A Novel Splicing Mutation in the Gene Encoding the Chromatin-Associated Factor ATRX Associated with Acquired Hemoglobin H Disease in Myelodysplastic Syndrome (ATMDS).
Authors: Steensma David P. , Allen Steven L. , Gibbons Richard J. , Fisher Chris A. , Higgs Douglas R. 
Published: October 2019

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.