Case presentation
An 11 years old male patient has been followed up due to chronic non-immune, non-spherocytic hemolytic anemia of unknown origin since one year old. He was born by C/S as a term baby and second pregnancy. His parents were consanguineous. His one brother has been following up due to an unknown cause of hemolytic anemia. On the examination, he had jaundice, pallor appearance, and 3 cm splenomegaly, and 2 cm hepatomegaly. At the last laboratory results, his blood count was hemoglobin 9.0 gr/dl, mean corpuscular volume 80 fL, mean corpuscular hemoglobin 23 pg, mean corpuscular hemoglobin concentration (MCHC) 33 g/dL, red cell distribution width 17.7 % (n=11-14 %), platelet 255000/mm3, leukocyte 6200/mm3, absolute neutrophil count 4400/mm3, absolute reticulocyte number 210.0 x103/μL. The direct antiglobulin test was negative. Levels of hepatic enzymes were unconjugated bilirubin 2.1 mg/dl, conjugated bilirubin 0.7 mg/dl, lactate dehydrogenase 806 IU/L (n=180-430 IU/L). Folic acid and vitamin B12 levels were normal. Haptoglobin was below 30 mg/dl. Ferritin level was 450 ng/mL. His peripheral blood smear revealed severe polychromasia, anisocytosis, tear cells, a few fragmented erythrocytes, target cells, 5 % stomatocytes, increased normoblasts, and Howell-Jolly body in the erythrocyte. He presented with mild anemia, not needing frequent erythrocyte transfusions, jaundice, hepatosplenomegaly, high levels of unconjugated bilirubin and low levels of haptoglobin. Hemoglobin electrophoresis was normal. Cryohemolysis and osmotic fragility test were normal, as also CD 55 and CD 59 expression by flow cytometry. The flow cytometric EMA-binding test (expressed as % of decrease in fluorescence) was detected 10 % (normal values 11 %). Due to difficulty in reaching a diagnosis, the patient’s blood sample was tested in collaboration with Fondazione Ospedale Maggiore Policlinico(Milan, Italy). The activity of the most common red cell enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, hexokinase, glucose-6-phosphate isomerase, phosphofructokinase, glyceraldehyde-P-dehydrogenase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase, adenylate kinase, purine/pyrimidine ratio (to exclude the presence of pyrimidine 5’ nucleotidase deficiency) was normal. After receiving the patient’s informed consent, the DNA sample of the patient was analysed on an NGS targeted panel SureDesign software (Agilent), containing 40 genes associated with congenital hemolytic anemia. Libraries were obtained by HaloPlexHS Target Enrichment System Kit and sequenced on a MiSeq platform (Illumina).2 In our patient, new missense mutations (c.3364G> A, p.Glu1122Lys) were identified in PIEZO1 associated with DHS. Mutations were confirmed by the Sanger method, as previously reported. Written informed consent was obtained from the patient and parents.