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Whole-exome sequencing identifies a novel somatic mutation in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia

Leukemia volume 28, pages 945–948 (2014)Cite this article

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The quality of extracted genomic DNA from the fixed cells of the diagnostic sample using the Gentra Puregen kit (Qiagen, Germantown, MD, USA) was highly comparable to that of freshly thawed peripheral blood of the remission sample (A260/280=1.89 vs 1.86, leukemia and remission, respectively) and no difference in quality was seen in the further analyses, including exome capture, PCR amplification and sequencing. To delineate the breakpoints on chromosomes 1 and 22, PCR was performed as described.5 Multiplex PCR was performed with combinations of primers (as shown in Figure 1d) to narrow down the breakpoint region. When single primer pairs were then used for individual PCR reactions, no amplification occurred with OTT4 and In4.1–3, but 0.5 and 2.2 kb products were amplified with OTT4 and In4.4 and In4.5, respectively (Figure 1e) confirming that the translocation occurred between chromosome 1 and 22 with the breakpoint in intron 1 of OTT and intron 4 of MKL1.

To identify potential cooperating mutations in this patient sample, exome capture followed by deep sequencing was performed on the leukemia and remission blood samples from the patient. A total of 88 807 512 and 79 716 978 reads were obtained from the leukemic and remission samples, respectively (Supplementary Table S2). Picard software identified and removed PCR duplicates (14.0% of the leukemia sample and 9.4% of the blood sample). About 96% of the remaining unique reads from both samples mapped to only one location in the genome with ∼94% of targeted bases with >10 × coverage (Supplementary Table S2). Varscan identified 97 926 single nucleotide variations (SNVs) with a minimum variant frequency in the sample of >0.02 and minimum average quality score of >20. Of these variants, 22 326 were called somatic variations, which were filtered to require <0.2 P-value so that the SNV was different in leukemia vs remission, leaving 4580. Of the 4580 SNV, 2123 were in genic regions and 95.3% were classified as coding variants, with 75.2% being non-synonymous. SIFT (sorting intolerant from tolerant) predicted 36.0% and 62.9% of the coding variants as damaging and tolerated variations, respectively (Supplementary Table 3). Of 1956 coding variants, 1876 were novel SNPs based on dbSNP. Of the 1956, 377 SNVs remained after further filtering to require <0.05 P-value and damaging/tolerated SIFT predictions, and to exclude known dbSNPs. To overcome the low blast purity (15% of cells), we used a less stringent filter to maximize the chances of detecting variations within the leukemic cells, which resulted in false positive variants from the initial analysis. Therefore, it was necessary to carefully inspect the reads manually using the integrated genomics viewer. After manual inspection of the 377 SNVs for base quality displayed in the integrated genomics viewer (IGV v2.1,10), we found 12 variants to be likely candidates and performed further validation for these 12 variants (Table 1). Neither known AML-associated mutations nor mutations in tumor suppressor genes were found. However, small indels would not be detectable in our data owing to the low blast percentage.

Table 1 Somatic mutations identified by exome sequencing and ddPCR
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Acknowledgements

We thank Dr Yardena Samuels (Weizmann Institute of Science, Rehovot, Israel) for providing protocols for collagen zymography. This work was supported by the Connecticut Stem Cell grant no. 10SCB03 (DSK), NIH R01HL106184 (PGG), a National Research Service Award from NIH/NCI F32CA165683 (YK), Hyundai Hope on Wheels Award (NS and TAG), the Eric Trump Foundation (TAG) and the Yale Stem Cell Center Core funded by the Connecticut Stem Cell Research Fund.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA

    Y Kim, A M Teixeira & D S Krause

  2. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Y Kim, A M Teixeira & D S Krause

  3. Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA

    V P Schulz & P G Gallagher

  4. Department of Pediatrics, Section of Hematology/Oncology, University of California, Davis, Comprehensive Cancer Center, Sacramento, CA, USA

    N Satake

  5. Departments of Oncology, St Jude Children’s Research Hospital, Memphis, TN, USA

    T A Gruber

  6. Department of Pathology, St Jude Children’s Research Hospital, Memphis, TN, USA

    T A Gruber

  7. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    A M Teixeira

  8. Division of Hematology, Department of Internal Medicine, Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    S Halene

  9. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    P G Gallagher

  10. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    D S Krause

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Kim, Y., Schulz, V., Satake, N. et al. Whole-exome sequencing identifies a novel somatic mutation in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia. Leukemia 28, 945–948 (2014). https://doi.org/10.1038/leu.2013.314

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