Isolated Granulocytopenia in Patients with Unclassifiable MDS May Be Related to a Mutation of PIG-A Gene Leading to GPI-APs Deficiency Expressed Predominantly on Granulocytes.
Authors: Cermak Jaroslav , Pekova Sona , Marinov Iuri , Mikulenkova Dana 
Published: October 2019

Abstract
An acquired somatic mutation of PIG-A gene leads to a deficiency in glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) in affected cells. A typical clinical manifestation of the GPI-APs deficiency is paroxysmal nocturnal hemoglobinuria (PNH) with intravascular hemolysis, various degree of cytopenia reflecting marrow failure and trombophilia. Besides so-called „classic“ PNH, clinical and laboratory features of PNH may also be present in some patients with aplastic anemia (AA) or myelodysplasia (MDS). Moreover, a small subset of AA or MDS patients may exhibit in peripheral blood small populations of GPI-APs deficient red blood cells (RBC) and granulocytes, not accompanied by clinical and laboratory signs of hemolysis. This subset of patients with a small PNH clone may respond to immunosuppressive therapy. Herein, we report on three patients who were referred to our hospital with the diagnosis of unclassified MDS (RA unclassifiable according to the WHO classification). In all patients, bone marrow aspiration and biopsy revealed normocellular marrow with only minimal to mild dysplasia with no excess of blasts and no karyotype abnormalities. The main laboratory feature was moderate to severe granulocytopenia (absolute neutrophil count: 0.23x109/l, 1.39x109/l and 0.81x109/l, respectively). All patients had normal Hb level, RBC and platelet counts and none of them exhibited any clinical and laboratory signs of hemolysis (reticulocyte count, serum bilirubin, LDH and haptoglobin were within normal limits). Flow cytometric analysis of peripheral blood revealed a significant number of GPI-AP-deficient granulocytes in all patients, but no CD59 or CD55 deficient RBC except for a small PNH RBC clone in patient Nr.3. In patient Nr.1 (50 years old male) who exhibited 30–35% of GPI-AP-deficient granulocytes, sequencing analysis of PIG-A gene discovered transition C/T in exon 3, causing a missense substitution at codon 248 (P248L). Patient Nr.2 was a 28 years female with 20% of GPI-AP-deficient granulocytes, with sequentially identified transition G/A in exon 6 of PIG-A gene resulting in replacement of glutamic acid by lysine at codon 482 (E482K). In patient Nr.3 (34 years old male), flow cytometric analysis revealed 40–45% of PNH granulocytes and 8–12% of CD59 and CD55 deficient RBC. In this patient, sequencing analysis of PIG-A gene revealed transversion A/T in exon 5, leading to a non-sense mutation at codon 367, forming a preterminal STOP codon. This mutation resulted in the loss of 117 aminoacids from the C-terminus of the protein. Our results suggest that unexplained profound granulocytopenia may be in some patients related to mutation of PIG-A gene leading to GPI-APs deficiency expressed predominantly on granulocytes. MDS patients with isolated granulocytopenia who are mostly diagnosed as unclassifiable RA according to the WHO criteria should be screened by sensitive flow cytometry methods for presence of GPI-APs deficient granulocytes. Evidence of a PNH clone may have a prognostic value and may be helpful for decision of an optimal treatment strategy, including immunosuppression.