Contributions
Abstract: EP698
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
RBC morphology is a key diagnostic feature for hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions and does not predict disease course or severity. However, RBC deformability measured by osmotic gradient ektacytometry (OGE) allows a clear differentiation between HS and HE, where the truncated osmoscan curve reflects the inability of the already elliptical cells to deform further under shear stress. In Hereditary Pyropoikilocytosis (HPP), the RBCs appear to have a significantly decreased ability to maintain deformability in the face of hypotonicity and the classical trapezoidal profile of HE is less evident
Aims
We aim to demonstrate that in patients with HPP and SPTA1 variants, the implication of a canonical splicing site, may explain not only the marked defect of spectrin chains tetramerization and elliptocytosis, but also the weakness of skeleton attachment to the lipid bilayer and the loss of RBC membrane, microvesiculation and spherocytosis. As most of the SPTA1 gene variants described, so far, in patients with HE or HPP, only one or few nucleotides are affected, the resulting protein involvement could be variable, ranging from a slight clinical form (the most frequent) associated with moderate elliptocytosis to a more severe form (the rarest) leading to poikilocytosis, depending on the degree of tetramerization failure
Methods
The diagnosis of HHA was performed via a step-wise process including RBC morphology, Hb electrophoresis, and measurement of common RBC enzyme activities. RBC deformability and other rheological parameters were studied by OGE, using the osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca; MaxSis. RR Mechatronics) as previously described. The genetic diagnosis of membranopathies was performed by gene capture followed by t-NGS that includes a panel of 35 genes responsible for membranopathy hemoglobinopathy, enzymopathy and CDA. NGS and Whole exome sequencing (WES) have been performed using an Illumina HiSeq2000 device .
Results
Both patients studied here showed severe neonatal hemolytic anemia and jaundice, that required phototherapy and transfusion. One patient was a typical case of HPP splenectomized at the age of 30 years, and the other patient a newborn with a severe clinical form of HS. In both cases, the t-NGS study demonstrated a complex set of five variants in the SPTA1 gene. Three of these variants were novel, two leading to abnormal splicing, and one to a microdeletion of chromosome 1, confirmed by WES, that removes the entire gene. In the patient with HPP, we have observed that, in addition to ellyptocytes and other bizarre forms, a high number of typical spherocytes are present. This effect is also demonstrated by the Osmoscan profile that is characteristic of HS, probably due to the masking of the HE trapezoid shape of the curve characteristic of ellyptocytes by the more severe rheological abnormalities of spherocytes.
Conclusion
Our study demonstrates that in HPP, associated with a high percentage of spherocytes and typical HS osmoscan profile, the interactions between both SPTA1 gene variants, in addition to the loss of shape recovery after elongation (ellyptocytes), a membrane instability leading to spherocytosis can also be observed, depending on the severity of tetramerization failure. This highlights the importance of molecular diagnostic in patients with no family history of RBC cytoskeleton disorders.
Keyword(s): Anemia, Erythrocyte, Hemolysis, Peripheral blood
Abstract: EP698
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
RBC morphology is a key diagnostic feature for hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions and does not predict disease course or severity. However, RBC deformability measured by osmotic gradient ektacytometry (OGE) allows a clear differentiation between HS and HE, where the truncated osmoscan curve reflects the inability of the already elliptical cells to deform further under shear stress. In Hereditary Pyropoikilocytosis (HPP), the RBCs appear to have a significantly decreased ability to maintain deformability in the face of hypotonicity and the classical trapezoidal profile of HE is less evident
Aims
We aim to demonstrate that in patients with HPP and SPTA1 variants, the implication of a canonical splicing site, may explain not only the marked defect of spectrin chains tetramerization and elliptocytosis, but also the weakness of skeleton attachment to the lipid bilayer and the loss of RBC membrane, microvesiculation and spherocytosis. As most of the SPTA1 gene variants described, so far, in patients with HE or HPP, only one or few nucleotides are affected, the resulting protein involvement could be variable, ranging from a slight clinical form (the most frequent) associated with moderate elliptocytosis to a more severe form (the rarest) leading to poikilocytosis, depending on the degree of tetramerization failure
Methods
The diagnosis of HHA was performed via a step-wise process including RBC morphology, Hb electrophoresis, and measurement of common RBC enzyme activities. RBC deformability and other rheological parameters were studied by OGE, using the osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca; MaxSis. RR Mechatronics) as previously described. The genetic diagnosis of membranopathies was performed by gene capture followed by t-NGS that includes a panel of 35 genes responsible for membranopathy hemoglobinopathy, enzymopathy and CDA. NGS and Whole exome sequencing (WES) have been performed using an Illumina HiSeq2000 device .
Results
Both patients studied here showed severe neonatal hemolytic anemia and jaundice, that required phototherapy and transfusion. One patient was a typical case of HPP splenectomized at the age of 30 years, and the other patient a newborn with a severe clinical form of HS. In both cases, the t-NGS study demonstrated a complex set of five variants in the SPTA1 gene. Three of these variants were novel, two leading to abnormal splicing, and one to a microdeletion of chromosome 1, confirmed by WES, that removes the entire gene. In the patient with HPP, we have observed that, in addition to ellyptocytes and other bizarre forms, a high number of typical spherocytes are present. This effect is also demonstrated by the Osmoscan profile that is characteristic of HS, probably due to the masking of the HE trapezoid shape of the curve characteristic of ellyptocytes by the more severe rheological abnormalities of spherocytes.
Conclusion
Our study demonstrates that in HPP, associated with a high percentage of spherocytes and typical HS osmoscan profile, the interactions between both SPTA1 gene variants, in addition to the loss of shape recovery after elongation (ellyptocytes), a membrane instability leading to spherocytosis can also be observed, depending on the severity of tetramerization failure. This highlights the importance of molecular diagnostic in patients with no family history of RBC cytoskeleton disorders.
Keyword(s): Anemia, Erythrocyte, Hemolysis, Peripheral blood