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The most commonly reported variant in ASXL1 (c.1934dupG;p.Gly646TrpfsX12) is not a somatic alteration

Leukemia volume 24, pages 1656–1657 (2010)Cite this article

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We read with great interest the recent reports of recurrent mutations in the putative tumor suppressor gene ASXL1 that were published in this journal, as well as others. Several groups have reported the identification of missense, nonsense and frameshift mutations in ASXL1 in a wide spectrum of myeloid malignancies, including 11% of myelodysplastic syndrome, 17% acute myelogenous leukemia, 8% of myeloproliferative neoplasm, 43% of chronic myelomonocytic leukemia and 15% of chronic myelogenous leukemia patients.1, 2, 3, 4, 5

As part of our efforts to delineate the frequency of somatic ASXL1 mutations in myeloid malignancies, we have sequenced the entire open reading frame of the ASXL1 gene in paired tumor DNA and DNA extracted from buccal tissue from individuals with a variety of myeloid malignancies and from patients without hematologic disease. The c.1934dupG; pGly646TrpfsX12 variant is readily apparent in paired tumor and normal DNA (Figure 1a). This finding in combination with the fact that this candidate frameshift alteration occurs within an 8 base-pair guanine mononucleotide repeat sequence makes this reported variant suspicious for an artifact of PCR amplification rather than a true somatic mutation. This is a well-known phenomenon that is commonly seen as an artifact after PCR amplification of a region of DNA with homopolymer runs.6, 7 Moreover, slipped-strand mispairing of the PCR polymerase resulting in this in vitro frameshift does not necessarily occur in every PCR amplification product,6, 7 which explains the variable presence of this allele in different patients as assessed by Sanger resequencing, which has limited sensitivity. To improve our ability to detect this variant, we perfomed amplification of this region of ASXL1 followed by sensitive mass spectrometry (Sequenom, San Diego, CA, USA) to distinguish between PCR products with 8 versus 9 guanine nucleotides in paired tumor and normal DNA (Figure 1b). When we performed PCR amplification followed by (Seqeunom, San Diego, CA, USA) mass spectrometry for this variant in 10 paired samples from samples with myelodysplastic syndrome, myeloproliferative neoplasm and chronic myelomonocytic leukemia, this variant was detected in tumor and normal DNA in every instance. This again strongly suggests that this alteration is not a somatic mutation. Finally, we performed Sanger sequencing of ASXL1 in granulocyte DNA extracted from 96 individuals with no evidence of any hematologic disorder. The c.1934dupG p.Gly646TrpsfsX12 variant was readily apparent in >25% of samples from patients without hematologic disease (Figure 1c). Although mutations in ASXL1 have been reported in individuals without clinical evidence of a hematologic disorder at the time of DNA acquisition, the fact that this sample is found repeatedly in paired tumor and normal DNA makes this unlikely to be a somatic mutation.8

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Authors and Affiliations

  1. Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    O Abdel-Wahab, O Kilpivaara, J Patel & R L Levine

  2. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    O Abdel-Wahab & R L Levine

  3. Research Centre, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada

    L Busque

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Correspondence to R L Levine.

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Abdel-Wahab, O., Kilpivaara, O., Patel, J. et al. The most commonly reported variant in ASXL1 (c.1934dupG;p.Gly646TrpfsX12) is not a somatic alteration. Leukemia 24, 1656–1657 (2010). https://doi.org/10.1038/leu.2010.144

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