A Novel 5′ ATRX Point Mutation with Splicing Consequences in Chronic Myelomonocytic Leukemia with Immune Dysregulation and Acquired Alpha Thalassemia.
Authors: Nelson Maria E. , Thurmes Paul J. , Hoyer James D. , Steensma David P. 
Published: April 2020

Abstract
BACKGROUND: Acquired hemoglobin disorders including α thalassemia (hemoglobin H (HbH) disease) can complicate neoplastic myeloid disorders, especially myelodysplastic syndrome (MDS). Acquired α thalassemia in MDS has recently been linked to mutations in the trans-acting ATRX at Xq13, a gene previously associated with inherited ATR-X (α thalassemia-mental retardation-X linked) syndrome. In the 3 instances where acquired ATRX point mutations predicted to affect splicing have been encountered, mRNA has been unavailable for assessment or unexpressed; in the 3 cases where ATRX splicing changes were described in MDS, the responsible genomic DNA mutation could not be located. Here we report a novel genomic point mutation in an ATRX splice donor motif and define the mutation’s splicing and expression consequences.
METHODS AND RESULTS: A 59 year old anemic Swiss man with chronic myelomonocytic leukemia (CMML) and multiple autoimmune complications including granulomatous uveitis, diabetes insipidus, and pleuropericarditis, developed anemia (Hb 10 g/dL) with MCH of 16.7 pg and MCV of 54 fL. We detected 50% HbH-containing cells by supravital staining and 7.5% HbH by weak cation chromatography, confirmed as HbH by isoelectric focusing. Peripheral blood granulocytes, mononuclear cells and bone marrow mononuclear cells were isolated using dual density Ficoll-Hypaque, and DNA and RNA extracted for mutation screening. ATRX coding region (35 exons) and splice site DNA from mononuclear cells was amplified and subjected to denaturing high performance liquid chromatography (DHPLC). A heteroduplex peak was encountered in the amplicon centered on exon 4. Sequencing demonstrated thymidine to cytidine point mutation with high proportional clonality in the canonical GT splice donor motif 3′ to ATRX exon 4 (IVS 4+2 T&gt;C), also detectable in patient granulocytes. Electrophoresis of raw PCR product from the 5′ end of ATRX cDNA demonstrated multiple abnormal bands. Subcloning and sequencing of cDNA demonstrated 2 splicing consequences of this mutation: exon 4 skipping (i.e., exon 3 was spliced directly to exon 5), and a second isoform with intact exons 3, 4, and 5 but retaining 43 base pairs of intron 4 (including the T&gt;C point mutation in the splice donor site), activating a previously unrecognized cryptic splice site. Since exon 4 is 53 base pairs in length, both aberrant splicing consequences result in a frameshift and generation of a premature stop codon in the 5′ end of exon 5. Exon 6 is normally alternatively spliced, and this variation was retained, so there were two subforms of each aberrant spliceoform. Expression of ATRX mRNA in the patient’s monocytes, measured by RT-PCR probing the 3′ helicase domain, was 11.1% of healthy controls; in the granulocytes, expression was 15.3% of normal.
CONCLUSION: Pre-mRNA splicing abnormalities such as that described here are increasingly recognized in association with human disease and particularly neoplasia, and may represent a potential therapeutic target. Point mutations in general are sought in myeloid disorders as they can provide proof of clonality in ambiguous cases and allow tracking of clonality with therapy and disease evolution. Germline 5′ ATRX splicing abnormalities are only rarely associated with inherited ATR-X syndrome, presumably because of the devastating consequences on gene expression and fetal development, but may be more common in acquired thalassemia. In future analyses of acquired HbH disease, when seeking ATRX mutations, splicing abnormalities should be routinely considered.