ACQUIRED Α-THALASSEMIA (HBH DISEASE) IN A PATIENT WITH MYELODYSPLASTIC
SYNDROME WITH EXCESS BLASTS - 1 (AT-MDS-EB-1) REVEALS A NOVEL SPLICE-DONOR
SITE ATRX VARIANT ON NGS
Prashant Sharma1, Nabhajit Mallik2, Manu Jamwal1, Namrata Singh2, Reena Das1, Sanjeev Chhabra3,
Jasbir Kaur Hira2 and Pankaj Malhotra4
(1)Department of Hematology, Postgraduate Institute of Medical Education and Research,
Chandigarh, India
(2)Hematology Department, PGIMER, Chandigarh, India
(3)Hematology Department, Postgraduate Institute of Medical Education and Research (PGIMER),
Chandigarh, India
(4)Adult Clinical Hematology Unit, Department of Internal Medicine, PGIMER, Chandigarh, India
Background: Acquired HbH disease is an ultra-rare cause of microcytic hypochromic anemia in the
myelodysplastic syndromes (MDS) that may be possibly underdiagnosed.
Case Report: A 60-year-old male, with preexisting coronary artery disease, developed progressive
fatigability over 6 months, requiring 2 units packed erythrocytes. Hb was 72 g/L, TLC 12.6 x109/L
and platelets 278 x109/L. Peripheral smear revealed marked anisopoikilocytosis with microcytic
hypochromic erythrocytes, elliptocytes, teardrop and pencil cells, a few fragmented cells and
polychromatophils. There was leucoerythroblastosis 04% blasts, 04% myelocytes, 02%
metamyelocytes and 42 nucleated erythrocytes per 100 leukocytes with pseudo-Pelger-Huet
neutrophils and circulating megakaryocytic fragments. Bone marrow was hypercellular with
trilineage dysplasia and 02% blasts. Dysgranulopoiesis (48%) was noted in the form of cytoplasmic
hypogranularity, irregular chromatin clumping and abnormal nuclear segmentation. 15% erythroid
precursors showed nuclear budding and membrane irregularity, while 12% ring sideroblasts were
seen on Perls’ stain. 66% megakaryocytes had hypolobate nuclei. A diagnosis of MDS with excess
blasts–1 (MDS-EB-1) was made.
To investigate the cause of microcytic hypochromic anemia with the marked erythrocyte changes,
supravital new methylene blue staining of the peripheral blood was done. Strikingly, ~20% of the
erythrocytes showed golf-ball like inclusions. Hemoglobin high-performance liquid
chromatography (Hb-HPLC) showed pre-integration peaks, corroborating HbH disease. In addition,
he had HbD-Punjab trait, a common asymptomatic hemoglobinopathy in north India. Multiplex-gap
PCR for 8 common α-thalassemia deletions and sequencing of both the α-globin genes were
normal. A careful family history also revealed that he had maintained good health all his life, and
never required transfusions before the current illness. Hence, it was concluded that the patient
had acquired HbH disease secondary to MDS-EB-1.
For targeted NGS, DNA library was prepared using HaloPlex HS Target Enrichment System (Agilent
Technologies, Santa Clara) for genomic analysis of 78 genes implicated in non-malignant
hematological disorders. Sequencing was done on a MiSeq sequencer (Illumina, San Diego). Data
analysis (SureCall 4.0 software, Agilent) revealed a hemizygous, missense/splice site novel variant
NM_000489.5: c.6848A>C, (p. Lys2283Thr) in exon 31 of ATRX with a variant allele frequency of
83.3% (30/36 reads, corroborating its acquired nature). In-silico pathogenicity prediction tools
revealed this variant to be probably pathogenic/deleterious. It was not found in ExAC or gnomAD
databases. The highly conserved position is a splicing donor site hence, the substitution possibly
leads to a splicing defect. Sanger sequencing validated this variant in the index case and 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 HbH
disease.
Conclusion: Acquired α-thalassemia has been very rarely described in myeloid malignancies,
predominantly MDS as the acquired α-thalassemia–MDS syndrome (AT-MDS) syndrome. The