Whole-Exome Resequencing Identifies Somatic Mutations Of BCOR and BCORL1 Transcriptional Corepressor Genes and Major Cohesin Complex Component Genes In Pediatric Acute Myeloid Leukemia
Authors: Shiba Norio , Yoshida Kenichi , Nagata Yasunobu , Kon Ayana , Okuno Yusuke , Shiraishi Yuichi , Kato Motohiro , Park Myoung-ja , Ohki Kentaro , Takita Junko , Kanazawa Takashi , Kudo Kazuko , Ito Etsuro , Sanada Masashi , Tomizawa Daisuke , Tawa Akio , Adachi Souichi , Miyano Satoru , Ogawa Seishi , Hayashi Yasuhide 
Published: October 2019

Abstract

Background
Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML.

Methods
To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases.

Results and Discussion
A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%).

Conclusion
Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations.

Disclosures:
No relevant conflicts of interest to declare.