Putative RNA-splicing gene LUC7L2 on 7q34 represents a candidate gene in pathogenesis of myeloid malignancies

Acute myeloid leukemia (AML) is a myeloid malignancy that arises spontaneously or that may evolve from myelodysplastic syndrome (MDS). AML is characterized by somatic cytogenetic and molecular mutations associated with distinct clinical outcomes. In patients with normal cytogenetics, genetic techniques have been used to discover novel mutations. In order to identify new candidate mutations involved in AML disease progression and pathogenesis, we conducted whole exome sequencing (WES) on DNA from a patient with cytogenetically normal-acute myeloid leukemia (CN-AML) evolving from MDS. A patient in the seventh decade of life with CN-AML evolving from MDS was identified for exome sequencing. The patient initially presented with anemia and thrombocytopenia, and a bone marrow biopsy (BMBx) showed refractory anemia with excess blasts-1 (RAEB-1). Cytopenias worsened and a second BMBx was done 4 months after diagnosis, which also revealed RAEB-1. Six months later, cytopenias worsened and myeloblasts were detected in the peripheral blood. A third BMBx showed AML with 26% blasts. The cytogenetics were normal, and the FLT3 internal tandem duplication (ITD), tyrosine kinase domain (TKD) and characteristic NPM1 mutations were not detected. WES was performed on DNA obtained from bone marrow mononuclear cells (BMMNCs), representative of tumor sample. In addition, buccal mucosa and serially passaged bone marrow stromal cells (BMSCs), serving as germline controls, were also sequenced. Details of WES, data analysis and primers used are presented in the Supplementary Methods section. We identified 16 single-nucleotide variations (SNVs) in the tumor sample, which were absent in the buccal and stroma DNA, and which were also absent in the dbSNP database. Fourteen SNVs were validated by Sanger sequencing (Table 1) while two were false positives. No insertions or deletions (indels) were identified in this sample. SNVs in two genes, IDH2 and RUNX1, had been previously identified in patients with AML. The stroma did not contain unique SNVs or indels when compared with the buccal tissue. We examined two prior bone marrow samples from the same patient (collected at initial diagnosis and 4 months after initial diagnosis, both showing RAEB-1) using Sanger sequencing for the presence of the 14 mutations found in the secondary AML. The initial diagnostic sample did not carry the IDH2 and LUC7L2 mutations (Table 1). The second sample, which was obtained 6 months prior to the diagnosis of AML and which contained 5% blasts (RAEB-1), exhibited all 14 SNVs. Homogeneous mass extension (hME, Sequenom) genotyping was performed on a panel of patients to rapidly screen for the presence of the novel SNVs. (PCPGM, Partners Center for Personalized Genetic Medicine; details in Supplementary Methods). IDH2 and RUNX1 were excluded from this analysis. HMe confirmed the presence of the remaining 11 mutations in all three of the index patient’s samples with the exception that the LUC7L2 nonsense mutation was absent in the initial sample, as previously confirmed by Sanger sequencing, and indicating that this mutation was acquired coincident with disease progression. Allelic frequencies of the SNVs found in the index AML patient ranged from 45 to 51% (as assessed by Sequenom), and were