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No parental origin bias for the rearranged chromosomes in myeloid leukemias associated with t(9;22), t(8;21) and t(15;17)
Leukemia Research 22 (1998) 793 – 796
No parental origin bias for the rearranged chromosomes in myeloid leukemias associated with t(9;22), t(8;21) and t(15;17) Hideo Nakamura a,*, Takahiro Itoyama d, Norio Niikawa c, Naoki Sadamori b, Masao Tomonaga b a Department of Internal Medicine, Nagasaki Municipal Medical Center, 20 -5 Fuchi-machi, Nagasaki 852, Japan Department of Hematology, Atomic Bomb Disease Institute, Nagasaki Uni6ersity School of Medicine, Nagasaki, Japan c Department of Human Genetics, Atomic Bomb Disease Institute, Nagasaki Uni6ersity School of Medicine, Nagasaki, Japan d Department of Technology, Toyama Uni6ersity, Toyama, Japan b
Received 10 November 1997; accepted 12 March 1998
Abstract We investigated parental origin of rearranged chromosomes 9 and 22 (9q + and 22q− ) in five patients with Ph-positive chronic myeloid leukemia (CML) using the C-banding and silver-staining methods of nucleolus organizer regions, respectively; of rearranged chromosome 21 (21q +) in seven patients with t(8;21)-positive acute myeloid leukemia (AML); and of rearranged chromosome 15 (15q +) in six patients with t(15;17)-positive AML. It was found that these rearranged chromosomes can be of either paternal or maternal origin. Although the number of patients examined was small, these results indicate that the genes rearranged as a result of these chromosome translocations (ABL, BCR, AML-1 and PML) are not genomically imprinted. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Genomic imprinting; t(9;22), t(8;21), t(15;17); Heterochromatin polymorphism
1. Introduction A reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;q11), is characteristic of chronic myeloid leukemia (CML). Based on heterochromatin polymorphisms in these chromosomes, Haas et al. [1] reported that the origins of the rearranged chromosomes 9 and 22 (9q+and 22q−) in CML patients were exclusively paternal and maternal, respectively. This parent of origin bias would be an example of genomic imprinting. This result led to the hypothesis that the genes disrupted and rearranged by the translocation, i.e. ABL and BCR, were themselves imprinted. However, several later reports, using molecular methods, have shown that the ABL and BCR genes are not Abbre6iations: Ag-NOR, silver-staining method of nucleolus organizer regions; AML, acute myeloid leukemia; BM, bone marrow; CML, chronic myeloid leukemia; DAPI, 4%-6-diaminido-2-phenylindole; PB, peripheral blood. * Present address: Heart Center 6F, Nagasaki A-Bomb Casualty Council, 2-41 Mori-machi, Nagasaki 852-8104, Japan. Fax: + 81 95 8439255; e-mail: [email protected]. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00069-1
imprinted [2–6]. In this study, we tried to reproduce the Haas et al. investigation with the same method employed by that original group [1]. Furthermore, we cytogenetically investigated the origin of rearranged chromosome 21 (21q+ ) in acute myeloid leukemia (AML) with t(8;21)(q22;q22) and of rearranged chromosome 15 (15q + ) in AML with t(15;17)(q22;q21), since chromosomes 15 and 21 are acrocentric and their heterochromatin polymorphisms are distinguishable by the silver-staining patterns of nucleolus-organizing regions (NORs) on the short arms.
2. Materials and methods We examined five CML patients with t(9;22), seven AML patients with t(8;21) and six AML patients with t(15;17). Blood samples were obtained from all parents with informed consent. The cell samples for cytogenetic studies were obtained from bone marrow (BM) of the patients and from peripheral blood (PB) of the parents. Whenever possible, PB samples from the patients were
H. Nakamura et al. / Leukemia Research 22 (1998) 793–796
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Table 1 A list of 10 leukemia patients with rearranged chromosomes whose parental origins were determined by C-banding or Ag-NOR-staining methods Case no.
Age/sex
Diagnosis
Karyotype
Origin of rearranged chromosome
1 2 3 4 5 6 7 8 9 10
41/F 37/F 38/F 17/F 12/M 8/M 38/F 20/F 6/F 28/M
CML CML CML AML AML AML AML AML AML AML
t(9;22) t(9;22) t(3;9;22) t(8;21) t(8;21) t(8;21) t(8;21) t(15;17) t(15;17) t(15;17)
Paternal (9q+) Maternal (22q−) Maternal (9q+) Paternal (22q−) Maternal (22q−) Paternal (21q+) Maternal (21q+) Maternal (21q+) Maternal (21q+) Paternal (15q+) Paternal (15q+) Maternal (15q+)
also examined to confirm that the heterochromatin polymorphism patterns between the PB and BM cells were identical. BM samples were harvested from a 24 or 48 h unstimulated culture and PB samples were harvested using a 72 h culture stimulated with phytohemagglutinin. For the analyses of C-banded polymorphisms of heterochromatin regions on chromosome 9, we used a sequential Q and C staining method [7] and for the analyses of heterochromatin polymorphisms of chromosomes 15, 21 and 22, a silver-staining method of NORs (Ag-NORs) was used [8]. In the Q and C staining method, photographs were taken of at least 30 metaphases on Q-stained slides with actinomycin D and 4%-6-diaminido-2-phenylindole (DAPI) for each patient and parent. After being rinsed with water, the slides were C-stained with distamycin A and DAPI. We photographed the same metaphases and compared the C-banding patterns of chromosome 9 between CML patients and their parents. In the silverstaining method, photographs were taken of at least 30 metaphases on Q-stained slides with quinacrine, for each patient and parent. After being rinsed with water, the slides were Ag-NOR-stained. We photographed the same metaphases and then compared the Ag-NORstaining patterns of chromosomes 15, 21 and 22 between patients and their parents.
were of maternal origin (Fig. 2). Among the six AML patients with t(15;17), two had paternal 15q+ and one was of maternal origin (Fig. 3). For each rearranged chromosome, its parental origin could not be deter-
3. Results The parental origins of the rearranged chromosomes were found by C-banding or Ag-NOR-staining method in 10 of the 18 patients examined (Table 1). Among the five patients with CML, one had paternal 9q+ and maternal 22q−, one maternal 9q+ and paternal 22q− and one undetermined 9q+ and maternal 22q−. The parental origins of the rearranged chromosomes in patient 2 contradict the results of Haas et al. [1]. The partial karyotypes of the patient and her parents are shown in Fig. 1. Among the seven AML patients with t(8;21), one had paternal 21q+ and three
Fig. 1. Partial karyotypes from a CML patient with maternal 9q + and paternal 22q − chromosomes and her parents. (A) Normal (left) and rearranged (right) chromosomes 9 from the patient are compared with the corresponding paternal and maternal chromosomes. A pair of the chromosomes of the patient in the upper row was Q-banded and the remaining three pairs were C-banded. The 9q + chromosome was suggestive of maternal origin because of its polymorphic size of the centromeric heterochromatin region. (B) Normal (left) and rearranged (right) chromosomes 22 from the patient are compared with the corresponding paternal and maternal chromosomes. Three pairs of the chromosomes in the upper row were Q-banded and the remaining three pairs were Ag-NOR-stained. The 22q − chromosome was suggestive of paternal origin because of its polymorphic size of the NOR on the short arm.
H. Nakamura et al. / Leukemia Research 22 (1998) 792–796
Fig. 2. Partial karyotypes from two AML patients with t(8;21) and their parents. Normal (left) and rearranged (right) chromosomes 21 from the patients are compared with the corresponding paternal and maternal chromosomes. The chromosomes in the upper rows were Q-banded and the remaining ones were Ag-NOR-stained. The 21q + chromosome was suggestive of paternal origin in one patient (A) and of maternal origin in the other (B), because of the polymorphic sizes of the NORs on the short arms.
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rearranged allele of BCR was paternal in origin in three CML cases by using P6uII and MaeII restriction site polymorphisms of the BCR gene. For the ABL gene, Melo et al. [5,6], using a BstNI restriction fragment length polymorphism of the ABL gene, showed that the two alleles were expressed both in normal subjects and in CML patients and that the translocated ABL gene can be either of paternal or maternal origin. Although these molecular results have provided conclusive evidence that the BCR and ABL genes are not imprinted, no other studies have tried to reproduce the reported cytogenetic results [1]. Our results are consistent with the molecular results, but not with the findings of Haas et al. despite using the same method employed by the original group. A possible explanation for the discrepancy between the results using molecular methods and those of Haas et al. [1], is a high frequency of somatic recombination, or gene conversion events, taking place between the polymorphic regions of chromosomes 9 and 22 and the ABL and BCR gene loci, respectively. This hypothesis must be proven by using combined molecular and cytogenetic studies. Haas et al. were able to determine parental origins of 9q+ and 22q− chromosomes in almost all t(9;22)positive leukemia patients examined, but we would like to stress the fact that it was difficult to determine the parental origins of rearranged chromosomes based on
mined in about one-half of the cases examined. This was because C-banded polymorphism patterns were often homogeneous in size among the two homologous chromosomes 9 and because it was more difficult to stain NORs of acrocentric chromosomes with silver for leukemic cells than for normal PB cells. Among the 10 patients listed in Table 1, only patient 6 had BM cells with normal karyotypes, in addition to those with t(8;21). There were no differences in the heterochromatin polymorphism patterns of the NORs of chromosomes 21 between the BM cells with and without t(8;21) in the patient.
4. Discussion In this study, we attempted to reproduce the results of Haas et al. using the same method that they employed [1] and found that some CML patients may have maternal 9q+ and paternal 22q− chromosomes. Haas et al. showed a marked parent of origin bias in the chromosomes involved in the t(9;22) by using heterochromatin polymorphisms [1]. In 1994, however, Riggins et al. [2] and Fioretos et al. [3] reported that the normal BCR gene is expressed from both alleles by using a polymorphic CGG-repeat and a BamHI polymorphism in the untranslated regions of BCR. Subsequently, Litz and Copenhaver [4] showed that the
Fig. 3. Partial karyotypes from two AML patients with t(15;17) and their parents. Normal (left) and rearranged (right) chromosomes 15 from the patients are compared with the corresponding paternal and maternal chromosomes. The chromosomes in the upper rows were Q-banded and the remaining ones were Ag-NOR-stained. The 15q + chromosome was suggestive of paternal origin in one patient (A) and of maternal origin in the other (B), because of the polymorphic sizes of the NORs on the short arms.
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H. Nakamura et al. / Leukemia Research 22 (1998) 793–796
heterochromatin polymorphisms in about one-half of cases we examined, thus causing us to doubt the accuracy of the original findings. Our results showed that 21q+ in AML with t(8;21) and of 15q+ in AML with t(15;17) may also lack parent of origin biases, which suggests that the genes rearranged as a result of these chromosome translocations (AML-1 and PML) are not genomically imprinted. To resolve this issue, however, the parental origin of the rearranged alleles of the genes must be identified using molecular methods.
Acknowledgements The authors thank Professor Yoshiro Tsuji and Dr Masahiko Nakayama (Department of Pediatrics, Nagasaki University School of Medicine) for providing clinical data of some patients. This study was supported by Grant-in-Aid for Exploratory Research from the Ministry of Education, Japan.
.
References [1] Haas OA, Argyriou-Tirita A, Lion T. Parental origin of chromosomes involved in the translocation t(9;22). Nature 1992;359:414. [2] Riggins GJ, Zhang F, Warren ST. Lack of imprinting of BCR. Nat Genet 1994;6:226. [3] Fioretos T, Heisterkamp N, Groffen J. No evidence for genomic imprinting of the human BCR gene. Blood 1994;83:3441. [4] Litz CE, Copenhaver CM. Parental origin of the rearranged major breakpoint cluster region in chronic myeloid leukemia. Blood 1994;83:3445. [5] Melo JV, Yan X-H, Diamond J, Goldman JM. Lack of imprinting of the ABL gene. Nat Genet 1994;8:318. [6] Melo JV, Yan X-H, Diamond J, Goldman JM. Balanced parental contribution to the ABL component of the BCR-ABL gene in chronic myeloid leukemia. Leukemia 1995;9:734. [7] Taniwaki M. New chromosome banding techniques with base specific antibodies and fluorochromes for qualified identification of marker chromosomes in leukemia and related disorders. Acta Haematol Jpn 1985;48:1423. [8] Howell WM, Black DA. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1step method. Experimentia 1980;36:1014.
No parental origin bias for the rearranged chromosomes in myeloid leukemias associated with t(9;22), t(8;21) and t(15;17) Hideo Nakamura a,*, Takahiro Itoyama d, Norio Niikawa c, Naoki Sadamori b, Masao Tomonaga b a Department of Internal Medicine, Nagasaki Municipal Medical Center, 20 -5 Fuchi-machi, Nagasaki 852, Japan Department of Hematology, Atomic Bomb Disease Institute, Nagasaki Uni6ersity School of Medicine, Nagasaki, Japan c Department of Human Genetics, Atomic Bomb Disease Institute, Nagasaki Uni6ersity School of Medicine, Nagasaki, Japan d Department of Technology, Toyama Uni6ersity, Toyama, Japan b
Received 10 November 1997; accepted 12 March 1998
Abstract We investigated parental origin of rearranged chromosomes 9 and 22 (9q + and 22q− ) in five patients with Ph-positive chronic myeloid leukemia (CML) using the C-banding and silver-staining methods of nucleolus organizer regions, respectively; of rearranged chromosome 21 (21q +) in seven patients with t(8;21)-positive acute myeloid leukemia (AML); and of rearranged chromosome 15 (15q +) in six patients with t(15;17)-positive AML. It was found that these rearranged chromosomes can be of either paternal or maternal origin. Although the number of patients examined was small, these results indicate that the genes rearranged as a result of these chromosome translocations (ABL, BCR, AML-1 and PML) are not genomically imprinted. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Genomic imprinting; t(9;22), t(8;21), t(15;17); Heterochromatin polymorphism
1. Introduction A reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;q11), is characteristic of chronic myeloid leukemia (CML). Based on heterochromatin polymorphisms in these chromosomes, Haas et al. [1] reported that the origins of the rearranged chromosomes 9 and 22 (9q+and 22q−) in CML patients were exclusively paternal and maternal, respectively. This parent of origin bias would be an example of genomic imprinting. This result led to the hypothesis that the genes disrupted and rearranged by the translocation, i.e. ABL and BCR, were themselves imprinted. However, several later reports, using molecular methods, have shown that the ABL and BCR genes are not Abbre6iations: Ag-NOR, silver-staining method of nucleolus organizer regions; AML, acute myeloid leukemia; BM, bone marrow; CML, chronic myeloid leukemia; DAPI, 4%-6-diaminido-2-phenylindole; PB, peripheral blood. * Present address: Heart Center 6F, Nagasaki A-Bomb Casualty Council, 2-41 Mori-machi, Nagasaki 852-8104, Japan. Fax: + 81 95 8439255; e-mail: [email protected]. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00069-1
imprinted [2–6]. In this study, we tried to reproduce the Haas et al. investigation with the same method employed by that original group [1]. Furthermore, we cytogenetically investigated the origin of rearranged chromosome 21 (21q+ ) in acute myeloid leukemia (AML) with t(8;21)(q22;q22) and of rearranged chromosome 15 (15q + ) in AML with t(15;17)(q22;q21), since chromosomes 15 and 21 are acrocentric and their heterochromatin polymorphisms are distinguishable by the silver-staining patterns of nucleolus-organizing regions (NORs) on the short arms.
2. Materials and methods We examined five CML patients with t(9;22), seven AML patients with t(8;21) and six AML patients with t(15;17). Blood samples were obtained from all parents with informed consent. The cell samples for cytogenetic studies were obtained from bone marrow (BM) of the patients and from peripheral blood (PB) of the parents. Whenever possible, PB samples from the patients were
H. Nakamura et al. / Leukemia Research 22 (1998) 793–796
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Table 1 A list of 10 leukemia patients with rearranged chromosomes whose parental origins were determined by C-banding or Ag-NOR-staining methods Case no.
Age/sex
Diagnosis
Karyotype
Origin of rearranged chromosome
1 2 3 4 5 6 7 8 9 10
41/F 37/F 38/F 17/F 12/M 8/M 38/F 20/F 6/F 28/M
CML CML CML AML AML AML AML AML AML AML
t(9;22) t(9;22) t(3;9;22) t(8;21) t(8;21) t(8;21) t(8;21) t(15;17) t(15;17) t(15;17)
Paternal (9q+) Maternal (22q−) Maternal (9q+) Paternal (22q−) Maternal (22q−) Paternal (21q+) Maternal (21q+) Maternal (21q+) Maternal (21q+) Paternal (15q+) Paternal (15q+) Maternal (15q+)
also examined to confirm that the heterochromatin polymorphism patterns between the PB and BM cells were identical. BM samples were harvested from a 24 or 48 h unstimulated culture and PB samples were harvested using a 72 h culture stimulated with phytohemagglutinin. For the analyses of C-banded polymorphisms of heterochromatin regions on chromosome 9, we used a sequential Q and C staining method [7] and for the analyses of heterochromatin polymorphisms of chromosomes 15, 21 and 22, a silver-staining method of NORs (Ag-NORs) was used [8]. In the Q and C staining method, photographs were taken of at least 30 metaphases on Q-stained slides with actinomycin D and 4%-6-diaminido-2-phenylindole (DAPI) for each patient and parent. After being rinsed with water, the slides were C-stained with distamycin A and DAPI. We photographed the same metaphases and compared the C-banding patterns of chromosome 9 between CML patients and their parents. In the silverstaining method, photographs were taken of at least 30 metaphases on Q-stained slides with quinacrine, for each patient and parent. After being rinsed with water, the slides were Ag-NOR-stained. We photographed the same metaphases and then compared the Ag-NORstaining patterns of chromosomes 15, 21 and 22 between patients and their parents.
were of maternal origin (Fig. 2). Among the six AML patients with t(15;17), two had paternal 15q+ and one was of maternal origin (Fig. 3). For each rearranged chromosome, its parental origin could not be deter-
3. Results The parental origins of the rearranged chromosomes were found by C-banding or Ag-NOR-staining method in 10 of the 18 patients examined (Table 1). Among the five patients with CML, one had paternal 9q+ and maternal 22q−, one maternal 9q+ and paternal 22q− and one undetermined 9q+ and maternal 22q−. The parental origins of the rearranged chromosomes in patient 2 contradict the results of Haas et al. [1]. The partial karyotypes of the patient and her parents are shown in Fig. 1. Among the seven AML patients with t(8;21), one had paternal 21q+ and three
Fig. 1. Partial karyotypes from a CML patient with maternal 9q + and paternal 22q − chromosomes and her parents. (A) Normal (left) and rearranged (right) chromosomes 9 from the patient are compared with the corresponding paternal and maternal chromosomes. A pair of the chromosomes of the patient in the upper row was Q-banded and the remaining three pairs were C-banded. The 9q + chromosome was suggestive of maternal origin because of its polymorphic size of the centromeric heterochromatin region. (B) Normal (left) and rearranged (right) chromosomes 22 from the patient are compared with the corresponding paternal and maternal chromosomes. Three pairs of the chromosomes in the upper row were Q-banded and the remaining three pairs were Ag-NOR-stained. The 22q − chromosome was suggestive of paternal origin because of its polymorphic size of the NOR on the short arm.
H. Nakamura et al. / Leukemia Research 22 (1998) 792–796
Fig. 2. Partial karyotypes from two AML patients with t(8;21) and their parents. Normal (left) and rearranged (right) chromosomes 21 from the patients are compared with the corresponding paternal and maternal chromosomes. The chromosomes in the upper rows were Q-banded and the remaining ones were Ag-NOR-stained. The 21q + chromosome was suggestive of paternal origin in one patient (A) and of maternal origin in the other (B), because of the polymorphic sizes of the NORs on the short arms.
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rearranged allele of BCR was paternal in origin in three CML cases by using P6uII and MaeII restriction site polymorphisms of the BCR gene. For the ABL gene, Melo et al. [5,6], using a BstNI restriction fragment length polymorphism of the ABL gene, showed that the two alleles were expressed both in normal subjects and in CML patients and that the translocated ABL gene can be either of paternal or maternal origin. Although these molecular results have provided conclusive evidence that the BCR and ABL genes are not imprinted, no other studies have tried to reproduce the reported cytogenetic results [1]. Our results are consistent with the molecular results, but not with the findings of Haas et al. despite using the same method employed by the original group. A possible explanation for the discrepancy between the results using molecular methods and those of Haas et al. [1], is a high frequency of somatic recombination, or gene conversion events, taking place between the polymorphic regions of chromosomes 9 and 22 and the ABL and BCR gene loci, respectively. This hypothesis must be proven by using combined molecular and cytogenetic studies. Haas et al. were able to determine parental origins of 9q+ and 22q− chromosomes in almost all t(9;22)positive leukemia patients examined, but we would like to stress the fact that it was difficult to determine the parental origins of rearranged chromosomes based on
mined in about one-half of the cases examined. This was because C-banded polymorphism patterns were often homogeneous in size among the two homologous chromosomes 9 and because it was more difficult to stain NORs of acrocentric chromosomes with silver for leukemic cells than for normal PB cells. Among the 10 patients listed in Table 1, only patient 6 had BM cells with normal karyotypes, in addition to those with t(8;21). There were no differences in the heterochromatin polymorphism patterns of the NORs of chromosomes 21 between the BM cells with and without t(8;21) in the patient.
4. Discussion In this study, we attempted to reproduce the results of Haas et al. using the same method that they employed [1] and found that some CML patients may have maternal 9q+ and paternal 22q− chromosomes. Haas et al. showed a marked parent of origin bias in the chromosomes involved in the t(9;22) by using heterochromatin polymorphisms [1]. In 1994, however, Riggins et al. [2] and Fioretos et al. [3] reported that the normal BCR gene is expressed from both alleles by using a polymorphic CGG-repeat and a BamHI polymorphism in the untranslated regions of BCR. Subsequently, Litz and Copenhaver [4] showed that the
Fig. 3. Partial karyotypes from two AML patients with t(15;17) and their parents. Normal (left) and rearranged (right) chromosomes 15 from the patients are compared with the corresponding paternal and maternal chromosomes. The chromosomes in the upper rows were Q-banded and the remaining ones were Ag-NOR-stained. The 15q + chromosome was suggestive of paternal origin in one patient (A) and of maternal origin in the other (B), because of the polymorphic sizes of the NORs on the short arms.
796
H. Nakamura et al. / Leukemia Research 22 (1998) 793–796
heterochromatin polymorphisms in about one-half of cases we examined, thus causing us to doubt the accuracy of the original findings. Our results showed that 21q+ in AML with t(8;21) and of 15q+ in AML with t(15;17) may also lack parent of origin biases, which suggests that the genes rearranged as a result of these chromosome translocations (AML-1 and PML) are not genomically imprinted. To resolve this issue, however, the parental origin of the rearranged alleles of the genes must be identified using molecular methods.
Acknowledgements The authors thank Professor Yoshiro Tsuji and Dr Masahiko Nakayama (Department of Pediatrics, Nagasaki University School of Medicine) for providing clinical data of some patients. This study was supported by Grant-in-Aid for Exploratory Research from the Ministry of Education, Japan.
.
References [1] Haas OA, Argyriou-Tirita A, Lion T. Parental origin of chromosomes involved in the translocation t(9;22). Nature 1992;359:414. [2] Riggins GJ, Zhang F, Warren ST. Lack of imprinting of BCR. Nat Genet 1994;6:226. [3] Fioretos T, Heisterkamp N, Groffen J. No evidence for genomic imprinting of the human BCR gene. Blood 1994;83:3441. [4] Litz CE, Copenhaver CM. Parental origin of the rearranged major breakpoint cluster region in chronic myeloid leukemia. Blood 1994;83:3445. [5] Melo JV, Yan X-H, Diamond J, Goldman JM. Lack of imprinting of the ABL gene. Nat Genet 1994;8:318. [6] Melo JV, Yan X-H, Diamond J, Goldman JM. Balanced parental contribution to the ABL component of the BCR-ABL gene in chronic myeloid leukemia. Leukemia 1995;9:734. [7] Taniwaki M. New chromosome banding techniques with base specific antibodies and fluorochromes for qualified identification of marker chromosomes in leukemia and related disorders. Acta Haematol Jpn 1985;48:1423. [8] Howell WM, Black DA. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1step method. Experimentia 1980;36:1014.