U/l, respectively) patients. In contrast, the concentrations of serum haptoglobin were significantly lower in PNH patients (12.9 ± 27.6 mg/dl) than AA (84.6 ± 81.9 mg/dl) and MDS (77.7 ± 47.3 mg/dl) patients. When comparing PNH patients (n=9; minimal PNH) with less than 5% of CD59− erythrocytes with those (n=19; bulky PNH) with over 5% of CD59− erythrocytes, the values of AST (70.7 ± 58.2U/l) and LDH (1021 ± 1083U/l) of the latter were significantly higher than those of the former (21.4 ± 6.2U/l and 220.3 ± 45.1U/l, respectively), but the concentrations of haptoglobin were similar between the latter (11.8 ± 28.9mg/dl) and the former (15.8 ± 26.0mg/dl). In addition, all the PNH patients, but not AA and MDS patients, with over 1% of CD59− erythrocytes had significant decrease of haptoglobin concentration, suggesting that low concentrations of serum haptoglobin may predict occurrence of classic PNH during the clinical course in AA and MDS patients. In conclusion, over 1% of CD59− erythrocytes in PNH patients certainly cause clinical hemolysis and serum haptoglobin is a useful marker which can predict the occurrence of classic PNH.

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