Recently, Mueller et al1 found PU.1 mutations in 7% of the 126 cases of acute myeloid leukemia (AML) they analyzed. DNA binding and transactivation of the M-CSF receptor promoter, a direct PU.1 target gene, were deficient in the 7 mutants that affected the DNA-binding domain of the PU.1. Those mutations also decreased the ability of PU.1 to synergize with proteins such as AML1 or c-Jun, possibly contributing to the differentiation block found in AML. We looked for study analysis of PU.1 gene mutations in 77 cases of primary AML (excluding therapy-related AML and AML following MDS): 30 M0, 10 M1, 11 M2, 11 M4, 13 M5, 1 M6 AML, and 1 M7 AML, according to FAB classification.2 In all blood and marrow samples analyzed, more than 80% of blasts were present. Detection of PU.1 gene mutations was made on DNA by single-strand conformation polymorphism (SSCP) analysis3 and direct sequencing of the 5 exons corresponding to the entire coding region of the PU.1 gene. The size of the polymerase chain reaction (PCR) products was 190 bp to 440 bp (Table1). Except for a polymorphism in intron 3 (G>A transversion), no abnormal SSCP profile and no variation of sequence of the PU.1 gene was observed in the 77 de novo AML cases studied.

Table 1.

Primers used for SSCP and direct sequencing analysis

Forward primers*Reverse primers*Hybridization temperature, °CSize of PCR product, bpRestriction enzyme used (sizes of products after digestion, bp)
(EX1S) 5′-GTTGGGCTGGTGGAGGAGT-3′ (EX1AS) 5′-GACACCCCAAGGGGACTATC-3′ 64 310 N/A  
(EX2S) 5′-CTCTCTCCAGACCCCAGGA-3′  (EX2AS) 5′-CCTGACACCTCCTCTCTCA-3′ 64 190 N/A  
(EX3S) 5′-ACAGGCCTGGCAGTCTC-3′ (EX3AS) 5′-CTGAGCTCACCCCATACTG-3′ 64 270 N/A  
(EX4S) 5′-TGGCTGCTGGGTCAGTT-3′ (EX4AS) 5′-CTTCCTCCAGTCTCCTGTGC-3′ 60 400 RsaI (180 and 220)  
(EX5S) 5′-GGCGAGGGCTTAATGCTAT-3′ (EX5AS) 5′-GGGGCGGCTCCCATGTGG-3′ 60 440 AluI (140 and 300) 
Forward primers*Reverse primers*Hybridization temperature, °CSize of PCR product, bpRestriction enzyme used (sizes of products after digestion, bp)
(EX1S) 5′-GTTGGGCTGGTGGAGGAGT-3′ (EX1AS) 5′-GACACCCCAAGGGGACTATC-3′ 64 310 N/A  
(EX2S) 5′-CTCTCTCCAGACCCCAGGA-3′  (EX2AS) 5′-CCTGACACCTCCTCTCTCA-3′ 64 190 N/A  
(EX3S) 5′-ACAGGCCTGGCAGTCTC-3′ (EX3AS) 5′-CTGAGCTCACCCCATACTG-3′ 64 270 N/A  
(EX4S) 5′-TGGCTGCTGGGTCAGTT-3′ (EX4AS) 5′-CTTCCTCCAGTCTCCTGTGC-3′ 60 400 RsaI (180 and 220)  
(EX5S) 5′-GGCGAGGGCTTAATGCTAT-3′ (EX5AS) 5′-GGGGCGGCTCCCATGTGG-3′ 60 440 AluI (140 and 300) 
*

EX indicates exon, followed by exon number; S, sense; and AS, antisense.

Our results are in contrast to those of Mueller et al1 but in agreement with results of Vegesna et al,4 who found no mutation in 60 AML patients tested. There are no clear explanations for these discrepancies. Vegesna et al4 only used SSCP analysis for screening of mutations, but we also used, like Mueller et al, direct sequencing. We and Vegesna et al4 used intronic primers and performed analyses on DNA, whereas Mueller et al1 performed direct sequencing on cDNA of the PU.1 gene. As 2 of the 9 mutations reported by Mueller et al1corresponded to large deletions, probably not detectable by techniques using DNA, such mutations could have been overlooked in our study.

Mutations reported by Mueller et al1 predominantly occurred in undifferentiated AML (M0 AML) or in AML of the monocytic lineage (M4 and M5 AML), but we included a large proportion of those cases in our study and none of them were mutated.

Other hypotheses for those discrepancies include ethnic differences, as patients studied by Mueller et al1 were, to a large extent, part of the Japanese cohort studied by Osato et al5 for AML1 mutations. Finally, we and Vegesna et al4 included only cases of primary AML (excluding therapy-related MDS and AML following MDS), and 3 of the 9 mutated patients reported by Mueller et al1corresponded to AML after MDS. Vegesna et al4 found no PU.1 mutations in 60 MDS patients, but PU.1 mutations could possibly occur during the progression of MDS to AML in some patients.

In conclusion, we were unable to identify a significant number of PU.1 mutations in our patient population with AML, although their occurrence in some patients during the progression of MDS to AML cannot be excluded.

Supported by the Centre Hospitalier Universitaire of Lille (PHRC 1997), the Ligue Nationale contre le cancer (Comitédu Nord et de l'Aisne), and the fondation de France (ComitéLeucémie)

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