Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Transient erythroblastopenia due to a GATA1 variant in an infant female.

Diamond-Blackfan anemia (DBA) is a congenital anemia with erythroid cell aplasia. Most of the causative genes are ribosomal proteins. GATA1, a hematopoietic master transcription factor required for erythropoiesis, also causes DBA. GATA1 is located on Xp11.23; therefore, DBA develops only in males in an X-linked inheritance pattern. Here, we report a case of transient erythroblastopenia and moderate anemia in a female newborn infant with a de novo GATA1 variant. In this patient, increased methylation of the GATA1 wild-type allele was observed in erythroid cells. Skewed lyonization of GATA1 may cause mild transient erythroblastopenia in a female patient.

A Case of Congenital Leukemia With MYB-GATA1 Fusion Gene in a Female Patient.

We report a female newborn with acute myelogenous leukemia (AML) associated with a MYB-GATA1 fusion gene. Morphologic findings of myeloid lineage were obtained using light microscopy. Cytogenetic analysis of peripheral blood showed a complex karyotype: 46,X,-X,add(3)(q21),der(6)add(6)(q21)del(6)(q?), +mar1[5]/46,XX[15]. Targeted RNA sequencing revealed a MYB-GATA1 fusion gene. Reduced-dose AML-type chemotherapy resulted in remission and survival for >3 years without relapse. The present case demonstrated the feasibility of carrying out targeted RNA sequencing for identifying MYB-GATA1 and supports the notion that neonatal AML with MYB-GATA1 with reduced chemotherapy may show better prognosis than other highly toxic therapies.

De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

Association of Multiple Gene Polymorphisms Including Homozygous NUDT15 R139C With Thiopurine Intolerance During the Treatment of Acute Lymphoblastic Leukemia.

Although thiopurine is a crucial drug for treating acute lymphoblastic leukemia, individual variations in intolerance are observed due to gene polymorphisms. A 3-year-old boy with B-cell precursor acute lymphoblastic leukemia who was administered thiopurine developed mucositis, sepsis, and hemophagocytic lymphohistiocytosis due to prolonged hematologic toxicity, chronic disseminated candidiasis, and infective endocarditis that triggered multiple brain infarctions. The patient was found to harbor 3 gene polymorphisms associated with thiopurine intolerance including homozygous NUDT15 R139C, heterozygous ITPA C94A, and homozygous MTHFR C677T and heterozygous RFC1 G80A. Thus, the combined effect of intolerance via multiple gene polymorphisms should be considered in case of unexpected adverse reactions.

Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia.

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in RPS19.

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.

Highly sensitive detection of GATA1 mutations in patients with myeloid leukemia associated with Down syndrome by combining Sanger and targeted next generation sequencing.

Myeloid leukemia associated with Down syndrome (ML-DS) is characterized by a predominance of acute megakaryoblastic leukemia, the presence of GATA1 mutations and a favorable outcome. Because DS children can also develop conventional acute myeloid leukemia with unfavorable outcome, detection of GATA1 mutations is important for diagnosis of ML-DS. However, myelofibrosis and the significant frequency of dry taps have hampered practical screening of GATA1 mutations using bone marrow (BM) samples. In response to those problems, 82 patients were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D11 study. GATA1 mutations were analyzed by Sanger sequencing (SS) using genomic DNA (gDNA) from BM and cDNA from peripheral blood (PB) followed by targeted next-generation sequencing (NGS) using pooled diagnostic samples. BM and PB samples were obtained from 71 (87%) and 82 (100%) patients, respectively. GATA1 mutations were detected in 46 (56%) and 58 (71%) patients by SS using BM gDNA and PB cDNA, respectively. Collectively, GATA1 mutations were identified in 73/82 (89%) patients by SS. Targeted NGS detected GATA1 mutations in 74/82 (90%) patients. Finally, combining the results of SS with those of targeted NGS, GATA1 mutations were identified in 80/82 (98%) patients. These results indicate that SS using BM gDNA and PB cDNA is a rapid and useful method for screening for GATA1 mutations in ML-DS patients. Thus, a combination of SS and targeted NGS is a sensitive and useful method to evaluate the actual incidence and clinical significance of GATA1 mutations in ML-DS patients.

[New insights into inherited bone marrow failure syndrome].

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by bone marrow failure, congenital anomalies, and increased risk of malignant disease. Next generation sequencing methods have greatly facilitated the discovery of genetic etiology in IBMFS. Recently, de novo mutations activating TP53 were detected in patients with BMFS, mimicking Diamond-Blackfan anemia (DBA), using whole exome sequencing, and these patients were recognized as having a novel disorder. This discovery provides important insights into the previously postulated connection between p53 activation and IBMFS. Furthermore, a novel IBMFS, aldehyde degradation deficiency syndrome, was found in patients with aplastic anemia resembling Fanconi anemia (FA). This disorder is caused by combined inactivating mutations in ADH5 and ALDH2 coding formaldehyde-detoxifying enzymes. In this review, we highlight recent studies on DBA, FA, and their related diseases in Japan.

Clinical, cytogenetic, and molecular analyses of 17 neonates with transient abnormal myelopoiesis and nonconstitutional trisomy 21.

BACKGROUND: Transient abnormal myelopoiesis (TAM) is a unique myeloproliferative disorder that occurs in neonates with constitutional trisomy 21/Down syndrome (DS). Although TAM also develops in neonates without constitutional trisomy 21, the clinical, cytogenetic, and molecular characteristics of those patients are not fully understood. PROCEDURE: We retrospectively evaluated the clinical and cytogenetic findings and GATA1 mutation status of 17 neonates with TAM and nonconstitutional trisomy 21 tested for GATA1 mutations at our institute, and compared the findings with those of 64 neonates with TAM and constitutional trisomy 21/DS. RESULTS: DS clinical features were observed in five of the 17 (29%) patients. In all patients, both trisomy 21 and GATA1 mutations were detected in diagnostic samples. Over a median follow-up of 33 (range, 0-139) months, early death (< 6 months of age) occurred in four patients (24%). Overall and event-free survivals were not significantly different between the patients with TAM and nonconstitutional trisomy 21 and those with TAM and constitutional trisomy 21/DS (five-year overall survival: 76% ± 10% vs 53% ± 13%, P = 0.40; five-year event-free survival: 55% ± 13% vs 48% ± 12%, P = 0.90). The five-year cumulative incidence of progression to myeloid leukemia of DS was also similar between the groups (21% vs 24%, P = 0.80). CONCLUSIONS: Patients with TAM and nonconstitutional trisomy 21 exhibited similar biology and outcomes to those with TAM and constitutional trisomy 21/DS. The possibility of TAM should be considered even in phenotypically normal neonates with TAM symptoms, for appropriate management.

Integrated genetic and epigenetic analysis revealed heterogeneity of acute lymphoblastic leukemia in Down syndrome.

Children with Down syndrome (DS) are at a 20-fold increased risk for acute lymphoblastic leukemia (ALL). Compared to children with ALL and no DS (non-DS-ALL), those with DS and ALL (DS-ALL) harbor uncommon genetic alterations, suggesting DS-ALL could have distinct biological features. Recent studies have implicated several genes on chromosome 21 in DS-ALL, but the precise mechanisms predisposing children with DS to ALL remain unknown. Our integrated genetic/epigenetic analysis revealed that DS-ALL was highly heterogeneous with many subtypes. Although each subtype had genetic/epigenetic profiles similar to those found in non-DS-ALL, the subtype distribution differed significantly between groups. The Philadelphia chromosome-like subtype, a high-risk B-cell lineage variant relatively rare among the entire pediatric ALL population, was the most common form in DS-ALL. Hypermethylation of RUNX1 on chromosome 21 was also found in DS-ALL, but not non-DS-ALL. RUNX1 is essential for differentiation of blood cells, especially B cells; thus, hypermethylation of the RUNX1 promoter in B-cell precursors might be associated with increased incidence of B-cell precursor ALL in DS patients.

Two siblings with familial neuroblastoma with distinct clinical phenotypes harboring an ALK germline mutation.

The authors report two siblings with familial neuroblastoma with a germline R1275Q mutation of the tyrosine kinase domain of ALK. Whole exome sequencing and copy number variation assay were performed to investigate genetic alterations in the two cases. No common somatic mutations or gene polymorphisms related to the tumorigenesis of neuroblastoma were detected. A distinct pattern involving both segmental chromosomal alteration and MYCN amplification was detected. The diversity of biological behavior of familial neuroblastoma harboring a germline ALK mutation may depend on conventional prognostic factors, such as segmental chromosomal alterations and MYCN amplification, rather than additional acquired mutations.

[Diagnostic targets and exosome sequence analysis of Diamond-Blackfan anemia in Japan].

Diamond-Blackfan anemia (DBA) is a rare, inherited, congenital bone marrow failure syndrome, which typically manifests in infancy. In addition, the disease is often accompanied by normochromic macrocytic anemia and acute reticulocytopenia in the peripheral blood and by selective deficiency of erythroid precursors in the normocellular bone marrow. Approximately 50% of patients with DBA exhibit growth retardation and multiple congenital anomalies, which primarily include craniofacial dysmorphism, upper-limb abnormalities, urogenital malformations, and congenital heart defects. In addition, heterozygous mutations in genes encoding ribosomal proteins (RP) that lead to a loss of function of the genes are detected in nearly 60% of patients with DBA. As a majority of identified causative genes belong to RP genes, the impairment of ribosome biogenesis is considered accountable for the disease. This study aims to outline the molecular pathology of DBA and the causative gene RPS15A isolated using our exosome analysis.

Erythrocyte glutathione is a novel biomarker of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with mutations in ribosomal protein (RP) genes. Elevated activity of erythrocyte adenosine deaminase (eADA) has been utilized as a biomarker of DBA. We examined erythrocyte reduced glutathione (GSH) as well as eADA in 22 patients in 18 DBA families, in whom RP gene mutations had been identified. Simultaneous evaluation of both eADA and GSH demonstrated that all examined DBA patients showed elevated values of either eADA or GSH, whereas presence of both eADA and GSH elevation was able to distinguish DBA patients from 34 normal controls and 14 unaffected members of the DBA families. Furthermore, a support vector machines analysis using both eADA and GSH levels yielded a formula to differentiate DBA from both normal controls and non-DBA family members. To confirm the usefulness of the formula, we analyzed additional 7 patients diagnosed by the clinical criteria. Although eADA showed within normal values in 3 patients, all of these patients were diagnosed as 'DBA' by use of the formula. Because extensive analysis of the RP genes failed to detect no causative mutation in approximately 40% of clinically diagnosed DBA patients, GSH may be useful an additional biomarker for diagnosis of DBA.

Preserved High Probability of Overall Survival with Significant Reduction of Chemotherapy for Myeloid Leukemia in Down Syndrome: A Nationwide Prospective Study in Japan.

BACKGROUND: On the basis of results of previous Japanese trials for myeloid leukemia in Down syndrome (ML-DS), the efficacy of risk-oriented therapy was evaluated in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D05 study. PROCEDURE: All patients received induction chemotherapy that consisted of pirarubicin, intermediate-dose cytarabine, and etoposide. Patients who achieved complete remission (CR) after initial induction therapy were stratified to the standard risk (SR) group and received four courses of reduced-dose intensification therapy. Patients who did not achieve CR were stratified to the high risk (HR) group and received intensified therapy that consisted of continuous or high-dose cytarabine. RESULTS: A total of 72 patients were eligible and evaluated. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and two patients to HR. No therapy-related deaths were observed during intensification therapy. The 3-year event-free and overall survival rates were 83.3% ± 4.4% and 87.5% ± 3.9%, respectively. Age at diagnosis less than 2 years was a significant favorable prognostic factor for risk of relapse (P = 0.009). CONCLUSIONS: The attempt of risk-oriented prospective study for ML-DS was unsuccessful, but despite the dose reduction of chemotherapeutic agents, the overall outcome was good, and further dose reduction might be possible for specific subgroups.

Recent advances in inherited bone marrow failure syndrome research.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by bone marrow failure, congenital anomalies, and an increased risk of malignancies. Diagnosis is often difficult due to the wide variety of clinical expressions. The representative diseases are Diamond Blackfan anemia (DBA), Fanconi anemia (FA), congenital sideroblastic anemia (CSA), congenital dyserhthropoietic anemia, Shwachman Diamond syndrome, and dyskeratosis congenita. Next-generation sequencing technologies have facilitated the discovery of germline mutations that cause IBMFS. Recently, Japanese groups have identified novel causative genes for DBA, FA and congenital thrombocytopenia by applying whole exome-sequencing. In this review, we will highlight recent studies on DBA, FA and CSA in Japan, which have employed next-generation sequencing technologies to elucidate the genetic etiology of IBMFS.

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.

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.