Rika Kanezaki

The landscape of somatic mutations in Down syndrome–related myeloid disorders

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome–related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

Cloning and expression of human B cell-specific transcription factor BACH2 mapped to chromosome 6q15

The transcription factor Bach2, a member of the BTB-basic region leucine zipper (bZip) factor family, binds to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and the related Maf-recognition element (MARE) by forming homodimers or heterodimers with Maf-related transcription factors. Bach2 regulates transcription by binding to these elements. To understand the function in hematopoiesis, we isolated a cDNA clone for human Bach2 (BACH2) encoding a protein of 841 amino acid residues with a deduced amino acid sequence having 89.5% identity to mouse homolog. Among human hematopoietic cell lines, BACH2 is expressed abundantly only in some B-lymphocytic cell lines. RT–PCR analysis of hematopoietic cells revealed that BACH2 mRNA is expressed in primary B-cells. Enforced expression of BACH2 in a human Burkitt cell line, RAJI that does not express endogenous BACH2, resulted in marked reduction of clonogenic activity, indicating that BACH2 possesses an inhibitory effect on cell proliferation. By fluorescent in situ hybridization, the BACH2 gene was localized to chromosome 6q15. Because deletion of the long arm of chromosome 6 (6q) is one of the commonest chromosomal alterations in human B-cell lymphoma, we examined for the loss of heterozygosity (LOH) of the BACH2 gene in human B-cell non-Hodgkins lymphomas (NHL). Among 25 informative cases, five (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Functional analysis of JAK3 mutations in transient myeloproliferative disorder and acute megakaryoblastic leukaemia accompanying Down syndrome

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS‐AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS‐AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient‐derived JAK3 mutation (JAK3I87T), as well as two novel JAK3 mutations (JAK3Q501H and JAK3R657Q) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof‐of‐principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS‐AMKL patients carrying activating JAK3 mutations.

Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome.

Mutations of the GATA1 gene on chromosome X have been found in almost all cases of transient myeloproliferative disorder and acute megakaryoblastic leukemia (AMKL) accompanying Down syndrome (DS). Although most GATA1 mutations lead to the expression of GATA1s lacking the N-terminal activation domain, we recently found two novel GATA1 proteins with defects in another N-terminal region. It has been suggested that loss of the N-terminal portion of GATA1 might interfere with physiological interactions with the critical megakaryocytic transcription factor RUNX1, and this would imply that GATA1s is not able to interact properly with RUNX1. However, the interaction domain of GATA1 remains controversial. In this study, we show that GATA1 binds to RUNX1 through its zinc-finger domains, and that the C-finger is indispensable for synergy with RUNX1. All of the patient-specific GATA1 mutants interacted efficiently with RUNX1 and retained their ability to act synergistically with RUNX1 on the megakaryocytic GP1bα promoter, whereas the levels of transcriptional activities were diverse among the mutants. Thus, our data indicate that physical interaction and synergy between GATA1 and RUNX1 are retained in DS-AMKL, although it is still possible that increased RUNX1 activity plays a role in the development of leukemia in DS.

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia

Diamond‐Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond‐Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond‐Blackfan anaemia, we performed whole‐exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond‐Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond‐Blackfan anaemia. In vitro knockdown of gene expression disturbed pre‐ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond‐Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond‐Blackfan anaemia.

Identification of TRIB1 R107L gain-of-function mutation in human acute megakaryocytic leukemia

Trib1 has been identified as a myeloid oncogene in a murine leukemia model. Here we identified a TRIB1 somatic mutation in a human case of Down syndrome-related acute megakaryocytic leukemia. The mutation was observed at well-conserved arginine 107 residue in the pseudokinase domain. This R107L mutation remained in leukocytes of the remission stage in which GATA1 mutation disappeared, suggesting the TRIB1 mutation is an earlier genetic event in leukemogenesis. The bone marrow transfer experiment showed that acute myeloid leukemia development was accelerated by transducing murine bone marrow cells with the R107L mutant in which enhancement of ERK phosphorylation and C/EBPα degradation by Trib1 expression was even greater than in those expressing wild-type. These results suggest that TRIB1 may be a novel important oncogene for Down syndrome-related acute megakaryocytic leukemia.

The key role of stem cell factor/KIT signaling in the proliferation of blast cells from Down syndrome-related leukemia.

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

Naturally occurring oncogenic GATA1 mutants with internal deletions in transient abnormal myelopoiesis in Down syndrome

Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.

Three novel mutations of the fibrillin-1 gene and ten single nucleotide polymorphisms of the fibrillin-3 gene in Marfan syndrome patients

AbstractMarfan syndrome (MFS) is an autosomal dominant disorder of the extracellular matrix. Allelic variations in the gene for fibrillin-1 (FBN1) have been shown to cause MFS. To date, over 550 mutations have been identified in patients with MFS and related connective tissue diseases. However, about a half of MFS cases do not possess mutations in the FBN1 gene. These findings raise the possibility that variants located in other genes cause or modify MFS. To explore this possibility, firstly we analyzed FBN1 allelic variants in 12 Japanese patients with MFS, and secondly we analyzed fibrillin-3 gene (FBN3) in patients without FBN1 mutations using conformation sensitive gel electrophoresis (CSGE) and direct sequencing analysis. We identified three novel FBN1 mutations and ten FBN3 single nucleotide polymorphisms (SNPs). In this report, we could not detect a responsible mutation of the FBN3 gene for MFS. Although the number of the cases in this report is small, at least these results suggest that disease-causing mutations in exon regions of the FBN3 gene are very rare in MFS.