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A new dic(7;12)(p12.21;p12.2) chromosome aberration in a case of acute myeloid leukemia
Cancer Genetics and Cytogenetics 185 (2008) 102e105
Short communication
A new dic(7;12)(p12.21;p12.2) chromosome aberration in a case of acute myeloid leukemia Cinzia Tapinassia,b,1, Elvira Gerbinoa,b,1, Omar Malazzia,b, Carla Micuccia,b, Patrizia Gasparinia,b,2, Maria J. Najerac, Maria J. Calasanzd, Maria D. Oderod,e, Pier Giuseppe Peliccia,b, Elena Bellonia,b,* a
Department of Molecular Medicine, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy b IFOM-IEO Campus, Milan, Italy c Department of Hematology, Hospital San Milla´n, La Rioja, Spain d Department of Genetics, University of Navarra, Pamplona, Spain e Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain Received 21 March 2008; received in revised form 21 May 2008; accepted 27 May 2008
Abstract
A new dic(7;12)(p12.21;p12.2) chromosome aberration was identified in an acute myeloid leukemia with FAB-M1 morphology and was cloned. Fluorescence in situ hybridization and genomic quantitative polymerase chain reaction mapping experiments revealed the precise localization of the breakpoints, at 7p12, just 50 to the GRB10 gene, and 12p11, within a genomic region containing no known genes. As a result, a new dicentric chromosome is created, dic(7;12), with the consequent deletion of 50 Mb at 7p, from the telomere to the GRB10 region, and of 30 Mb at 12p, from the telomere to p11. Several genes are included in the affected areas. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Chromosomal rearrangements leading to the creation of aberrant fusion proteins are a common feature in acute myeloid leukemia (AML), observed in ~45% of all reported cases [1]. Normal karyotypes are also frequently found (~50% of the cases), and it has been shown that more subtle defects, resulting in gene sequence alterations (mainly in NPM1 and FLT3), direct the leukemogenic process in such instances [2,3]. The remaining 5% of cases are characterized by rare rearrangements, mostly translocations, leading to the aberrant juxtaposition of chromosomal sequences and to either the creation of aberrant new rare fusion products or the deregulation or truncation of specific genes involved in the rearrangement [1]. Chromosome 7 can be considered a main target for chromosomal rearrangements in AML, with del(7q) and monosomy 7 representing quite common cytogenetic abnormalities in both adult and childhood AML [4]. In a few cases, the region 7p15 * Corresponding author. Tel.: þ39-02-57489396; fax: þ39-0257489851. E-mail address: [email protected] (E. Belloni). 1 These authors contributed equally to the work. 2 Present address: Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy. 0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2008.05.015
has been found to be rearranged, with involvement of the HOXA9 gene in the t(7;11)(p15;p15) resulting in the NUP98eHOXA9 fusion product [5]. Chromosome 12 also has been implicated in AML, mainly because of translocations affecting the ETV6 gene, with various partner chromosomes and as many partner genes, such as MN1, MNX1, MDS1 and EVI1, and CHIC2 [6]. Here, we describe a dic(7;12)(p12.21;p12.2) translocation, a new chromosomal aberration identified in an AML-M1 case.
2. Materials and methods 2.1. Patient A 53-year-old man was admitted to the hospital with fever, hematemesis, and melena. Laboratory findings were as follows: hemoglobin 16 g/dL, white blood cell count 94.4 109/L with 76% blasts, and platelet count 17 109/ L. Cytochemical testing revealed positivity for myeloperoxidase. Immunophenotyping performed on bone marrow cells revealed positivity for CD45 (96.4%), CD34 (62%), and CD33 (78.4%) antigens and negativity for CD14. A diagnosis of AML-M1 was made. Induction treatment was initiated, but 1 month later the patient died because of heart failure.
C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
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Fig. 1. (a). Left: FISH experiment with probes specific for the chromosome 7 (in red) and 12 (in green) centromeres. The dic(7;12), with signals corresponding to both centromeres is clearly visible and indicated with a white asterisk. Right, top: An enlargement of the GTG banded dic(7;12). Right, bottom: Schematic representation of the dic(7;12), with retained portion of chromosome 7 represented in pink and the portion of chromosome 12 represented in green. (b) FISH mapping of the 7p and 12p breakpoints (BPs). Left: A schematic representation of chromosomes 7p and 12p. The genomic clones selected for the initial FISH experiments are indicated, with vertical ordering from telomere to centromere. Right: Physical maps of the refined 7p and 12p BP regions are shown. Maps are derived from the Web site of the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/), with adaptations. Genomic BAC clones selected for FISH experiments are indicated as horizontal bars. Genes located in the BP regions are indicated as blue horizontal bars: genomic structure, showing exons as vertical bars, is given. Blue arrows on top of genes names indicate the 50 e30 orientation of genes. The position of the two BPs is given. The black asterisk indicates the position of the predicted LOC390299 locus, as derived from the U.S. National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/). Deleted clones are indicated in red, retained clones in green.
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C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
2.2. Fluorescence in situ hybridization FISH was performed as described [7] with selected genomic clones, obtained from either the Roswell Park Cancer Institute (http://bacpac.chori.org/) or the California Institute of Technology (http://www.tree.caltech.edu/) bacterial artificial chromosome (BAC) libraries. Each clone has been tested on metaphase spreads from a normal control, to confirm the chromosomal location and check for its specificity. 2.3. Real-time quantitative polymerase chain reaction Real-time quantitative PCR (RQ-PCR) was performed using the SYBR Green technology on an ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). PCR reactions were prepared in a final volume of 25 mL (1 SYBR Green PCR master mix; 5 ng DNA). Results were normalized to a control genomic sequence, corresponding to the MYB gene (primer sequences available upon request). The GRB10/MYB ratio for the studied case was also normalized to the same ratio concomitantly measured in the total human DNA control sample (as a calibrator). 3. Results and discussion The karyotype interpretation from the conventional cytogenetic analysis was a 45,XY,e7,der(12)t(7;12)(q11;p11) [30]/46,XY,del(7)(p11),del(12)(p11)[5] karyotype. We started our analysis performing FISH with probes specific for both chromosome 7 and chromosome 12 centromeres. This analysis revealed the presence of a dicentric chromosome, dic(7;12) (Fig. 1a). Moreover, FISH experiments with the genomic BAC probe RP11-417D8 (7q11) showed
hybridization signals on the normal chromosome 7 and on the dic(7;12), indicating that the long arm of chromosome 7 had not been affected by the rearrangement, which instead involved 7p. More precisely, the rearrangement happened within 7p and 12p, leading to the fusion of the two derivative chromosomes 7 and 12, and also the loss of the more telomeric portions of 7p and 12p (Fig. 1a). To exactly map the position of the two breakpoints (BPs), we used FISH mapping with selected genomic probes for 7p and 12p (Fig. 1b). For chromosome 12, the BAC clone RP1177I22 was deleted, whereas the contiguous BAC clone RP11960L23 hybridized both the normal chromosome 12 and the dic(7;12), precisely indicating the BP position within 12p11.21, in a genomic region containing no known genes (Fig. 1b). The genes IPO8, CAPRIN2, and TSPAN11 lie in this area; given the FISH results, however, these genes do not appear to be directly affected by the rearrangement. Moreover, the predicted locus LOC390299 has been mapped to this area (Fig. 1b), but its possible role in the rearrangement could not be taken into consideration, because we were unsuccessful in our efforts to amplify even the annotated wild-type sequence from the patient cDNA and three reference controls (total human bone marrow, placental, and fetal brain cDNA) (BD Biosciences, Palo Alto, CA) (data not shown). On chromosome 7, we could demonstrate that the BAC clone RP11-875O24 is deleted on the dic(7;12), while the BAC clone RP11-1006N4 is not, showing that the chromosome 7 BP lies within 7p12.2, in a region containing the GRB10 gene (Fig. 1b). These results allowed a redefinition of the patient’s karyotype as 45,XY,dic(7;12)(p12.21; p12.2)[30]/46,XY,del(7)(p11),del(12)(p11)[5]. To further
Fig. 2. Summary of the genomic real-time quantitative polymerase chain reaction (RQ-PCR) mapping. Top: The 7p BP genomic region, containing the three GRB10 variants. Maps are derived from the Web site of the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/), with adaptations. Primer pairs used for RQ-PCR experiments are indicated; primer sequences are available upon request. Bottom: Results of RQ-PCR on the total genomic DNA from the present case and a normal control (Human Genomic DNA, Roche, Indianapolis, IN). The inferred BP position is indicated.
C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
refine the position of the chromosome 7 BP, we performed RQ-PCR mapping on the genomic DNA from the patient, with the primer pairs indicated in Figure 2. Three GRB10 isoforms have been described [8] (Fig. 2). We could position the BP within a 16-kb region between the primer pairs GRB10-5F/5R and GRB10-0F/0R, around the first exon of the isoform g, which is untranslated. Figure 2 shows a summary of the genomic Q-PCR results. Given these findings, we can conclude that the immediate effect of this rearrangement is the haploinsufficiency of GRB10 and all the other genes, located in the 50-Mb region telomeric to the 7 p BP, as well as those on 12 p in the 30Mb portion, from the BP to the telomere. These two regions contain several genes, some of which have already been implicated in leukemia, such as HOXA9 and ETV6, and it is reasonable to assume that the loss of one copy of 7p12.2~pter and 12p11.21~pter is an AML-causing event in this case.
Acknowledgments The authors would like to thank Mirko Riboni and Sara Volorio for technical assistance. This work has been supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) and Eredita` ‘‘Benilde Viotti.’’
105
References [1] Mitelman F, Johansson B, Mertens F, editors. Mitelman database of chromosome aberrations in cancer [Internet]. Updated February 2008. Available at http://cgap.nci.nih.gov/Chromosomes/Mitelman [2] Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF. GIMEMA Acute Leukemia Working Party. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005;352:254e66. [Erratum in: N Engl J Med 2005;352:740]. [3] Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplication in patients with acute myeloid leukaemia. Blood 2007;110:1262e70. [4] Johnson E, Cotter FE. Monosomy 7 and 7q associated with myeloid malignancy. Blood Rev 1997;11:46e55. [5] Lam DH, Aplan PD. NUP98 gene fusions in hematologic malignancies. Leukemia 2001;15:1689e95. [6] Bohlander SK. ETV6: a versatile player in leukemogenesis. Semin Cancer Biol 2005;15:162e74. [7] Minucci S, Monestiroli S, Giavara S, Ronzoni S, Marchesi F, Insinga A, Diverio D, Gasparini P, Capillo M, Colombo E, Matteucci C, Contegno F, Lo-Coco F, Scanziani E, Gobbi A, Pelicci PG. PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors. Blood 2002;100:2989e95. [8] Lim MA, Riedel H, Liu F. Grb10: more than a simple adaptor protein. Front Biosci 2004;9:387e403.
Short communication
A new dic(7;12)(p12.21;p12.2) chromosome aberration in a case of acute myeloid leukemia Cinzia Tapinassia,b,1, Elvira Gerbinoa,b,1, Omar Malazzia,b, Carla Micuccia,b, Patrizia Gasparinia,b,2, Maria J. Najerac, Maria J. Calasanzd, Maria D. Oderod,e, Pier Giuseppe Peliccia,b, Elena Bellonia,b,* a
Department of Molecular Medicine, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy b IFOM-IEO Campus, Milan, Italy c Department of Hematology, Hospital San Milla´n, La Rioja, Spain d Department of Genetics, University of Navarra, Pamplona, Spain e Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain Received 21 March 2008; received in revised form 21 May 2008; accepted 27 May 2008
Abstract
A new dic(7;12)(p12.21;p12.2) chromosome aberration was identified in an acute myeloid leukemia with FAB-M1 morphology and was cloned. Fluorescence in situ hybridization and genomic quantitative polymerase chain reaction mapping experiments revealed the precise localization of the breakpoints, at 7p12, just 50 to the GRB10 gene, and 12p11, within a genomic region containing no known genes. As a result, a new dicentric chromosome is created, dic(7;12), with the consequent deletion of 50 Mb at 7p, from the telomere to the GRB10 region, and of 30 Mb at 12p, from the telomere to p11. Several genes are included in the affected areas. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Chromosomal rearrangements leading to the creation of aberrant fusion proteins are a common feature in acute myeloid leukemia (AML), observed in ~45% of all reported cases [1]. Normal karyotypes are also frequently found (~50% of the cases), and it has been shown that more subtle defects, resulting in gene sequence alterations (mainly in NPM1 and FLT3), direct the leukemogenic process in such instances [2,3]. The remaining 5% of cases are characterized by rare rearrangements, mostly translocations, leading to the aberrant juxtaposition of chromosomal sequences and to either the creation of aberrant new rare fusion products or the deregulation or truncation of specific genes involved in the rearrangement [1]. Chromosome 7 can be considered a main target for chromosomal rearrangements in AML, with del(7q) and monosomy 7 representing quite common cytogenetic abnormalities in both adult and childhood AML [4]. In a few cases, the region 7p15 * Corresponding author. Tel.: þ39-02-57489396; fax: þ39-0257489851. E-mail address: [email protected] (E. Belloni). 1 These authors contributed equally to the work. 2 Present address: Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy. 0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2008.05.015
has been found to be rearranged, with involvement of the HOXA9 gene in the t(7;11)(p15;p15) resulting in the NUP98eHOXA9 fusion product [5]. Chromosome 12 also has been implicated in AML, mainly because of translocations affecting the ETV6 gene, with various partner chromosomes and as many partner genes, such as MN1, MNX1, MDS1 and EVI1, and CHIC2 [6]. Here, we describe a dic(7;12)(p12.21;p12.2) translocation, a new chromosomal aberration identified in an AML-M1 case.
2. Materials and methods 2.1. Patient A 53-year-old man was admitted to the hospital with fever, hematemesis, and melena. Laboratory findings were as follows: hemoglobin 16 g/dL, white blood cell count 94.4 109/L with 76% blasts, and platelet count 17 109/ L. Cytochemical testing revealed positivity for myeloperoxidase. Immunophenotyping performed on bone marrow cells revealed positivity for CD45 (96.4%), CD34 (62%), and CD33 (78.4%) antigens and negativity for CD14. A diagnosis of AML-M1 was made. Induction treatment was initiated, but 1 month later the patient died because of heart failure.
C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
103
Fig. 1. (a). Left: FISH experiment with probes specific for the chromosome 7 (in red) and 12 (in green) centromeres. The dic(7;12), with signals corresponding to both centromeres is clearly visible and indicated with a white asterisk. Right, top: An enlargement of the GTG banded dic(7;12). Right, bottom: Schematic representation of the dic(7;12), with retained portion of chromosome 7 represented in pink and the portion of chromosome 12 represented in green. (b) FISH mapping of the 7p and 12p breakpoints (BPs). Left: A schematic representation of chromosomes 7p and 12p. The genomic clones selected for the initial FISH experiments are indicated, with vertical ordering from telomere to centromere. Right: Physical maps of the refined 7p and 12p BP regions are shown. Maps are derived from the Web site of the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/), with adaptations. Genomic BAC clones selected for FISH experiments are indicated as horizontal bars. Genes located in the BP regions are indicated as blue horizontal bars: genomic structure, showing exons as vertical bars, is given. Blue arrows on top of genes names indicate the 50 e30 orientation of genes. The position of the two BPs is given. The black asterisk indicates the position of the predicted LOC390299 locus, as derived from the U.S. National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/). Deleted clones are indicated in red, retained clones in green.
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C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
2.2. Fluorescence in situ hybridization FISH was performed as described [7] with selected genomic clones, obtained from either the Roswell Park Cancer Institute (http://bacpac.chori.org/) or the California Institute of Technology (http://www.tree.caltech.edu/) bacterial artificial chromosome (BAC) libraries. Each clone has been tested on metaphase spreads from a normal control, to confirm the chromosomal location and check for its specificity. 2.3. Real-time quantitative polymerase chain reaction Real-time quantitative PCR (RQ-PCR) was performed using the SYBR Green technology on an ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). PCR reactions were prepared in a final volume of 25 mL (1 SYBR Green PCR master mix; 5 ng DNA). Results were normalized to a control genomic sequence, corresponding to the MYB gene (primer sequences available upon request). The GRB10/MYB ratio for the studied case was also normalized to the same ratio concomitantly measured in the total human DNA control sample (as a calibrator). 3. Results and discussion The karyotype interpretation from the conventional cytogenetic analysis was a 45,XY,e7,der(12)t(7;12)(q11;p11) [30]/46,XY,del(7)(p11),del(12)(p11)[5] karyotype. We started our analysis performing FISH with probes specific for both chromosome 7 and chromosome 12 centromeres. This analysis revealed the presence of a dicentric chromosome, dic(7;12) (Fig. 1a). Moreover, FISH experiments with the genomic BAC probe RP11-417D8 (7q11) showed
hybridization signals on the normal chromosome 7 and on the dic(7;12), indicating that the long arm of chromosome 7 had not been affected by the rearrangement, which instead involved 7p. More precisely, the rearrangement happened within 7p and 12p, leading to the fusion of the two derivative chromosomes 7 and 12, and also the loss of the more telomeric portions of 7p and 12p (Fig. 1a). To exactly map the position of the two breakpoints (BPs), we used FISH mapping with selected genomic probes for 7p and 12p (Fig. 1b). For chromosome 12, the BAC clone RP1177I22 was deleted, whereas the contiguous BAC clone RP11960L23 hybridized both the normal chromosome 12 and the dic(7;12), precisely indicating the BP position within 12p11.21, in a genomic region containing no known genes (Fig. 1b). The genes IPO8, CAPRIN2, and TSPAN11 lie in this area; given the FISH results, however, these genes do not appear to be directly affected by the rearrangement. Moreover, the predicted locus LOC390299 has been mapped to this area (Fig. 1b), but its possible role in the rearrangement could not be taken into consideration, because we were unsuccessful in our efforts to amplify even the annotated wild-type sequence from the patient cDNA and three reference controls (total human bone marrow, placental, and fetal brain cDNA) (BD Biosciences, Palo Alto, CA) (data not shown). On chromosome 7, we could demonstrate that the BAC clone RP11-875O24 is deleted on the dic(7;12), while the BAC clone RP11-1006N4 is not, showing that the chromosome 7 BP lies within 7p12.2, in a region containing the GRB10 gene (Fig. 1b). These results allowed a redefinition of the patient’s karyotype as 45,XY,dic(7;12)(p12.21; p12.2)[30]/46,XY,del(7)(p11),del(12)(p11)[5]. To further
Fig. 2. Summary of the genomic real-time quantitative polymerase chain reaction (RQ-PCR) mapping. Top: The 7p BP genomic region, containing the three GRB10 variants. Maps are derived from the Web site of the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/), with adaptations. Primer pairs used for RQ-PCR experiments are indicated; primer sequences are available upon request. Bottom: Results of RQ-PCR on the total genomic DNA from the present case and a normal control (Human Genomic DNA, Roche, Indianapolis, IN). The inferred BP position is indicated.
C. Tapinassi et al. / Cancer Genetics and Cytogenetics 185 (2008) 102e105
refine the position of the chromosome 7 BP, we performed RQ-PCR mapping on the genomic DNA from the patient, with the primer pairs indicated in Figure 2. Three GRB10 isoforms have been described [8] (Fig. 2). We could position the BP within a 16-kb region between the primer pairs GRB10-5F/5R and GRB10-0F/0R, around the first exon of the isoform g, which is untranslated. Figure 2 shows a summary of the genomic Q-PCR results. Given these findings, we can conclude that the immediate effect of this rearrangement is the haploinsufficiency of GRB10 and all the other genes, located in the 50-Mb region telomeric to the 7 p BP, as well as those on 12 p in the 30Mb portion, from the BP to the telomere. These two regions contain several genes, some of which have already been implicated in leukemia, such as HOXA9 and ETV6, and it is reasonable to assume that the loss of one copy of 7p12.2~pter and 12p11.21~pter is an AML-causing event in this case.
Acknowledgments The authors would like to thank Mirko Riboni and Sara Volorio for technical assistance. This work has been supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) and Eredita` ‘‘Benilde Viotti.’’
105
References [1] Mitelman F, Johansson B, Mertens F, editors. Mitelman database of chromosome aberrations in cancer [Internet]. Updated February 2008. Available at http://cgap.nci.nih.gov/Chromosomes/Mitelman [2] Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF. GIMEMA Acute Leukemia Working Party. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005;352:254e66. [Erratum in: N Engl J Med 2005;352:740]. [3] Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplication in patients with acute myeloid leukaemia. Blood 2007;110:1262e70. [4] Johnson E, Cotter FE. Monosomy 7 and 7q associated with myeloid malignancy. Blood Rev 1997;11:46e55. [5] Lam DH, Aplan PD. NUP98 gene fusions in hematologic malignancies. Leukemia 2001;15:1689e95. [6] Bohlander SK. ETV6: a versatile player in leukemogenesis. Semin Cancer Biol 2005;15:162e74. [7] Minucci S, Monestiroli S, Giavara S, Ronzoni S, Marchesi F, Insinga A, Diverio D, Gasparini P, Capillo M, Colombo E, Matteucci C, Contegno F, Lo-Coco F, Scanziani E, Gobbi A, Pelicci PG. PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors. Blood 2002;100:2989e95. [8] Lim MA, Riedel H, Liu F. Grb10: more than a simple adaptor protein. Front Biosci 2004;9:387e403.