Lab Med Qual Assur.  2022 Jun;44(2):76-81. 10.15263/jlmqa.2022.44.2.76.

Detection of ASXL1 Codon 646 Variant Using Amplicon-Based Next-Generation Sequencing

  • 1Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
  • 2allym Institute of Translational Genomics and Bioinformatics, Hallym University Medical Center, Anyang, Korea
  • 3Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea
  • 4Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea


The ASXL1 codon 646 variant is the most common ASXL1 variant that negatively impacts the prognoses of patients with myeloid malignancies, particularly those with myelodysplastic syndromes and acute myeloid leukemia. However, it has been suggested that this mutation is not somatic but rather an artifact of next-generation sequencing (NGS) owing to its location in an 8 bp guanine mononucleotide repeat. In this study, we evaluated the performance of amplicon-based NGS in discriminating the ASXL1 codon 646 variant.
Amplicon-based NGS was performed on the Myeloid DNA Reference Standard HD829 in varying reference material dilution ratios using the TruSight Myeloid panel and a MiSeqDx system.
The expected and measured variant allele frequencies (VAFs) of the ASXL1 codon 646 mutation in the reference material were 40.00% and 18.65%, respectively. The measured VAFs in reference materials serially diluted at 1:1, 1:2, 1:4, and 1:8 were 9.09%, 5.82%, 1.92%, and 2.87%, respectively (y=0.4391x+0.8642; r2 =0.9846). Most of the other variants showed VAFs comparable to expected VAFs.
The measured allele frequencies of the ASXL1 codon 646 variant in the serially diluted reference materials were approximately half their expected values, suggesting difficulties in the correct detection of the variant using amplicon-based NGS.


ASXL1; Amplicon; Variant allele frequency; Next-generation sequencing
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