A novel ATRX splice variant causing acquired HbH disease in myelodysplastic syndrome with excess blasts-1

Abstract

A 60-year-old male with myelodysplastic syndrome with excess blasts-1 had unexplained microcytic hypochromic anemia. The cause of his anemia was revealed on supravital staining, hemoglobin studies and next-generation sequencing to be a novel hemizygous potentially pathogenic missense/splice site variant NM_000489.5:c.6848A>C, (p.Lys2283Thr) in exon 31 of the ATRX gene.

Introduction

A majority of patients with the myelodysplastic syndromes (MDS) present with fatigue and weakness associated with chronic anemia, which is predominantly macrocytic or normocytic [1]. Microcytic anemia, especially in the absence of iron deficiency, is rare in MDS. Acquired HbH disease is a rare, but possibly underdiagnosed, cause of microcytic hypochromic anemia in MDS and rarely, other myeloid neoplasms [2], [3], [4].

A 60-year-old north-Indian gentleman, a known case of coronary artery disease, presented with progressive fatigability for 6 months, requiring 2 units of packed erythrocytes. Investigations revealed Hb 72 g/L, total leucocyte count 12.6 × 10⁹/L and platelet count 278 × 10⁹/L. His peripheral smear (Fig. 1) revealed marked anisopoikilocytosis with markedly microcytic hypochromic erythrocytes, elliptocytes, teardrop and pencil cells, a few fragmented cells and polychromatophils. There was leucoerythroblastosis with 04% blasts, 04% myelocytes, 02% metamyelocytes and 42 nucleated red cells per 100 leukocytes. Pseudo-Pelger-Huet neutrophils and circulating megakaryocytic fragments were noted.

In view of the circulating blasts, a bone marrow evaluation was done. The marrow was hypercellular with trilineage dysplasia (Fig. 2): 48% dysgranulopoiesis in the form of cytoplasmic hypogranularity, irregular chromatin clumping and abnormal nuclear segmentation; 15% dyserythropoiesis in the form of nuclear budding and membrane irregularity with 12% ring sideroblasts on Perls’ stain; and 66% megakaryocytes showing hypolobate nuclei. Blasts were not increased (02%). Reverse transcriptase PCR was negative for t(8;21), t(15;17) and inv(16). A diagnosis of myelodysplastic syndrome with excess blasts–1 was made.

To investigate the cause of erythrocytic changes, supravital staining of the peripheral blood was done with new methylene blue dye. Strikingly, ~30% of the red cells showed golf-ball like inclusions (Fig. 3). Hemoglobin high performance liquid chromatography (Hb-HPLC) showed two pre-integration peaks, further corroborating HbH disease. In addition, HbD-Punjab trait, a common asymptomatic hemoglobinopathy in north India, was also found (Fig. 3).

Multiplex-gap PCR for eight common α-thalassemia deletions [–α^3.7 (rightward) and – α^4.2 (leftward), – –^SEA (Southeast Asian), – –^MED (Mediterranean), – –^SA (South African), – –^THAI (Thailand), – –^FIL(Filipino) and –(α)^20.5], two common α-globin triplications [ααα^{anti 3.7} and ααα^{anti 4.2} triplications] and sequencing of both the α-globin genes (HBA1 and HBA2) were all normal. A careful family history revealed that he had maintained good health all his life, never suffered jaundice, and never required transfusions before the current illness. Hence, it was concluded that the patient had an acquired HbH disease secondary to MDS-EB-1.

DNA library preparation was done using HaloPlex HS Target Enrichment System (Agilent Technologies, Santa Clara, USA) for genomic analysis of 53 genes implicated in non-malignant hematological disorders. Sequencing was done on a MiSeq sequencer (Illumina, San Diego, USA). Data analysis (SureCall 4.0 software, Agilent Technologies, Santa Clara, USA) revealed a hemizygous potentially pathogenic missense/splice site novel variant NM_000489.5:c.6848A>C, (p.Lys2283Thr) in exon 31 of the ATRX gene with a variant allele frequency of 83.3% (30/36 reads), corroborating its acquired nature (Fig. 4).

In-silico pathogenicity prediction tools revealed this variant to be probably pathogenic/deleterious. It was not found in ExAC and gnomAD databases. The highly conserved position is a splicing donor site hence, the substitution possibly leads to a splicing defect (Table 1). Sanger sequencing validated this variant in the index case. It was absent in his asymptomatic daughter, corroborating the somatic/sporadic event. We classified this variant to be likely pathogenic based on the ACMG guidelines and the cause of his acquired α-thalassemia (Fig. 5).

The patient's Hb remained low despite erythropoietin and G-CSF therapy. He subsequently developed pancytopenia and succumbed to the consequences of marrow failure one year after diagnosis.