- Email: [email protected]
P-54 Telomere length control bytelomere binding proteins, TRF1, TRF2 and POT1, in myelodysplastic syndrome
Posters
$44
respectively, in AA patients and base substitutions or missense mutations, respectively, in MDS patients. Our results suggest that PIG-A gene analysis in AA and MDS patients is a useful tool to know the process of growth or selection of PNH clones.
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STUDY OF DNA REPAIR GENE EXPRESSION IN MDS PATIENTS WITH SHORTENED TELOMERES AND MULTIPLE C H R O M O S O M A L ABNORMALITIES BY ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION (A-CGH)
L. Gondek*, C. O'Keefe, M. Wlodarski, LR Maciejewski.
Cleveland Clinic Foundation, Cleveland, USA *E-maih [email protected] f.org A predisposition to DNA and genomic damage, including large defects in chromosome structure, is a frequent feature of MDS and has been hypothesized to be caused by age-related changes in the DNA repair machinery. Loss of telomeric sequences may represent one of the possible mechanisms rendering the chromosomes susceptible to rearrangement and loss of material. However, the relationship between telomere length and karyotypic instability is complex. The sequence and structure of telomeres are recognized by number of DNA-binding proteins, including DNA repair proteins that have direct effects on telomere maintenance. Thus, telomere shortening may itself reflect impairment of the DNA repair machinery. Here, we have studied the relationship between telomere length as measured by a novel quantitative telomere quantitative PCR and other parameters indicating defects in the maintenance of genomic integrity. They include the frequency of discrete chromosomal defects measured by a high resolution A-CGH, and the expression levels of DNA repair machinery genes using a targeted expression array and quantitative PCR. First, we established the expression levels of DNA repair proteins in CD34+ cells derived from healthy individuals and compared it to MDS patients with increased numbers of scattered chromosomal aberrations detected by A-CGH. Our study revealed a distinct expression pattern of various DNA repair proteins in patients with multiple chromosomal defects and excessively shortened telomeres.
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EVALUATION OF CD4 AND CD8 LYMPHOCYTES AS NORMAL CONTROLS FOR HUMARA CLONALITY ASSAY IN MDS
T. Mitsui1 *, A. Yamane 2, H. Koiso 1, H. Handa 3, T. Matsushima 1, N. Tsukamoto 1, H. Murakami 3, Y. Nojima 1, M. Karasawa 2. 1Department of Medicine
and Clinical Science, Gunma University Graduate School of Medicine, 2Division of Blood Transfusion Service, University Ho~Tital, Faculty of Medicine, Gunma University, 3Faculty of Medicine, Gunma University School of Health Science, Japan *E-maih [email protected] dp The HUMARA based clonal assay has been applied to evaluate clonality. However, this method has the main drawback of biased X chromosome inactivation, by which extremely skewed results are seen even in normal females. In most previous reports, clonality is assessed using whole T-lymphocyte as a normal control. Since it has been reported that frequent clonal expansion of CD8 cells occur with age, we speculated that CD4 cells are a better control for the assay. To evaluate this possibility, we examined X inactivation status in 34 heterozygous healthy females and 13 MDS patients (12 RA and 1 RARS) in separated peripheral CD4 and CD8 lymphocytes. In healthy subjects, CD4 cells from only 4 cases and CD8 cells from 9 cases had biased X inactivation. In MDS patients, 2 patients showed skewing both in CD4 and CD8 cells. The mean complexity score of TCR V[5 repertoire for CD8 + lymphocytes was significantly lower than that for CD4 + lymphocytes in control (86.7 to 139.6, P <0.0001) and in MDS patients (82.0 to 144.3, P < 0.0001). Moreover, the skewing associated with decreased the naive cell compartment, reducing the TCR repertoire. Analogous results were also obtained in the patients with MDS. In conclusion, CD4 ÷ lymphocytes might be better control than CD8 + lymphocytes for methylation-based clonal assay in MDS patients.
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TELOMERE LENGTH CONTROL BY TELOMERE BINDING PROTEINS, TRF1, TRF2 AND POT1, IN MYELODYSPLASTIC SYNDROME
S. Honda 1 *, LH. Ohyashiki 2, T. Takaku 1, Y. Ishii1, G. Sashida 1, T. Tauchi 1, K. Ohyashiki 1. 1First Department
of Internal Medicine, Tokyo Medical University, 2Intractable Immune System Diseases Research Cente~ Tokyo Medical University, Japan *E-maih [email protected] .ne.j p Telomeres consist of DNA sequences, tandemly repeated at the end of chromosomes, which bind protein factors and make a "cap", securing each end of every chromosome.
Molecular Study While telomere binding proteins, TRF-1, TRF-2 and POT-1 play crucial roles in maintaining telomeres, little is known about the regulatory system of telomere binding proteins in myelodysplastic syndrome (MDS), despite the fact that it frequently exhibits telomere attrition. To investigate the possible role of telomere maintenance in MDS, we analyzed the expression of telomerase components (TERC and hTERT), and telomere binding proteins (TRF-1, TRF-2 and POT-l) by real-time RT-PCR in 25 patients with MDS as well as in healthy volunteers, then compared the telomere length measured by either terminal restriction fragment analysis or flow-FISH analysis. In the current study, telomere attrition was obvious in 10 of the 25 patients. Multiple regression analysis revealed that the telomere length was significantly associated with the expression level of telomere binding proteins (y2 0.552, p 0.043 ) in healthy volunteers, but there was no association between telomere length and telomere binding proteins in MDS patients (y2 0.08, p 0.81). Our results suggest that telomere homeostasis mediated by the interaction of telomere binding proteins is disrupted in MDS.
IP~5
AZACYTIDINE IN MYELODYSPLASTIC SYNDROMES: CORRELATION BETWEEN CLINICAL RESPONSES AND CDKN2B PROMOTER METHYLATION
K. Raj *, A. John, A. Ho, N.S. Thomas, G. Mufti.
Department of Haematological and Molecular Medicine, King~ College London, UK *E-maih kavita.raj @kcl.ac.uk Whether superior responses to 5 azacytidine in Myelodysplastic syndromes occur due to its role as a demethylating agent remain unclear. We studied the clinical responses to 5 azacytidine and associated changes in promoter methylation of the CDKN2B gene (encodes pl5INK4B). Twenty-four patients (IPSS 0 n 1, Int-1 n 6, Int-2 n 8, High n 9) were treated with 1 11 (median 4) courses of 5 azacytidine (75 mg/m2 for 7 days). Complete remission was achieved in 5 patients (2: trisomy 8, 3: monosomy 7) after 2 5 cycles of treatment and haematological improvement (HI) in platelets occurred in 6 patients, HI-Hb in 2 patients and HI-N in 3 patients. The CDKN2B promoter (+115 ~ +220) was methylated in 15 of 16 patients prior to treatment by MS-PCR. Bisulfite Genomic sequencing of the CDKN2B promoter ( 3 6 ~ + 2 5 1 ; 29 CpG) in 10 of these patients showed a variegated methylation pattern in the CD34+, CD33+ and dual negative fractions. Following azacytidine treatment (3 5 cycles) 4 patients demethylated CDKN2B by MS-PCR. Confirmatory Bisulfite genomic sequencing done on the same unmethylated samples for 2 patients showed no change in the variegated methylation previously observed. Moreover the demethylation observed did not correlate with clinical response. These preliminary results suggest that
$45
other mechanisms may be responsible for the clinical effects of 5 azacytidine. This work is fianded by the MRC and LRF, UK.
[P~
CDKN2B (P151NK4B) AND CDKNIA (P21CIP1) PROMOTER HYPERMETHYLATION OCCURS IN LYMPHOCYTES AND NOT IN CD34+ OR CD33+ MYELOID CELLS OF MDS PATIENTS
A. John*, R. Kavita, N.S. Thomas, G. Mufti. Department
of Haematological arid Molecular Medicine, Kingk College London, UK *E-mail: kavita.raj @kcl.ac.uk Epigenetic gene silencing of the cell cycle genes CDKN2B and CDKN1A, encoding pl5INK4B and p21Cipl, has been reported to occur in high-risk MDS and ALL respectively. We detected CDKN2B promoter hypermethylation by MS-PCR in 100% (12/12) and CDKN1A in 50% (3/6) high-risk patients at presentation. MS-PCR is highly sensitive, detecting 1 hypermethylated promoter in 1,000. Therefore, we investigated the CpG methylation pattern of CDKN2B ( 36 )+251; 29 CpGs) and CDKN1A ( ~ 2 9 ~ + 3 4 7 ; 58 CpGs) by bisulfite genomic sequencing, using unbiased primers without CpGs. No hypermethylated CDKN2B promoter was detected in any of the clones sequenced from CD34+ or CD33+ cells of 17 patients. However, the CDKN2B promoter was hypermethylated in the lymphocytes of all 5 patients analysed (10 55% of clones sequenced with 93 100% CpGs methylated). The promoter was unmethylated in normal T cells and CD34+ cells (n 3 each). Similarly, the CDKN1A promoter was only hypermethylated in lymphocytes, but not CD34+ cells of 3 patients analysed. We also noted an age-related characteristic pattern of 34 methylated CpGs in normal T cells (16/19 clones sequenced). Our data show that abnormal, hypermethylation of CDKN2B and CDKN1A promoters occurs in lymphocytes of MDS patients. Since T cells from CDKN2B ~ and CDKN1A ~ mice have a proliferative advantage, epigenetic silencing of these cell cycle genes provides a mechanism for T cell expansion in MDS. Funding: MRC and LRF, UK.
$44
respectively, in AA patients and base substitutions or missense mutations, respectively, in MDS patients. Our results suggest that PIG-A gene analysis in AA and MDS patients is a useful tool to know the process of growth or selection of PNH clones.
•
STUDY OF DNA REPAIR GENE EXPRESSION IN MDS PATIENTS WITH SHORTENED TELOMERES AND MULTIPLE C H R O M O S O M A L ABNORMALITIES BY ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION (A-CGH)
L. Gondek*, C. O'Keefe, M. Wlodarski, LR Maciejewski.
Cleveland Clinic Foundation, Cleveland, USA *E-maih [email protected] f.org A predisposition to DNA and genomic damage, including large defects in chromosome structure, is a frequent feature of MDS and has been hypothesized to be caused by age-related changes in the DNA repair machinery. Loss of telomeric sequences may represent one of the possible mechanisms rendering the chromosomes susceptible to rearrangement and loss of material. However, the relationship between telomere length and karyotypic instability is complex. The sequence and structure of telomeres are recognized by number of DNA-binding proteins, including DNA repair proteins that have direct effects on telomere maintenance. Thus, telomere shortening may itself reflect impairment of the DNA repair machinery. Here, we have studied the relationship between telomere length as measured by a novel quantitative telomere quantitative PCR and other parameters indicating defects in the maintenance of genomic integrity. They include the frequency of discrete chromosomal defects measured by a high resolution A-CGH, and the expression levels of DNA repair machinery genes using a targeted expression array and quantitative PCR. First, we established the expression levels of DNA repair proteins in CD34+ cells derived from healthy individuals and compared it to MDS patients with increased numbers of scattered chromosomal aberrations detected by A-CGH. Our study revealed a distinct expression pattern of various DNA repair proteins in patients with multiple chromosomal defects and excessively shortened telomeres.
•
EVALUATION OF CD4 AND CD8 LYMPHOCYTES AS NORMAL CONTROLS FOR HUMARA CLONALITY ASSAY IN MDS
T. Mitsui1 *, A. Yamane 2, H. Koiso 1, H. Handa 3, T. Matsushima 1, N. Tsukamoto 1, H. Murakami 3, Y. Nojima 1, M. Karasawa 2. 1Department of Medicine
and Clinical Science, Gunma University Graduate School of Medicine, 2Division of Blood Transfusion Service, University Ho~Tital, Faculty of Medicine, Gunma University, 3Faculty of Medicine, Gunma University School of Health Science, Japan *E-maih [email protected] dp The HUMARA based clonal assay has been applied to evaluate clonality. However, this method has the main drawback of biased X chromosome inactivation, by which extremely skewed results are seen even in normal females. In most previous reports, clonality is assessed using whole T-lymphocyte as a normal control. Since it has been reported that frequent clonal expansion of CD8 cells occur with age, we speculated that CD4 cells are a better control for the assay. To evaluate this possibility, we examined X inactivation status in 34 heterozygous healthy females and 13 MDS patients (12 RA and 1 RARS) in separated peripheral CD4 and CD8 lymphocytes. In healthy subjects, CD4 cells from only 4 cases and CD8 cells from 9 cases had biased X inactivation. In MDS patients, 2 patients showed skewing both in CD4 and CD8 cells. The mean complexity score of TCR V[5 repertoire for CD8 + lymphocytes was significantly lower than that for CD4 + lymphocytes in control (86.7 to 139.6, P <0.0001) and in MDS patients (82.0 to 144.3, P < 0.0001). Moreover, the skewing associated with decreased the naive cell compartment, reducing the TCR repertoire. Analogous results were also obtained in the patients with MDS. In conclusion, CD4 ÷ lymphocytes might be better control than CD8 + lymphocytes for methylation-based clonal assay in MDS patients.
•
TELOMERE LENGTH CONTROL BY TELOMERE BINDING PROTEINS, TRF1, TRF2 AND POT1, IN MYELODYSPLASTIC SYNDROME
S. Honda 1 *, LH. Ohyashiki 2, T. Takaku 1, Y. Ishii1, G. Sashida 1, T. Tauchi 1, K. Ohyashiki 1. 1First Department
of Internal Medicine, Tokyo Medical University, 2Intractable Immune System Diseases Research Cente~ Tokyo Medical University, Japan *E-maih [email protected] .ne.j p Telomeres consist of DNA sequences, tandemly repeated at the end of chromosomes, which bind protein factors and make a "cap", securing each end of every chromosome.
Molecular Study While telomere binding proteins, TRF-1, TRF-2 and POT-1 play crucial roles in maintaining telomeres, little is known about the regulatory system of telomere binding proteins in myelodysplastic syndrome (MDS), despite the fact that it frequently exhibits telomere attrition. To investigate the possible role of telomere maintenance in MDS, we analyzed the expression of telomerase components (TERC and hTERT), and telomere binding proteins (TRF-1, TRF-2 and POT-l) by real-time RT-PCR in 25 patients with MDS as well as in healthy volunteers, then compared the telomere length measured by either terminal restriction fragment analysis or flow-FISH analysis. In the current study, telomere attrition was obvious in 10 of the 25 patients. Multiple regression analysis revealed that the telomere length was significantly associated with the expression level of telomere binding proteins (y2 0.552, p 0.043 ) in healthy volunteers, but there was no association between telomere length and telomere binding proteins in MDS patients (y2 0.08, p 0.81). Our results suggest that telomere homeostasis mediated by the interaction of telomere binding proteins is disrupted in MDS.
IP~5
AZACYTIDINE IN MYELODYSPLASTIC SYNDROMES: CORRELATION BETWEEN CLINICAL RESPONSES AND CDKN2B PROMOTER METHYLATION
K. Raj *, A. John, A. Ho, N.S. Thomas, G. Mufti.
Department of Haematological and Molecular Medicine, King~ College London, UK *E-maih kavita.raj @kcl.ac.uk Whether superior responses to 5 azacytidine in Myelodysplastic syndromes occur due to its role as a demethylating agent remain unclear. We studied the clinical responses to 5 azacytidine and associated changes in promoter methylation of the CDKN2B gene (encodes pl5INK4B). Twenty-four patients (IPSS 0 n 1, Int-1 n 6, Int-2 n 8, High n 9) were treated with 1 11 (median 4) courses of 5 azacytidine (75 mg/m2 for 7 days). Complete remission was achieved in 5 patients (2: trisomy 8, 3: monosomy 7) after 2 5 cycles of treatment and haematological improvement (HI) in platelets occurred in 6 patients, HI-Hb in 2 patients and HI-N in 3 patients. The CDKN2B promoter (+115 ~ +220) was methylated in 15 of 16 patients prior to treatment by MS-PCR. Bisulfite Genomic sequencing of the CDKN2B promoter ( 3 6 ~ + 2 5 1 ; 29 CpG) in 10 of these patients showed a variegated methylation pattern in the CD34+, CD33+ and dual negative fractions. Following azacytidine treatment (3 5 cycles) 4 patients demethylated CDKN2B by MS-PCR. Confirmatory Bisulfite genomic sequencing done on the same unmethylated samples for 2 patients showed no change in the variegated methylation previously observed. Moreover the demethylation observed did not correlate with clinical response. These preliminary results suggest that
$45
other mechanisms may be responsible for the clinical effects of 5 azacytidine. This work is fianded by the MRC and LRF, UK.
[P~
CDKN2B (P151NK4B) AND CDKNIA (P21CIP1) PROMOTER HYPERMETHYLATION OCCURS IN LYMPHOCYTES AND NOT IN CD34+ OR CD33+ MYELOID CELLS OF MDS PATIENTS
A. John*, R. Kavita, N.S. Thomas, G. Mufti. Department
of Haematological arid Molecular Medicine, Kingk College London, UK *E-mail: kavita.raj @kcl.ac.uk Epigenetic gene silencing of the cell cycle genes CDKN2B and CDKN1A, encoding pl5INK4B and p21Cipl, has been reported to occur in high-risk MDS and ALL respectively. We detected CDKN2B promoter hypermethylation by MS-PCR in 100% (12/12) and CDKN1A in 50% (3/6) high-risk patients at presentation. MS-PCR is highly sensitive, detecting 1 hypermethylated promoter in 1,000. Therefore, we investigated the CpG methylation pattern of CDKN2B ( 36 )+251; 29 CpGs) and CDKN1A ( ~ 2 9 ~ + 3 4 7 ; 58 CpGs) by bisulfite genomic sequencing, using unbiased primers without CpGs. No hypermethylated CDKN2B promoter was detected in any of the clones sequenced from CD34+ or CD33+ cells of 17 patients. However, the CDKN2B promoter was hypermethylated in the lymphocytes of all 5 patients analysed (10 55% of clones sequenced with 93 100% CpGs methylated). The promoter was unmethylated in normal T cells and CD34+ cells (n 3 each). Similarly, the CDKN1A promoter was only hypermethylated in lymphocytes, but not CD34+ cells of 3 patients analysed. We also noted an age-related characteristic pattern of 34 methylated CpGs in normal T cells (16/19 clones sequenced). Our data show that abnormal, hypermethylation of CDKN2B and CDKN1A promoters occurs in lymphocytes of MDS patients. Since T cells from CDKN2B ~ and CDKN1A ~ mice have a proliferative advantage, epigenetic silencing of these cell cycle genes provides a mechanism for T cell expansion in MDS. Funding: MRC and LRF, UK.