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Keywords:

  • acute myeloid leukaemia 1;
  • runt-related transcription factor 1;
  • myelodysplastic syndrome;
  • leukaemia;
  • transcription factor

Summary

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

AML1/RUNX1, which encodes a transcription factor essential for definitive haematopoiesis, is a frequent target of leukaemia-associated chromosome translocations. Point mutations of this gene have also recently been associated with leukaemia and myelodysplastic syndrome (MDS). To further define the frequency and biological characteristics of AML1 mutations, we have examined 170 cases of such diseases. Mutations within the runt-domain were identified in five cases: one of de novo acute myeloid leukaemia (AML) and four of MDS. Where multiple time point samples were available, mutations were detected in the earliest samples, which persisted throughout the disease course. Of the five mutations, one was a silent mutation, two were apparent loss-of-function mutations caused by N-terminal truncation, and two were insertions, I150ins and K168ins, which preserved most of the AML1 DNA-binding domain. Both AML1 molecules with insertion mutations were non-functional in that they were unable to rescue haematological defects in AML1-deficient mouse embryonic stem cells. In addition, activating mutations of N-ras, deletion of chromosome 12p, or inactivation of TP53 accompanied some of the AML1 mutations. Together, these observations strongly suggest that one-allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations.

Acute myeloid leukaemia 1 (AML1; also known as runt-related transcription factor 1: RUNX1) was first cloned from the chromosome 21 breakpoint for the reciprocal translocation, t(8;21)(q22;q22), which is associated with acute myeloid leukaemia (AML) of the French–American–British (FAB) classification-M2 subtype (Miyoshi et al, 1991; Erickson et al, 1992; Miyoshi et al, 1993). The gene encodes the DNA-binding subunit of the transcription factor complex, core binding factor (CBF; also known as polyomavirus enhancer binding protein 2: PEBP2) (Ogawa et al, 1993; Wang et al, 1993). Its DNA binding is mediated through the runt-domain, which comprises 128 amino acids near the N-terminus, and this domain is also responsible for heterodimer formation with CBFβ, which results in an increased DNA-binding affinity (Kagoshima et al, 1996). AML1 was later recognized as one of the most frequent targets of leukaemia-associated chromosomal translocations, including t(3;21)(q26;q22) (Mitani et al, 1994; Nucifora et al, 1994) and t(12;21)(p12;q22) (Golub et al, 1995; Romana et al, 1995). In addition, the gene that encodes its heterodimer partner, CBFβ, has also been shown to be the target of another leukaemia-associated chromosome aberration, inv(16) (Liu et al, 1993).

AML1-deficient (AML1−/−) mice die in utero due to the complete block of fetal liver haematopoiesis (Okuda et al, 1996; Wang et al, 1996), showing that the gene plays an essential role in the early development of definitive haematopoiesis. This phenotype has been replicated in vitro by means of an embryonic stem (ES) cell experimental system, in which AML1-deficient ES cells lose their ability to differentiate into definitive haematopoietic cells in culture (Okuda et al, 1996). Importantly, the AML1-deficient ES cells regain their differentiation ability once wild-type AML1 cDNA is exogenously expressed using a targeted-insertion (knock-in) approach (Okuda et al, 2000). Furthermore, this knock-in allele can even rescue the entire mouse from embryonic death if it is transmitted into the germline (Nishimura et al, 2004).

Leukaemogenic forms of AML1 caused by classic chromosome translocations, such as AML1-myeloid translocation gene on chromosome 8 (MTG8; also known as eight-twenty-one: ETO), formed by the t(8;21)(q22;q22), have been established to trans-dominantly repress normal AML1 function in the entire animal (Yergeau et al, 1997; Okuda et al, 1998) and to contribute to leukaemic transformation. In addition, point mutations of the AML1 gene locus were recently found to occasionally associate with cases of AML (Osato et al, 1999). Although the frequency of AML1 mutations in de novo AML is relatively low, they have been detected at a higher frequency in a subtype of AML with an undifferentiated phenotype, FAB-M0 (Osato et al, 1999; Preudhomme et al, 2000; Langabeer et al, 2002), and in patients with a history of exposure to radiation or cancer chemotherapy (Harada et al, 2003). Furthermore, it has been reported that an autosomal-dominant hereditary disease, familial platelet disorder, that has a propensity to develop AML (FPD/AML), is associated with genomic alterations of the AML1 gene locus which generally result in haplo-insufficient activity of AML1 (Song et al, 1999; Buijs et al, 2001; Michaud et al, 2002; Walker et al, 2002).

In contrast to AML, myelodysplastic syndrome (MDS) is a preleukaemic state that comprises a heterogeneous group of clonal stem cell disorders characterized by ineffective haematopoiesis with an overall poor prognosis due to insufficient response to any treatment protocols. Point mutations of the AML1 gene locus have also been recently reported to occur in about 5% of MDS cases (Imai et al, 2000; Harada et al, 2003). One such point mutation in a case of MDS was shown to have a dominant negative effect against wild-type AML1(Imai et al, 2000). The accumulation of combinations of gene and/or chromosomal aberrations is known to occur in MDS, and these are generally distinct from those found in de novo AML cases (reviewed in Fenaux, 2001; Hirai, 2003). However, the frequencies and types of AML1 mutations that are preferentially associated with these disorders are yet to be established.

In order to further define the frequency and biological characteristics of the mutation of AML1 in leukaemia and related disorders, we examined 170 cases in this study. We have identified four novel loss-of-function mutations of the AML1 gene locus, and we also found additional genomic alterations, thus identifying candidate genes that may co-operate with AML1 mutation in the eventual development of overt haematological malignancies.

Patients

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

A total of 170 patients treated at the Kyoto Prefectural University of Medicine and related hospitals were enrolled in the study after informed consent was obtained. They included 85 patients with MDS, 58 patients with de novo AML, 11 patients with AML with trilineage dysplasia (AML/TLD), and 16 patients with therapy-related MDS/AML (t-MDS/AML) (Table I). The MDS cases comprised 18 patients with refractory anaemia (RA), six with RA with ringed sideroblasts (RARS), eight with chronic myelomonocytic leukaemia (CMML), 18 with RA with excess of blasts (RAEB), 13 with RAEB in transformation (RAEBt), 16 with leukaemia secondary to MDS, and six with unknown characteristics. The AML cases were further classified into seven cases of M1, 13 cases of M2, 11 cases of M3, six cases of M4, eight cases of M5, two cases of M6, one case of M7, and 10 cases of unknown classification. Diagnosis was made according to FAB criteria (Bennett et al, 1982) (Table I).

Table I.  Profile of 170 patients in the study.
  1. MDS, myelodysplastic syndrome; RA, refractory anaemia; RARS, RA with ringed sideroblasts; RAEB, RA with excess of blasts; RAEBt, RAEB in transformation; CMML, chronic myelomonocytic leukaemia; AML, acute myeloid leukaemia; TLD, trilineage dysplasia.

De novo MDS
 RA18
 RARS6
 RAEB18
 RAEBt13
 CMML8
 MDS leukaemia16
 Unknown6
 Total85
De novo AML
 M17
 M213
 M311
 M46
 M58
 M62
 M71
 Unknown10
 Total58
AML/TLD
 M01
 M14
 M43
 M71
 Unknown2
 Total11
 t-MDS/AML16

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

Genomic DNA was extracted from bone marrow samples by conventional phenol-extraction methods (Sambrook & Russell, 2001). DNA mutations in the patient samples were assessed using PCR-SSCP for exons 3, 4, 5 and 6 of the AML1 gene locus, which correspond to the runt-domain of the protein product. PCR was performed with 100 ng of genomic DNA obtained from bone marrow, 1X PCR buffer with 1·5 mmol/l MgCl2 (Takara Bio Inc., Ohtsu, Shiga, Japan), a dNTP mixture (20 μmol/l dATP, dTTP, dGTP and 2 μmol/l dCTP), 100 pmol of each primer, 37 kBq [α-32P]dCTP (Amersham Biosciences Corp., Piscataway, NJ, USA) and 0·5 U of Taq DNA polymerase (Takara Bio Inc.) in a total reaction volume of 25 μl. The primer sequences used to amplify the genomic DNA fragments were as follows: 5′-ATCCCAAGCTAGGAAGACCGAC-3′/5′-TGTTTGCAGGGTCCTAACTCAATC-3′ for exon  3, 5′-CATTGCTATTCCTCTGCAACC-3′/5′-CCATGAAACGTGTTTCAAGC-3′ for exon  4, 5′-CCACCAACCTCATTCTGTTT-3′/5′-AGACATGGTCCCTGAGTATA-3′ for exon  5, and 5′-GGGGGCCCATTCTGCTGAGAGG-3′/5′-GAGCATCAAGGGGAAACCCC-3′ for exon  6. Thirty-five thermocycles were performed, each over 94°C for 45 s, 60°C (exons 4 and 5) or 55°C (exons 3 and 6) for 1 min, and 72°C for 1 min. The PCR products were then denatured by formamide and heat treatment, and electrophoresed on a 6% polyacrylamide gel containing 5% or 10% glycerol at 600 V for 6 h. The gel was then dried and exposed to Kodak X-Omat film (Eastman Kodak Company, Rochester, NY, USA) at room temperature (Fig 1).

image

Figure 1. AML1 mutations detected by PCR-SSCP and sequencing analyses in the present study. Genomic mutations of the AML1 gene locus were detected in five patients. Shifted PCR-SSCP migration bands are indicated by arrows. The time points from the initial diagnosis (left panel for each case) are shown, and each corresponding sequencing electrophoregram (right panel) is attached. In all cases, wild-type sequences (upper-right panel) were detected in the presence of the mutated sequence (lower-right panel). Diag., diagnosis; mo., months.

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PCR products showing a mobility shift by SSCP analysis were subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA, USA) and sequenced by the dideoxy termination method using an auto sequencer (Applied Biosystems, Foster City, CA, USA). For patients who showed an AML1 mutation, we tried to analyse DNA samples taken at earlier and later time points to investigate when the mutation occurred during disease progression.

Simultaneous analyses of chromosomal and other genetic alterations

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

Chromosomal analysis of the bone marrow cells was performed for all cases using the conventional short-term culture method, as described elsewhere (Misawa et al, 1986). The karyotypes, based on the trypsin–Giemsa-banded samples, were defined according to the International System for Human Cytogenetic Nomenclature (Mitelman, 1995).

NRAS gene mutation was screened by modified allele-specific restriction enzyme analysis for codons  12 and 13, as previously reported (Horiike et al, 1994). Alterations of NRAS at codon 61 and TP53 in exons  4–8 were examined by PCR-SSCP, as described elsewhere (Horiike et al, 1994; Kaneko et al, 1995). To identify FLT3 tandem duplication, PCR amplification of exon  11 of FLT3 followed by agarose gel electrophoresis was carried out, as previously described (Nakao et al, 1996; Horiike et al, 1997).

Introducing an AML1 mutation into murine ES cells by use of a knock-in approach

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

To examine the biological effect of two mutated AML1 genes, I150ins and K168ins, we introduced the mutations into ES cells using the cDNA knock-in method, as previously described (Okuda et al, 2000). First, we made an 82-base pair (bp) genomic DNA fragment containing a given mutation cleaved from subcloned PCR products of the patient samples, using restriction enzymes HindIII and ApaI, and then replaced the corresponding sequences of the human cDNA for AML1 (Fig 2A). A 153-bp DNA fragment of the mutant cDNA, which was excised by Bsu36I and BclI restriction enzymes, was used to construct the final replacement-type vectors (Fig 2B). The amino acid sequences for this part of the AML1 protein are identical in human and mouse. Mutated cDNA was inserted into exon  4 of the gene, in order to keep the reading frame open, followed by poly-adenylation (Fig 2B) signal sequences of the rabbit globin gene and a puromycin resistance cassette for positive selection. A diphtheria toxin-A suicide cassette added at the 3′-end of the vector was used for negative selection. Each targeting vector was linearized and transfected into 8 × 106 ES cells of AML1-deficient genotype, and puromycin-resistant clones were selected. ES cells that underwent homologous recombination were then identified by Southern blot analysis with sequential 5′- and 3′-outside probes, followed by an internal probe to detect the copy number of the integrated vector DNA, as previously described (Fig 2C) (Okuda et al, 2000).

image

Figure 2. Haematopoietic differentiation of the knock-in embryonic stem (ES) cells with either I150ins or K168ins mutation. (A) Mutations of the AML1 gene identified from patients were introduced into the corresponding sites of the mouse cDNA. Patched areas indicate regions from the human gene. Note that all amino acid sequences involved in the replacement are identical between the human and mouse cDNAs. (B) The AML1 loci of the ES cell line were disrupted by targeting exon  4 by insertion of a hygromycin resistance cassette [KO(hygr)] and a neomycin resistance cassette [KO(neo)] for both alleles. A knock-in vector was designed to create an artificial allele [KI(puro)] that expresses the AML1 protein with each of the mutations. Asterisk indicates mutation of the AML1 gene. Abbreviations: hygr, hygromycin  B resistance cassette; neo, neomycin resistance cassette; puro, puromycin resistance cassette; DT-A, diphtheria toxin-A suicide cassette; X, XbaI restriction sites. Arrows above the cassettes signify the direction of transcription. (C) Resultant targeted-insertion alleles were detected by sequential Southern blot analysis with either 5′- or 3′-outside probe. (D) Results of the ES cell differentiation experiments with the knock-in clones are shown. ES cells were cultured for 14 d to form embryoid bodies (EBs) under conditions for induction of haematopoietic differentiation. Representative results for the ES cell clones of AML1+/+ (WT), AML1+/−, AML1−/−, AML1−/− with a knock-in wild-type AML1 (AML1−/wt-KI), and AML1−/− with a knock-in I150ins (AML1−/150-KI) or I168ins (AML1−/168-KI) mutation are shown. Dark bars indicate the percentage of EBs with visible haematopoietic cells whereas light bars represent those without a haematopoietic element. Figures above the columns are mean and standard deviation of the EB numbers per 300 ES cells cultured. Both I150ins and K168ins mutations were non-functional for this haematopoietic rescue. (E) In situ appearance of the representative EBs developed from each of the ES cell clones (original magnification: ×20). Note that EBs derived from clones with intact AML1 function (WT, AML1+/−, and AML1−/wt-KI) develop haematopoietic components, while I150ins (AML1−/150-KI) and K168ins (AML1−/168-KI) do not develop such cells, as is the case for AML1−/−.

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The resultant ‘knock-in’ alleles were designed to express engineered 3′-sequences of wild type or each mutant AML1 cDNA downstream from exon  4 under control of the endogenous transcriptional cis-elements.

Haematopoietic differentiation of ES cell clones in culture

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

The procedure for the in vitro differentiation of embryoid bodies (EBs) was based on a previously described method (Okuda et al, 2000; Fujita et al, 2001). ES cells were cultured in semi-solid methylcellulose medium with appropriate cytokines to form EBs, which are cell aggregates comprising all three germ-layer-derived components. By visual examination with an inverted microscope, haematopoietic differentiation of EBs was scored for the presence of surrounding macrophages on day 14. The morphology of the component cells was further defined by microscopic examination of cytospin preparations with May–Grünwald–Giemsa staining.

Detection of AML1 mutations in MDS and other myeloid malignancies

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

To define the frequency of AML1 gene mutation in leukaemia and related disorders, the genomic sequences of exons 3–6, which correspond to the runt-domain of the AML1 protein, were examined using the PCR-SSCP approach for 170 disease cases (Table I). Abnormally migrating bands were detected in five of the 170 cases (2·9%), and the DNA mutations responsible for the abnormal migration were successfully identified by sequence analysis of subcloned PCR products amplified from patient samples (Table II, Fig 1).

Table II.  Clinical features of the patients with AML1 mutations.
Case no.Age/sexDiagnosisMutant/WTExonAML1 mutation
  1. MDS, myelodysplastic syndrome; AML, acute myeloid leukaemia; RA, refractory anaemia; RAEB, RA with excess of blasts; CMML, chronic myelomonocytic leukaemia.

1878/FMDS [RIGHTWARDS ARROW] AML (M2)3/853 bp insertion after codon 150 (I150ins) ATCACT[RIGHTWARDS ARROW]ATC ATC ACT
4881/MMDS (RA [RIGHTWARDS ARROW] RAEB)2/54Frame-shift due to 1 bp deletion at codon 93 GTG GCC CTA G[RIGHTWARDS ARROW]GTG CCC TAG
9450/MMDS (RA)3/154Silent mutation at codon 122 GCA[RIGHTWARDS ARROW]GCG (A122syn)
12363/MMDS (CMML)5/554 bp insertion at codon 168 (K168ins) ATC ACA[RIGHTWARDS ARROW]AAA AAT CAC
14859/MAML (M2)3/2419 bp* duplication after colon 120 *GGCTGAGCTGAGAAATGCT

Four of the five mutations occurred in de novo MDS cases, while only one was detected in a case of de novo AML. No mutations of the AML1 gene were detected in the t-MDS/AML or AML/TLD cases. AML1 mutations were identified in four of 85 (4·7%) cases of de novo MDS at a frequency similar to those described in previous reports (2·7–5·4%) (Imai et al, 2000; Harada et al, 2003a). In contrast, the frequency of AML1 mutations in the AML cases was only 1·7% (1/58), somewhat lower than those reported in previous studies (Osato et al, 1999; Preudhomme et al, 2000).

Of the five AML1 mutations, one was a silent mutation that resulted in no change in the encoded amino acid sequence (Table II, Fig 1, case 94), and another was a single-nucleotide deletion (case 48). The remaining three mutations were insertions (cases 18, 123 and 148). In all five cases, the mutated bands were detected with the co-presence of the germline band at levels close to the predicted proportion, based on the abnormal cell population, indicating that the genetic involvement of the AML1 gene locus in these cases was heterozygous (Fig 1).

Predicted influence of mutations on AML1 function

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

The silent mutation was found in a case of MDS of the RA subtype (case 94), and this A-to-G substitution (A122syn) may possibly be a polymorphism. The single-nucleotide deletion found in a case of MDS-RAEB secondary to RA (case 48) led to a frame shift at codon 93 (V93del) and consequent termination at codon 95 near the N-terminus. The predicted molecule that would be produced from this mutated allele would, therefore, preserve no functional subdomain. Similarly, a tandem duplication of 19 bp found in a case of AML-M2 (case 148) led to a frame shift at codon 120 (A120ins), and this mutation disrupts the runt-domain, which is responsible for DNA-binding. Thus, these two mutations (cases 48 and 148) were considered to be of an apparent loss-of-function type.

In contrast, the remaining two cases for which mutations were found had insertions near to the C-terminal region of the runt-domain. In a case of AML-M2 secondary to MDS-RAEB (case 19), a 3-bp insertion occurred at codon 150 (I150ins). This resulted in an extra isoleucine residue, but otherwise the open reading frame was maintained through to the original C-terminus. In a case of CMML (case 123), a 4-bp insertion occurred at codon 168 (K168ins). This introduced a frame shift followed by an artificial stop codon, and generated a truncated protein, but one that still retained most of the runt-domain. Thus, the structures of the two mutated proteins caused by the insertion mutations were not by themselves predictive of the functional consequences. To our knowledge, none of the five mutations above have been described elsewhere.

AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

Haematological malignancies are recognized to be generated through multi-step processes that include accumulation of multiple genetic alterations, as observed for solid tumours (Fearon & Vogelstein, 1990; Kinzler & Vogelstein, 1996). Some mutations, such as the TP53 mutation in MDS, are thought to be early events in overt diseases (Kaneko et al, 1995; Misawa & Horiike, 1996), and others, such as NRAS gene activation in MDS, are recognized as late events associated with disease progression (Horiike et al, 1994). Samples from multiple time points were available for two cases with a mutated AML1 allele: one case of MDS-RAEB (case 18) and one of MDS-RA (case 48). In both cases, AML1 mutations were detected in the earliest samples, and they persisted throughout the disease course (Table III). Importantly, additional mutations occurred in case 18; NRAS gene activation and interstitial deletion of the short arm of chromosome 12 occurred when the disease progressed to overt leukaemia (FAB-M2) 5 months after the initial diagnosis. In addition, a TP53 abnormality was found in a case with an AML1 mutation (Table III, case 48). In contrast, no example was found of a partial tandem duplication of the FLT3 gene that was associated with an AML1 mutation. These findings suggest that AML1 mutations are likely to be an early event in MDS onset, and that TP53, NRAS and gene(s) localized on chromosome 12p may be candidate genomic factors that co-operate with AML1 in leukaemogenesis.

Table III.  Additional genetic alterations in the patients with AML1 mutations.
 AML1TP53N-rasFLT3Karyotype
  1. RA, refractory anaemia; RAEB, RA with excess of blasts; NE, not examined; CMML, chronic myelomonocytic leukaemia.

Case 18
 RAEB at diagnosisI150insWildWildWildNormal
 M2 after 5 monthsSameWildCodon 13 GGT[RIGHTWARDS ARROW]GATWilddel(12)(p11p13)
 Died after 13 months
Case 48
 RA at diagnosisV93delCodon 205 TAT[RIGHTWARDS ARROW]TGTWildWildder(14)t(1;14) (p13;p12)
 RAEB after 4 monthsSameSameWildWildSame
 Died after 5 months
Case 94
 RA at diagnosisNENEWildNE+8,del(13)(q12q14)
 RA after 26 monthsA122synWildWildWildSame
Case 123
 CMML at diagnosisK168insWildWildWildNormal
Case 148
 M2 at diagnosisA120insNENENEt(9;22)

Both I150ins and K168ins were experimentally proven to be non-functional

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

It has already been established that ES cells of AML1-deficient genotype lose their ability to differentiate into definitive haematopoietic cells in culture, and that they regain haematopoietic potential if the cDNA of wild-type AML1 is exogenously expressed by means of a knock-in approach. Using this experimental system, the biological consequences of the I150ins (case 18) and K168ins (case 123) mutations were assessed. As outlined in the Patients and Methods and in Fig 2(A–C), each of the mutant cDNAs derived from patient samples (case 18 or 123) was introduced into an AML1 gene locus in murine ES cells of AML1-deficient genotype by use of homologous recombination. Although the knock-in ES cell clones with the mutant cDNAs were cultured under conditions optimal for haematopoietic induction, they reproducibly failed to develop into haematopoietic cells in culture (Fig 2D and E). This phenomenon was in sharp contrast to the positive control clones: knocked-in clones with wild-type AML1, which readily produced haematopoietic cells under these culture conditions (Fig 2D and E). Thus, both I150ins and K168ins were demonstrated to be biologically non-functional in terms of haematopoietic regulation.

Discussion

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

In this study, runt-domain mutations in the AML1 gene locus were screened by means of a genomic DNA-based PCR-SSCP analysis in 170 patients with leukaemia or related disorders. Mutations were identified in five cases, giving an overall frequency of 2·9% (5/170), which is comparable with those described in previous reports (Osato et al, 1999; Imai et al, 2000; Preudhomme et al, 2000; Harada et al, 2003). Only one mutation was identified in 58 cases of de novo AML. This low frequency (1·7%) may have been due to the lack of M0-subtypes in the present study, as M0 was strongly associated with AML1 mutations in previous studies (Osato et al, 1999; Preudhomme et al, 2000; Langabeer et al, 2002). In contrast, we found four AML1 mutations in 85 MDS cases, at a frequency of 4·7%, which is in accordance with previous studies (Imai et al, 2000; Harada et al, 2003).

In contrast to reported AML cases, the majority of which harbour point mutations of the AML1 gene, only insertion or deletion mutations were observed for the MDS cases in the present study. However, a very recent report has described frequent detection of insertion-type mutations in the C-terminal portion of the AML1 gene in MDS patients (Harada et al, 2004). Similarly, MDS-related mutations of another myeloid transcription factor, CCAAT/enhancer binding protein alpha, are known to be often caused by insertion-type mutations (Pabst et al, 2001; Kaeferstein et al, 2003). Hence, there may be an underlying mechanism in MDS that leads to the preferential development of insertion-type mutations in these gene loci.

All of the mutations that led to either missense or frame-shift changes in our study resulted in loss-of-function. Two of these were N-terminal truncations and the other two were demonstrated to be biologically incompetent for the in vitro haematopoietic rescue of AML1-deficient ES cells. In addition, all the mutations were heterozygous. In both cases of MDS for which we were able to obtain data at multiple time points, AML1 mutations were detected in the earliest samples and persisted throughout the course of the disease. The number of cases was too small to draw a definite conclusion, but this observation is compatible with the possibility that AML1 mutation is one of the early genetic alterations during disease onset.

Hot spots for point mutations in the AML1 gene in de novo AML and sporadic MDS cases have so far been confined to the runt-domain of the protein (Osato et al, 1999; Imai et al, 2000; Preudhomme et al, 2000; Langabeer et al, 2002; Harada et al, 2003), which comprises 128 amino acid residues near to the N-terminus and is responsible for both sequence-specific DNA-binding and heterodimerization with the beta subunit. Very recently, the three-dimensional structure of this functional domain has been determined by nuclear magnetic resonance-spectroscopy and X-ray crystallography (Nagata et al, 1999; Tang et al, 2000; Bravo et al, 2001; Nagata & Werner, 2001; Tahirov et al, 2001), and frequently mutated amino acids, such as arginines at positions 80, 174 and 177, were shown to make direct contacts between AML1 and DNA at the consensus sequence, 5′-TGT/cGGT-3′. Substitution of these residues is, therefore, expected to result in a loss of DNA-binding affinity, while allowing retention of heterodimerization with the beta subunit. Indeed, biochemical examinations of such mutated proteins have demonstrated that these molecules lose their function as a transcription factor, and that most of them also have dominant-negative effects on normal AML1 function (Osato et al, 1999; Imai et al, 2000; Michaud et al, 2002; Harada et al, 2003, 2004). It would be of value to determine whether the two mutations identified in the present study, I150ins and K168ins, which preserved most of the runt-domain but were experimentally proven to be biologically non-functional, also function as a trans-dominant repressors of wild-type AML1. On the contrary, it also needs to be experimentally confirmed if the biochemically characterized point mutation proteins behave as non-functional molecules with dominant-negative activity from a biological perspective.

The great majority of AML1 mutations that have been described and related to haematological malignancies, including the present cases, appear to result in a net loss of AML1 activity. First, an almost total absence of activity by both allele mutations are often observed in AML1-M0 cases (Osato et al, 1999; Preudhomme et al, 2000; Langabeer et al, 2002; Harada et al, 2004), whose leukaemic blasts are characterized by the absence of differentiation. Secondly, chimaeric AML1 proteins generated by classic chromosome translocations, such as AML1-MTG8 caused by t(8;21), generally result in strong dominant-negative alleles, such that genome-manipulated mice heterozygous for the knock-in allele of the chimaeric gene die in utero, thus manifesting a phenocopy of the simple knockout of this gene (Yergeau et al, 1997; Okuda et al, 1998). Finally, theoretically speaking, most point mutations lead to a haplo-insufficient status of AML1 activity, as in our present cases, with or without a dominant-negative effect (Osato et al, 1999; Imai et al, 2000; Michaud et al, 2002; Harada et al, 2003, 2004). However, it is largely unclear how the reduced AML1-activity affects the haematopoietic progenitor cells in their pathway to leukaemic transformation. One possibility is that the AML1 dosage influences the fate of the haematopoietic progenitor cells, determining whether they self-renew or commit to differentiation. To support this possibility, haematopoietic progenitors that harbour engineered AML1-MTG8 allele(s) have been shown to have an increased potential for self-renewal (Okuda et al, 1998; Rhoades et al, 2000; de Guzman et al, 2002; Mulloy et al, 2002; Schwieger et al, 2002). In addition, multipotent haematopoietic stem cells appeared at earlier stages of development in AML1-haplo-insufficient mice, compared with the time they appeared in wild-type mice (Cai et al, 2000). Thus, haematopoietic progenitor cells with increased potential for self-renewal may serve as the target population that accumulates additional genetic aberrations in the terminal development of overt diseases.

The mutation of the AML1 gene locus alone is not sufficient for the development of haematological malignancies. In some young-adult patients, leukaemic fusion proteins involving AML1 have been retrospectively identified in the Guthrie spots of infantile blood (Ford et al, 1998; Wiemels et al, 1999, 2002). Similarly, affected family members of FPD/AML pedigree with a haplo-insufficient AML1 allele generally have 20–40 years of latency prior to development of overt leukaemia (Song et al, 1999; Buijs et al, 2001; Michaud et al, 2002; Walker et al, 2002). In addition, inducible or conditional AML1-MTG8-expressing mice required additional genetic alterations induced by chemical mutagens to develop experimental myeloid leukaemia (Yuan et al, 2001; Higuchi et al, 2002). Although it has been reported that abnormality of c-Kit or FLT3 genes is occasionally associated with leukaemia cases with AML1 mutations (Beghini et al, 2000; Care et al, 2003), it essentially remains unknown what combination of gene mutations are required for the onset of overt diseases. In this respect, it seems instructive that the AML1 gene mutations in the present study were associated with alterations of TP53 or NRAS genes, and/or deletion of chromosome 12p, where the TEL and p27 genes are localized (Andreasson et al, 1998). These genes are thus included as candidates for co-operative genes that may play a role in the development of AML1-related MDS or AML. Obvious experiments would, therefore, be to construct all these mutations in the same mice by means of transgenic and/or knock-in approaches to see if these mice would then serve as animal models for the diseases.

In conclusion, the present study identified novel loss-of-function mutations of the AML1 gene locus that were associated with cases of AML and MDS. These observations strongly suggest that one-allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations. In addition, the findings on the candidate co-operative genes obtained through the present study will contribute to the further understanding of the molecular basis of the development of haematological malignancies.

Acknowledgments

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References

The authors would like to express their gratitude to Professor Takeshi Okanoue of the Department of Hepato-gastroenterology, and Hematology/Oncology, Kyoto Prefectural University of Medicine, for his critical reading of the manuscript and for his encouragement throughout the project. We also thank Toshiko Nakano, Ayumi Takahashi, Emi Saito and Kenji Iemura for their outstanding technical assistance. This work was supported by grants from the Ministry of Education, Sports, Culture, Science, and Technology, Japan, as well as by Grants-in-Aid for Cancer Research from the Ministry of Health, Welfare, and Labor, Japan. TO was also supported by grants from the Mitsubishi Pharma Research Foundation for the Advanced Medicine and from The Shimizu Foundation for the Promotion of Immunology Research.

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  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients
  5. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis
  6. Simultaneous analyses of chromosomal and other genetic alterations
  7. Introducing an AML1 mutation into murine ES cells by use of a knock-in approach
  8. Haematopoietic differentiation of ES cell clones in culture
  9. Results
  10. Detection of AML1 mutations in MDS and other myeloid malignancies
  11. Predicted influence of mutations on AML1 function
  12. AML1 mutations were found at the early stages of disease, and were often associated with other genetic or chromosomal abnormalities
  13. Both I150ins and K168ins were experimentally proven to be non-functional
  14. Discussion
  15. Acknowledgments
  16. References
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