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.