Acquired α-thalassaemia is a rare disorder that can arise in patients with myelodysplastic syndrome (α-thalassaemia myelodysplastic syndrome, ATMDS).1 In contrast to cis-acting variants of the α-gene cluster in inherited thalassaemia, ATMDS is caused by somatic variants in the X-linked ATRX gene that encodes a trans-acting chromatin-associated protein.2, 3 Consistent with this, germline ATRX variants cause the inherited ATR-X syndrome characterised by severe mental retardation and mild α-thalassaemia in males.4, 5 Despite advances in understanding ATRX protein function, the exact mechanism of α-globin gene repression is unknown, and several clinical observations remain unresolved: (1) the haematologic phenotype for a given variant is more severe in ATMDS compared to ATR-X syndrome, (2) the proportion of HbH can fluctuate with time, and (3) the α-thalassaemia often resolves after leukaemic transformation. Loss of HbH after leukaemic transformation suggests that the ATRX clone has either regressed, being outgrown by the neoplastic population, or gained additional variants that act to mitigate the thalassaemia phenotype.3 To the best of our knowledge, the proportion of ATRX variants at serial time-points has only been reported in one patient and demonstrated a positive correlation between clone size and thalassaemia severity.6 In contrast, we report a case of ATMDS with leukaemic transformation in which the thalassaemia resolved and ATRX clone size increased. Additionally, we identified a novel intronic ATRX variant resulting in abnormal splicing and predicted premature translation termination. A 78-year-old male of European ancestry presented with fatigue and cytopenias. His medical history consisted of hypertension and peptic ulcer disease. The full blood examination (FBE) demonstrated a haemoglobin of 97 g/L [mean corpuscular volume (MCV) 83], a white cell count of 8.3 × 109/L, and platelets of 47 × 109/L. The blood film demonstrated normocytic red blood cells, teardrops, rare blasts, and dysplastic neutrophils. Bone marrow examination revealed a hypercellular marrow with trilineage dysplasia, a normal karyotype, and U2AF1 and ASXL1 variants, overall diagnostic of myelodysplastic syndrome with multilineage dysplasia. Management was conservative with periodic transfusion support. A bone marrow biopsy performed 12 months later demonstrated disease progression with 5% blasts. At this time, the FBE revealed a progressive microcytosis and hypochromia in the absence of iron deficiency (Figure 1). ATMDS was subsequently confirmed by the detection of 2.5% HbH on capillary electrophoresis and 30% HbH-containing cells on supravital staining (Figure 2B). Three years after the initial diagnosis, the patient presented with functional decline and peripheral blood blasts. A bone marrow biopsy demonstrated transformation to acute myeloid leukaemia (AML) with 21% blasts. Cytogenetic testing was unsuccessful with no mitoses. Repeat molecular testing detected a new JAK2 V617F variant and the previous U2AF1, ASXL1 variants. Treatment was supportive, and he passed away from neutropenic sepsis 1 month later. Notably, the MCV normalised and HbH inclusions were reduced shortly after disease transformation, which could not be explained by transfusion therapy (Figure 1). To assess ATRX, stored bone marrow DNA was sequenced using a hybridisation capture-based next-generation sequencing (NGS) panel covering approximately 300 genes mutated in haematological malignancy.7 Using this approach, we detected a variant in ATRX in intron 33 (NM_000489.3:c.7071+14C>T). This variant has not been reported in the literature or the genome aggregation database (gnomAD). The ATRX variant was present at progression and leukaemic transformation with a variant allele frequency (VAF) of 5% and 48%, respectively. Additionally, after manual inspection of alignments from diagnosis, four reads supportive of the variant were present (VAF 0.9%) suspicious for the presence of a low-burden ATRX variant at the panel's limit of detection. Functional significance was assessed using in-silico prediction with SpliceAI,8 which demonstrated a 0.99 probability of a donor splice site gain. RNA sequencing confirmed aberrant splicing due to a new GT consensus splice donor site and insertion of 12 nucleotides between exons 33 and 34 (Figure 2A). This insertion contains a premature stop codon that would be predicted to truncate the entire C-terminal amino acid sequence, resulting in loss of the highly conserved P- and Q-box domains. Interestingly, RNA sequencing at the point of leukaemic transformation demonstrated the presence of aberrant transcripts containing the 12bp insertion indicating at least partial escape from nonsense-mediated RNA decay. The amount of HbH has been shown to correlate with the size of the ATRX clone.6 In the Oxford ATMDS Registry, one patient demonstrated an ATRX clone size of 35% and 1.5% at two different time-points, corresponding to 25% and <1% HbH-containing cells by supravital staining respectively. In two other patients, ATRX clone size was 10% and 30%, corresponding to 10% and 40% HbH-containing cells respectively.6 Interestingly, the proportion of HbH can disappear entirely, especially during transformation to AML. In a registry of 54 patients with ATMDS, leukaemic transformation occurred in 17 patients with all but one demonstrating resolution of HbH.3 In our case, leukaemic transformation was associated with an increase in ATRX clone size from 5% to 48% and a decrease in HbH-containing cells from 30% to 5%. This suggests that clonal evolution, rather than regression of the ATRX clone, may act to mitigate the thalassaemia phenotype. Acquired ATRX variants are not always identified in ATMDS. In a recent literature review, 34% of patients with acquired α-thalassaemia had no variants identified.9 Similarly, ATRX variants were not identified in five of the 20 ATMDS registry patients with available genomic DNA.3 These unexplained cases may be due to detection methods that are insensitive to small ATRX clones or variants that occur outside the coding region. In our case, ATMDS was caused by an intronic ATRX variant that would have been missed in many of the published case series and reports. In the largest case series to date, ATRX variants were detected using polymerase chain reaction (PCR) primers designed to cover the 5′-untranslated region (UTR), the protein-coding region, and canonical splice sites.6 Intronic variants occurring between the canonical donor and acceptor sites may not have been detected by this approach. Similarly, individual case reports have focused on coding regions and canonical splice sites.10-13 Our case highlights the need to cover intronic regions in assessing for ATRX variants. While splicing variants account for approximately 15% of ATRX variants, this number is likely to be higher with coverage of intronic regions. Additionally, recognising the genomic spectrum of ATRX variants may help identify the molecular defect in unresolved ATMDS cases. In conclusion, we report a case of ATMDS with thalassaemic resolution and increased ATRX clone size at leukaemic transformation. Additionally, we identified a novel ATRX intronic variant with splicing consequences resulting in the introduction of a premature stop codon. These variants should be considered in the molecular evaluation of patients with suspected ATMDS. Phillip C. Nguyen designed the study, analysed data, and wrote the paper. David A. Westerman, Ing Soo Tiong and Piers Blombery acquired data and analysed data. All authors approved the final manuscript. The authors would like to gratefully acknowledge funding received by the Wilson Centre for Blood Cancer Genomics and the Snowdome foundation. The authors have no conflicts of interest to declare. The patient described in this case report has died and written consent was obtained from the next of kin.

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