Methemoglobinemia, Increased Deformability and Reduced Membrane Stability of Red Blood Cells in a Cat with a CYB5R3 Splice Defect

Abstract

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman’s assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca2+-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis.

Independent COL17A1 Variants in Cats with Junctional Epidermolysis Bullosa

“…The second case had a homozygous single base pair exchange 5 nucleotides after the exon 10/intron 11 boundary, c.769+5G>A. A corresponding splice region variant at the +5 position has recently been described in the CYB5R3 gene in a cat with methemoglobinemia [46]. The G at position +5 of the 5 -splice site occurs in 78% of human 5 -splice sites and normally forms a base-pair with a cytosine from the U1 snRNA [47].…”

Hemoglobin Binding to the Red Blood Cell (RBC) Membrane Is Associated with Decreased Cell Deformability

“…A major factor in this process is band-3 [ 26 ], which preferentially binds deoxy-Hb [ 27 , 28 , 29 , 30 ]. In physiological conditions, less than 1% of Hb can be oxidized to form methemoglobin (metHb) and FerriHb, which can also bind to the normal RBC membrane [ 31 , 32 ]. However, as their concentration in the cytosol is less than 1% [ 33 , 34 ], their presence on the membrane is insignificant.…”