scholarly journals TERC and TERT gene mutations in patients with bone marrow failure and the significance of telomere length measurements

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Hong-Yan Du ◽  
Elena Pumbo ◽  
Jennifer Ivanovich ◽  
Ping An ◽  
Richard T. Maziarz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Mononuclear Cells ◽  
Medical Center ◽  
Bone Marrow Failure ◽  
Screening Method ◽  
Gene Mutations ◽  
Telomere Shortening ◽  
Tert Gene ◽  
Length Measurements

Abstract Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (≪ 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

The Role of Telomerase Activity and Telomere Length in Cellular Sensitivity to DNA Damaging Agents.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4192-4192
Author(s):  
Greg T. Rice ◽  
Michael A. Beasley ◽  
Ike I. Akabogu ◽  
Erik R. Westin ◽  
Dale A. Winnike ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Skin Pigmentation ◽  
Cytotoxic Agents ◽  
Cell Surface Expression ◽  
Cell Transplant ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Increased Sensitivity

Abstract Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). While telomere shortening is a normal consequence of the aging process, DC patients display extremely short telomeres in many somatic cell types, including hematopoietic cells, and they often suffer from bone marrow failure. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents during myeloablative therapy. To test this hypothesis, we characterized lymphocytes from various AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol, Adriamycin, and Etoposide. Cell proliferation and viability were quantified by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. In addition to DC lymphocytes having a decreased proliferative capacity and higher basal apoptotic levels, an increased sensitivity to irradiation, Taxol, Adriamycin, and Etoposide was noted. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients. Further studies have been initiated to determine whether reconstitution of telomere length in DC cells alters response to cytotoxic agents.

Homozygous and Compound Heterozygous Mutations in the Telomerase Reverse Transcriptase Gene in Two Families with Spontaneous Dyskeratosis Congenita and Idiopathic Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1675-1675
Author(s):  
Hong-Yan Du ◽  
Elena Pumbo ◽  
Peter Manley ◽  
David B. Wilson ◽  
Philip Mason ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Autosomal Recessive ◽  
Amino Acid Change ◽  
Autosomal Dominant ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Dyskeratosis Congenita ◽  
Compound Heterozygous ◽  
The Family ◽  
Tert Gene

Abstract Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome. Classically, DC presents with progressive bone marrow failure, abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. The pattern of inheritance in families with DC suggests an X-linked recessive, an autosomal dominant, and an autosomal recessive form of DC. However, in the majority of patients the occurrence of the disease is sporadic or the family history is unknown. Mutations in four different genes have been associated with DC so far. Mutations in DKC1 have been shown to account for the X-linked form of DC and DKC1 de novo mutations account for about one third of male patients with sporadic disease. Mutations in the telomerase RNA TERC and in the catalytic subunit of telomerase, TERT, have been shown to be responsible for the autosomal dominant form of DC. Interestingly, patients with heterozygous mutations in TERC and TERT often show a milder form of disease and a later age of onset and often lack the classic mucocutaeous features, thus are classified as atypical DC. Very recently homozygosity for a mutation in NOP10 has been identified in one family with autosomal recessive disease. The products of the genes mutated in DC are all components of the telomerase complex, suggesting that disease in patients with DC is caused by a defect in telomere maintenance. Here we investigated two patients, one UPN # 199.001 presenting with the classic manifestations of DC and the other UPN# 284.001 presenting with progressive bone marrow failure but no other clinical features suggestive of DC. In both patients the telomeres measured in peripheral blood mononuclear cells were very short, being defined as being below the 1st percentile. Mutation analysis in the genes associated with DC revealed that patient 199.001 was homozygous for a novel TERT (C2110T) gene mutation, causing an amino acid change (P704S) within the RT domain of TERT. Both parents were heterozygous for the C to T transition. Interestingly however, the father was in addition heterozygote for a second mutation in TERT (C1234T; H412Y) a mutation which has previously been described and has been shown to reduce telomerase activity by 50%. Investigations of the family revealed that the parent’s were distantly related, explaining the same TERT sequence alteration in both parents. Both arms of the family contained members with pulmonary fibrosis. In the second patient 284.001 we identified two different novel TERT gene mutations. One A2537G causes the amino acid change Y846C in the RT domain of TERT whereas the other C2628G causes H876Q also in the RT domain. One of the mutations was inherited from each parent and the parent with the A2537G mutation also had very short telomeres. These two families illustrate that the pattern of inheritance in patients with DC may be complex and show for the first time that homozygous or compound heterozygous TERT gene mutation may be associated with DC. Co-dominance of the three different TERT gene mutations and the inheritance of short telomeres have possibly contributed to development of disease in these patients who were thought to have sporadic DC and idiopathic aplastic anemia.

Significance of Telomere Length Measurement in the Diagnosis of Dyskeratosis Congenita.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 836-836
Author(s):  
Hong-Yan Du ◽  
Elena Pumbo ◽  
Akiko Shimamura ◽  
Adrianna Vlachos ◽  
Jeffrey M. Lipton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Skin Pigmentation ◽  
Screening Method ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Healthy Controls ◽  
Mutation Carriers

Abstract Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome. The classical features of DC include nail dystrophy, abnormal skin pigmentation, and mucosal leukoplakia. The diagnosis of DC can be difficult. Originally, the diagnosis was based on the presence of the classical mucocutaneous features. However, the identification of four genes responsible for DC (DKC1, TERC, TERT, and NOP10) showed that these mucocutaneous features are only present in a proportion of patients with DC. Additionally, screening for mutations in the affected genes is expensive and is negative in about 50% of patients with classical features of DC. The products of the genes mutated in DC are the components of the telomerase ribonucleoprotein complex, which is essential for telomere maintenance. Therefore it has been postulated that DC is a disease arising from excessive telomere shortening. Here we examined whether the measurement of telomeres could be used as a screening test to identify individuals with DC. For this purpose we examined telomere length in peripheral blood mononuclear cells from 169 patients who presented with bone marrow failure including 17 patients with DC, diagnosed by the presence of classical cutaneous features or the identification of mutations in DKC1, TERC or TERT, 28 patients with paroxysmal nocturnal hemoglobinuria, 25 patients with Diamond Blackfan anemia, 5 patients with Shwachman-Diamond syndrome, 8 patients with myelodysplastic syndrome, and 74 patients with aplastic anemia of unknown cause classified as idiopathic aplastic anemia. In addition we measured telomere length in 12 patients with idiopathic pulmonary fibrosis and in 45 individuals with a de novo deletion of chromosome 5p including the TERT gene. Their telomere lengths were compared with those of 202 age-matched healthy controls. Moreover, mutations were screened in the genes associated with DC. In cases where a mutation was identified, telomere length and mutations were also examined in all the family members. Our results show that all patients with DC and bone marrow failure have very short telomeres far below the first percentile of healthy controls. Not all mutation carriers, including some carriers of apparently dominant mutations, have very short telomeres. What is more, very short telomeres could be found in healthy individuals in these families, some of whom were not mutation carriers. These findings indicate that in patients with BMF the measurement of telomere length is a sensitive screening method for DC, whether very short telomeres in this setting are also specific for DC remains to be determined. However, in contrast to a previous study, we find that telomere length does not always identify mutation carriers in the families of DC.

Longitudinal Changes In Telomere Length In Patients with Dyskeratosis Congenita

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2230-2230
Author(s):  
Blanche P Alter ◽  
Neelam Giri ◽  
Peter M. Lansdorp ◽  
Gabriela M. Baerlocher ◽  
Philip S Rosenberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Premature Aging ◽  
Bone Marrow Failure Syndrome ◽  
Cross Sectional ◽  
Telomere Shortening ◽  
Early Presentation ◽  
Telomere Attrition

Abstract Abstract 2230 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome with a complex clinical phenotype, including dysplastic nails, lacy reticular pigmentation, and oral leukoplakia (the diagnostic triad). Numerous other physical abnormalities may be present, in addition to cytopenias due to bone marrow failure, and a high risk of leukemia or solid tumors. However, many patients have no physical findings at diagnosis. Patients with DC have very short telomeres, and approximately one-half have a mutation in one of six genes important in telomere biology. Telomere length in leukocyte subsets, measured by automated flow fluorescence in situ hybridization (flow-FISH), is both sensitive and specific for identifying individuals with DC. Telomeres consist of nucleotide repeats and a protein complex at chromosome ends that are critical in chromosomal stability which shorten during normal cell division. Cross-sectional studies of normal individuals suggest that telomere length decreases with age in a sigmoid pattern from birth to old age. In a cross-sectional analysis of 26 patients with DC, we previously observed that telomere length appeared to be stable or even to slightly increase with age (BP Alter et al, Blood 110:149, 2007). Similar results were shown in 23 different DC patients by others (M Bessler et al, FEBS Lett 2010 in press). We speculated that these data were influenced by early presentation (or recognition) of clinically more severe patients, while patients with similar telomere length who were clinically milder were identified at older ages. In this pilot study, we examined, for the first time, the longitudinal age-association of telomere attrition in nine patients with DC who were followed for five to seven years (currently 8 – 50 years of age). These include three patients with mutations in TERC, and two each with TINF2, TERT, and DKC1 mutations. When first studied, four had normal hematopoiesis, three moderate cytopenias, one was receiving androgens, and one was on transfusions. Subsequently, one with normal hematopoiesis developed mild thrombocytopenia, one who was on transfusions responded to androgens, and one with moderate aplastic anemia became severe. In all cases, telomere length decreased with age. In a linear regression model, the average annual decrease in telomere length in lymphocytes was 167 base pairs/year (bp/yr) + 104, similar to the rate in granulocytes, 159 + 92 bp/yr. According to the literature, the rate of telomere attrition in longitudinal studies in normal blood is ∼45-50 bp/yr, albeit by methods other than flow-FISH; the rate of telomere shortening appears to decrease with increasing age. The average patient Z-scores at the beginning of the study were -3.9 standard deviations below the median for age in healthy normal controls, and were -4.3 at the end, consistent with the impression that DC patient telomeres shorten somewhat more than expected from normal aging. These data support the hypothesis that the earlier cross-sectional results for patients with DC indeed were influenced by the cross-sectional rather than longitudinal nature of the data. The current longitudinal data suggest that telomere shortening could possibly be accelerated in patients with DC, but larger studies are required. Our results indicate that patients with DC have telomeres that are much shorter than normal for their age, and that over time they continue to shorten, consistent with DC being classified as a disorder of premature aging. Disclosures: Lansdorp: Repeat Diagnostics: Equity Ownership.

Telomere Dysfunction in Bone Marrow Failure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-12-SCI-12
Author(s):  
Peter M. Lansdorp
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Aplastic Anemia ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Tumor Development ◽  
Dyskeratosis Congenita ◽  
Cell Numbers ◽  
Tert Gene

Abstract Abstract SCI-12 In order to distinguish a normal telomere from a double strand break, a minimum number of telomere repeats must “cap” each chromosome end. The length of each repeat array will reflect a unique history of addition and losses. Telomere losses are known to occur sporadic as well as with every replication cycle. Losses of telomeric DNA are countered by the telomerase enzyme containing telomerase RNA (encoded by the TERC gene) and a reverse transcriptase protein (encoded by TERT gene) as minimal components. Telomerase levels are high in cells of the germline and immortal cellines and the telomere length is typically maintained in such cells. In contrast, telomerase activity is limiting in most human somatic (stem) cells and as a result the average length of telomere repeats in most somatic cells shows a highly significant decline with age. The hypothesis that loss of telomere repeats acts as a “mitotic clock” and a tumor suppressor mechanism in stem cells is strongly supported by recent studies of patients with mild telomerase deficiency resulting from haplo-insufficiency for either the TERC or TERT gene. Such genetic defects can give rise to various disorders including autosomal dominant Dyskeratosis Congenita (DKC), aplastic anemia, liver fibrosis and pulmonary fibrosis. Other recent studies have revealed that amplification of the hTERT gene is one of the most common genetic abnormalities in various cancers. Paradoxically, it is becoming clear that SNPs within the TERT locus are among the most reproducible risk factors for the development of different types of cancer including lung cancer, acute myeloid leukemia and chronic lymphocytic leukemia. The links between hypo- and hyperproliferative consequences of inborn telomerase deficiencies and SNP's in the TERT gene are poorly understood. It seems plausible that the increased risk of leukemia development in aplastic anemia, myelodysplastic syndrome and Dyskeratosis Congenita, results from stem cell failure. Could reduced stem cell numbers by itself provide a risk factor for tumor development? More direct measures of stem cell numbers in vivo are needed to examine this possibility and the relationship between stem cell numbers and tumor development in patients with defective telomere maintenance as well as in normal individuals as a function of age. Measurements of the average telomere length as well as the length of telomere repeats at individual chromosome ends in specific cells and tissues will further calrify the involvement of telomeres in bone marrow failure, normal aging and tumor biology. Disclosures Lansdorp: Repeat Diagnostics Inc.: Equity Ownership.

Telomere Length Changes of Lymphocytes in Aplastic Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5160-5160
Author(s):  
Rong Fu ◽  
Jiangbo Zhang ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Telomere Length ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Predictive Biomarker ◽  
Cd34 Cells ◽  
Primary Disorder ◽  
Length Changes

Abstract Aplastic anemia (AA) is a primary disorder of severe bone marrow failure characterized by pancytopenia. The pathogenesis of AA is closely related to T cell hyperfunction. Abnormal telomere shortening of bone marrow mononuclear cells which has been reported in AA not only lead to genomic instability and apoptosis but also regulate T cells immunity to antigen. Interestingly,lymphocytes with shorter telomere length have undergone apoptosis escape in autoimmune disease. In our study, the relative telomere length (RTLs) of CD3+, CD3+CD4+, CD3+CD8+, CD19+ and CD34+ cells were investigated in 14 untreated AA patients and 32 controls. The RTLs of CD3+,CD3+CD4+ and CD3+CD8+T lymphocytes were shorter than those of the controls. Meanwhile, no differences in CD19+B and CD34+ cells were found between AA and controls. A change in telomere length may be involved in the pathogenesis of AA and could be considered as a predictive biomarker for the diagnose of AA. Disclosures No relevant conflicts of interest to declare.

Proaerolysin Multi Color Flow-Fish Reveals Shortened Telomere Length in GPI− as Compared to GPI+ Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 171-171
Author(s):  
Fabian Beier ◽  
Stefan Balabanov ◽  
Tom Buckley ◽  
M. Rojewski ◽  
T. Becker ◽  
...  
Keyword(s):  
Telomere Length ◽  
Genetic Instability ◽  
Bone Marrow Failure ◽  
Response To Treatment ◽  
Disease Stage ◽  
Healthy Individuals ◽  
Color Flow ◽  
Telomere Shortening ◽  
Replicative Stress ◽  
Bone Marrow Failure Syndromes

Abstract Objective: Replication-dependent telomere shortening can result in cellular senescence or genetic instability. Telomere length both reflects and limits the replicative potential of normal somatic cells. Shortened telomeres have been linked to disease stage, degree of cytopenia as well as to response to treatment in patients with bone marrow failure syndromes. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Pancytopenia in PNH is thought to result from an auto-immune mediated depletion of the GPI+ stem cell compartment. In the current study, we aimed to develop a methodology that allows the selective analysis of telomere length in granulocytes from patients with PNH dependent on GPI expression. The aim was to find out whether replicative stress posed on the residual GPI− HSC is reflected in shortened telomere length compared to their GPI+ counterparts. Methods: The telomere length of GPI+ and GPI− granulocytes of 12 patients with PNH and 22 healthy individuals was analysed. For this purpose, we developed Proearolysine Multi-color flow FISH which is based on the crosslinking of a fluorescence-labeled non-toxic derivative of the bacterial toxine Aerolysine (that selectively binds to the GPI anchor of cells) in combination with fluorescence in situ hybridization and flow cytometry. Results: We found significantly (p<0.05) shortened telomeres in GPI− granulocytes (6.68±0.3 telomere fluorescence unit (TFU), n=12) compared to age-matched healthy individuals (mean±S.E.: 7.73±0.8 TFU, n=22), but no significant shortening in GPI+ (7.06±0.3 TFU, n=12) granulocytes from PNH patients. Telomere length in GPI− granulocytes was found to be significantly shorter as compared to their GPI+ counterparts (deltaTEL: 0.38±0.1, p=0.002). These results were confirmed by Multi-color flow FISH using an anti-CD59 antibodies. Conclusion: Autoimmune-mediated damage to the GPI+ HSC compartment leads to compensatory hyperproliferation of predominantly residual GPI− HSC. In line with this hypothesis, coexisting GPI-negative HSC clones showed even more accelerated telomere shortening as compared to their GPI+ counterparts. Potentially, replicative exhaustion of individual HSC clones could contribute to disease progression in PNH. Whether acquired genetic instability due to progressive telomere shortening also accounts for an increased incidence of secondary clonal disorders will need to be evaluated in future prospective studies.

A Novel hTERC Deletion Manifesting with Features of Dyskeratosis Congenita and Genetic Anticipation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4157-4157
Author(s):  
Stan Benke ◽  
D. S. Houston ◽  
Inderjeet Dokal ◽  
Tom Vulliamy
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Peripheral Blood ◽  
Autosomal Dominant ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Phenotypic Expression ◽  
Dyskeratosis Congenita ◽  
Sequencing Analysis ◽  
Genetic Anticipation

Abstract The gene encoding the RNA component of human telomerase (hTERC) is mutated in families with the autosomal dominant form of dyskeratosis congenita (DC). The phenomenon of genetic anticipation has recently been reported to accompany this form of DC, with disease severity increasing in offspring of affected individuals. It has been postulated that anticipation in these families relates to the adverse impact of hTERC mutations on inherited telomere length, with progressive telomere shortening seen in succeeding generations (Nat Gen2004; 36:447). We describe here a novel hTERC mutation, with affected individuals presenting in adulthood with mild mucocutaneous abnormalities, bone marrow failure and a pattern of penetrance supporting the presence of disease anticipation. The proband in the family studied presented at age 49 with squamous cell carcinoma of the tongue and a history of oral leukoplakia which he had developed at age 30. Peripheral blood on presentation was remarkable only for a mild macrocytic anemia. During treatment of his malignancy, severe and irreversible bone marrow hypoplasia was precipitated by a single cycle of cisplatinum chemotherapy. The patient’s brother at age 25 had been previously diagnosed with severe aplastic anemia; this was refractory to standard immunosuppression with cyclosporine and antithymocyte globulin. No somatic abnormailites were identified in this patient. Testing for Fanconi anemia in both siblings was negative. Direct sequencing analysis of hTERC in these patients revealed both to be heterozygous for a novel hTERC mutation (79 deletion C). Further studies among family members documented heterozygosity for the mutation in the mother of these two siblings. At age 77, she displayed none of the mucocutaneous signs associated with DC, while the only abnormality seen in her peripheral blood was an elevated mean corpuscular volume. The hTERC mutation seen in this family most likely exerts its effects through disruption of the pseudoknot domain. The findings of an individual with normal longevity, minimal phenotypic expression and affected offspring are further evidence of genetic anticipation being an important feature of autosomal dominant DC. Correlation with determination of telomere length has been initiated.

of the Telomerase RNA (TERC) and Telomerase Reverse Transcriptase (TERT) in Japanese Children with Aplastic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3750-3750
Author(s):  
Juan Liang ◽  
Hirosi Yagasaki ◽  
Koji Kato ◽  
Kazuko Kudo ◽  
Seiji Kojima
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Unrelated Donor ◽  
Allogeneic Bone ◽  
Healthy Controls ◽  
Japanese Children ◽  
Ribonucleoprotein Complex ◽  
Base Substitutions ◽  
Tert Gene

Abstract It is well known that the incidence of aplastic anemia (AA) is much higher in Japan than in Western countries. However the reason for this finding is not known. Genetic backgrounds related to AA may be different between populations. Recent studies have shown that some patients with apparently “acquired” AA have mutations in telomerase ribonucleoprotein complex genes such as TERC and TERT. We studied 96 Japanese children with acquired AA (age range, 0–16 years; median, 7 years) and 59 healthy controls for mutations in TERC and TERT genes. Of these children, 35 were classified as having nonsevere AA, 39 as having severe AA, and 22 as having very severe AA. In 7 patients, AA was secondary to acute hepatitis. We extracted DNA samples from peripheral blood and all exons and flanking introns of TERT and TERC were amplified by PCR using 19 primer pairs (1 TERC, 18 TERT). To determine the sequence, the PCR products were analyzed by ABI/PRISM 3100 automated sequencer. Telomere lengths of leukocytes were assessed by flow-FISH. For the TERC gene, no mutation was found. One polymorphism (n514 G&gt;A) was observed in 57/96 (59.4%) of patients. The same substitutions were detected in 31/59 (52.5%) healthy controls. For the TERT gene, two novel heterozygous, nonsynonymous mutations were identified (exon5; n2045 G&gt;A, exon6; n2177 C&gt;T). These base substitutions introduce an amino acid change-G682D and T726M, respectively. Neither patient had any clinical characteristics suggesting constitutional bone marrow failure syndrome. The n2177C&gt;T substitution was identified in a 9-year-old girl with very severe AA who failed to respond to immunosuppressive therapy. She received an allogeneic bone marrow transplant (BMT) from an unrelated donor, but did not engraft. She was then treated by a second BMT from an HLA haploidentical her mother. Her blood cells had a very short telomere compared with that of age-matched controls. Another patient carrying the n2045G&gt;A substitution had nonsevere AA and did not require any specific medication for 8 years. Six polymorphisms in exons of the TERT gene were identified in 102 unrelated patients (n915 G&gt;A, n2097 C&gt;T, n2520 G&gt;A, n2946 T&gt;C, n3039 C&gt;T, and n3366 G&gt;A). The allele frequencies of these silent base substitutions were 38/192 (19.8%), 3/192 (1.6%), 1/192 (0.5%), 1/192 (0.5%), 57/192 (29.7%), 2/192 (1.0%), respectively. Additionally, we identified 5 polymorphisms in introns of the TERT genes in 64 patients (IVS4+143 A&gt;G, IVS9+11 C&gt;T, IVS13+45 C&gt;T, IVS15+136 G&gt;A, and IVS16+81 C&gt;T). The frequencies were 52/192 (27.1%), 3/192 (1.6%), 7/192 (3.6%), 1/192 (0.5%), and 1/192 (0.5%), respectively. Except for two substitutions (n915G&gt;A and IVS4+143A&gt;G), the other 9 were not listed in the SNP database. We found a few patients with AA carrying mutations of telomerase ribonucleoprotein complex genes. Because the incidence of these mutations is not higher than that in Western populations, this genetic difference does not explain the higher incidence of AA in Japanese children.

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