Gibbons, Richard J.

Abstract Introduction: The core-binding factor subunit RUNX1/CBFA2/AML1 (Runt-related transcription factor 1, Core-binding factor alpha 2 subunit, Oncogene AML1) is critical for generation of hematopoietic stem cells during embryogenesis and for normal megakaryopoiesis in adults. RUNX1/CBFA2/AML1 has long been recognized as an oncogene, and is frequently involved in leukemia-associated translocations that create aberrant fusion proteins (e.g., AML1-ETO). Recently, acquired somatic point mutations in the RUNX1/CBFA2/AML1 gene have been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). These point mutations are associated with the loss of normal RUNX1 trans-activation potential and/or altered binding to the non-DNA-binding subunit of core-binding factor, CBFB. Given the importance of core-binding factor in megakaryocytic differentiation and platelet production and the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), and in light of the myelofibrotic phenotype of GATA-1low mice (GATA-1 and RUNX1 are co-expressed and co-operate in megakaryocytic differentiation), we hypothesized that RUNX1 gene mutations might be common in MMM. In addition, we wanted to know whether patients with MDS-associated acquired alpha thalassemia (i.e., ATMDS), a special MDS subgroup with a very high incidence of point mutations in the X-linked ATRX gene, have an especially high incidence of RUNX1 mutations that might account for the more severe hematopoietic defect found in these patients compared with boys with inherited ATR-X syndrome (detailed in Steensma et al, Blood2004;103:2019). Methods and Results: We analyzed samples from 26 well-characterized patients with MMM and from 52 with MDS (11 RA, 3 RARS, 8 RCMD, 3 RAEB-1, 7 RAEB-2, 2 MDS in transition to AML-M6, 14 atypical/unclassifable MDS, 4 MDS subtype unknown - including 18 with ATMDS) for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC) followed by subcloning and sequencing of samples exhibiting heteroduplex peaks. We found 5 RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 (4 patients) or a history of treated AML (1 patient), but detected no mutations in MMM patients. All patients with RUNX1 mutations progressed to overt leukemia within 1 year. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18 patients with mutations, 11.1%) when compared with MDS without thalassemia (3/34 patients, 8.8%; p=0.58). Additionally, 1 MDS patient had a clonally restricted mutation in GATA-1 (c.1066 C>T) (also screened by DHPLC), but this is not predicted to change the GATA-1 amino acid sequence and is likely a random event reflecting the genomic instability characteristic of MDS. Conclusion: RUNX1 point mutations are common in high-risk MDS, but not in MMM, and that ATMDS patients do not have a risk of RUNX1 mutations in excess of that expected for MDS in general.

Abstract Acquired hemoglobin H (HbH) disease (α thalassemia) can arise in the setting of marrow neoplasia, especially the myelodysplastic syndrome (MDS). This association has been termed α thalassemia-MDS (ATMDS, MIM #300448). Recently, we have linked ATMDS to acquired point mutations in the ATRX gene, which encodes an X-linked chromatin-associated factor whose precise normal cellular function is unknown (Steensma et al, Blood2004;103:2019, and Gibbons et al, Nature Genetics2003;34:446). We now describe a novel acquired splicing mutation in the helicase domain of the ATRX gene in a 71 year-old man of southern Italian descent. The patient, who previously had normal blood indices, presented with a 6-month history of microcytic anemia (Hb 7.7 g/dL, MCV 73.7 fL, reticulocytes 0.2%; WBC 11,800/uL with 29% circulating blasts). Peripheral smear demonstrated marked anisopoikilocytosis and the presence of poorly hemoglobinized ghost cells, while a marrow aspirate and biopsy revealed dysplastic features including atypical megakaryocytes, and approximately 20% blasts, consistent with a diagnosis of MDS in leukemic transformation. On supravital staining, approximately 30% of circulating erythrocytes contained HbH inclusions. The α/β globin transcript ratio measured by quantitative real-time PCR ranged between 0.11 and 0.39, compared with the mean ratio for 11 normals (α/β ratios in normals ranged between 0.8–1.2 fold of the mean). Despite this evidence of α thalassemia, no gross rearrangements of the α globin cluster were detected on Southern blotting or by FISH, and sequencing of the α globin genes was unremarkable. Screening of the entire coding region of the ATRX gene by denaturing high-performance liquid chromatograpy showed no abnormalities. Amplification and sequencing of cDNA made from the patient’s blood, however, revealed loss of ATRX exon 28 (in the middle of the putative helicase domain), resulting in a frame shift and generation of a premature stop codon. The causative mutation at the genomic level remains unknown, despite sequencing of the consensus splice sites framing both ATRX exon 28 and the surrounding regions. A clonally restricted mutation in a distant cryptic splice enhancer or inhibitor is suspected. Following treatment with idarubicin and Ara-C, the patient achieved a complete remission with normalization of red cell morphology and indices, and HbH was no longer detectable. This case emphasizes the need to search carefully for abnormal mRNA splicing when screening for ATRX mutations and provides further support for a link between ATRX mutations and ATMDS. Among 20 ATMDS patients analyzed molecularly, we have now characterized ATRX missense, null, and nonsense mutations in 9 patients, found a codon substitution of uncertain significance in 1 patient, and detected ATRX splicing mutations in 3 patients, including the present case. Additionally, one patient had an acquired α globin deletion limited to the neoplastic clone (Steensma et al, Blood2004; 103:1518). The molecular defect remains unknown in 6 cases. Since RNA is not available from these 6 archival cases, it is possible that they have ATRX splicing mutations as well.

AbstractAbnormalities of hemoglobin synthesis are usually inherited but may also arise as a secondary manifestation of another disease, most commonly hematologic neoplasia. Acquired hemoglobin disorders can be seen in any population and are not restricted to areas of the world with high incidences of inherited hemoglobinopathies. In fact, the acquired hemoglobinopathies may be more readily recognized where inherited hemoglobin abnormalities are rare and less likely to cause diagnostic confusion. Acquired α-thalassemia is the best characterized of the acquired red blood cell disorders in patients with hematologic malignancy, and it is almost always associated with a myelodysplastic syndrome (MDS). At least 2 molecular mechanisms for acquired α-thalassemia are now recognized: acquired deletion of the α-globin gene cluster limited to the neoplastic clone and, more commonly, inactivating somatic mutations of the trans-acting chromatin-associated factor ATRX, which cause dramatic down-regulation of α-globin gene expression. Here we review the clinical, hematologic, and molecular genetic features of α-thalassemia arising in a clonal myeloid disorder, and we discuss howATRX might affect gene expression in normal and abnormal hematopoiesis through epigenetic mechanisms.

Abstract Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with α thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.

Abstract Rarely, myelodysplastic syndrome (MDS) is complicated by an acquired form of α-thalassemia (α-thalassemia in myelodysplastic syndrome [ATMDS]) characterized by hypochromic, microcytic, anisopoikilocytic red blood cells with hemoglobin H (HbH) inclusions. Acquired mutations in ATRX, a chromatin remodeling gene, have recently been found in 12 patients with typical features of ATMDS, though they have not been detected in MDS patients with similar red blood cell findings but little HbH. The α-globin genes themselves have appeared normal in all ATMDS patients studied to date. Here we characterize the molecular defect in a unique MDS patient with rare HbH inclusions in which an abnormal clone lost a greater than 1.9-Mb segment of the telomeric region of the short arm of one allele of chromosome 16, including both α-globin genes. Red blood cell changes associated with this acquired somatic genotype (––/αα) are surprisingly severe, demonstrating that a minor globin chain imbalance may be unexpectedly deleterious during the abnormal erythropoiesis that occurs in the context of MDS.