Erythrocytes must be durable and flexible to survive in situations of high shear stress as the pass through the microcirculation. The erythrocyte membrane lipid bilayer, transmembrane proteins, and the membrane skeletal network are essential to maintain erythrocyte integrity and homeostasis. Hemolytic anemia may occur when erythrocyte membrane properties are altered. Erythrocyte membrane disorders include inherited defects of membrane protein-protein and protein-lipid interactions.
Genetic mutations result in numerous erythrocyte membrane structural disorders in humans. Defects resulting in shape changes involve various proteins. Generally, vertical interactions involve the band 3-ankyrin-spectrin and glycophorin C-protein 4.1-spectrin binding, and horizontal interactions involve spectrin dimer-dimer association (tetramer formation) and contact of the distal ends of spectrin with F-actin, with the aid of protein 4.1 and adducin within the junctional complex.
Hereditary Spherocytosis (HS)
HS is a hereditary condition in spherocytosis is caused by genetic mutations. HS is characterized by the development of spherocytosis resulting in hemolytic anemia of varying severity, increased erythrocyte osmotic fragility, and splenomegaly. HS is a disorder of vertical interactions of the membrane proteins including spectrin, ankyrin, band 3, and protein 4.2. The lipid bilayer is destabilized with loss of membrane area that results in the formation of spherocytes.1 HS has been reported in humans, goats, cattle.2
Hereditary Band 3 Deficiency in Cattle
An intrinsic membrane deficiency of band 3 in Japanese Black Cattle is caused by a genetic mutation that disrupts the interaction of the erythrocyte membrane bilayer proteins.2 This defect destabilizes the membrane, predisposing the erythrocyte to membrane and surface area loss, leading to spherocyte formation. The band 3 deficiency leads to hemolytic anemia and premature removal and/or sequestration of the red blood cells by the spleen. The homozygous form typically presents with a severe hemolytic crisis early in life. The heterozygous forms are mild and compensation usually occurs.
Hereditary Spectrin Deficiency in Golden Retriever Dogs
Hereditary spherocytosis has been described in Dutch golden retriever dogs with severe hemolytic anemias, as well as in apparently healthy animals. In those cases, spherocytes were not recognized on initial stained blood films, but were seen as echinospherocytes by electron microscopy following 24 hour incubations. Spectrin deficiency in these dogs was confirmed by RIA. Spectrin deficiency is autosomal dominant, occurs frequently in Dutch golden retrievers, and also occurs frequently as an occult process in other breeds of dogs. It is not clear whether spectrin deficiency in golden retrievers results in hemolytic anemias it does in humans.3 Original canine cases recognized may have been complicated by concurrent immune-mediated hemolytic anemia.
Hereditary Stomatocytosis (HSt)
Stomatocytes are "bowl-shaped" erythrocytes in circulating blood. In stained blood films, they appear as erythrocytes with an elongated (mouth-like) area of central pallor. Stomatocytes are characterized by an increased cell volume to surface area ratio that correlates with increased osmotic fragility. This erythrocyte disorder is well-recognized in humans as a heterogeneous group of congenital disorders that cause hemolytic anemia. In humans, this erythrocyte membrane defect (also called hydrocytosis) may be associated with the lack of a cytoskeletal protein (band 7.2b or stomatin).2 HS have been recognized in four canine breeds: Alaskan malamute, Drentse partijshond, schnauzer, and Pomeranian. The specific erythrocyte defects are not established.
HSt in Chondrodysplastic Dwarf Alaskan Malamute Dogs
Stomatocytosis is associated with chondrodysplasia in Alaskan malamutes.4 This erythrocyte disorder is characterized by mild anemia, mild to moderate reticulocytosis, mild to moderate stomatocytosis, and a shortened erythrocytes life span. The pathogenesis of stomatocyte formation in malamutes appears to be similar to that reported in people and is attributed to an increase in intracellular monovalent cations, resulting in increased water content of the erythrocytes.2
HSt in Drentse Partijshond Dogs
In Drentse partijshond dogs, HSt is associated with the concurrent conditions of hypertrophic gastritis, polycystic renal disease, and regenerative macrocytic hypochromic hemolytic anemia.5 Studies of erythrocyte membranes and plasma of affected dogs indicate that abnormal fatty acid composition in plasma phospholipids leads to abnormal phospholipid composition in erythrocyte membranes and the correlated stomatocyte formation.6
Stomatocytosis in Miniature Schnauzers, Standard Schnauzers and Pomeranian Dogs
HSt has been reported as autosomal recessive defect in miniature schnauzers,2 standard schnauzers7 and Pomeranians8 without clinical signs of disease. The dogs were not anemic but erythrocytes appeared to be macrocytic and hypochromic.9 Unlike with the homologous disease in people, erythrocytes from affected standard schnauzer dogs do not lack in stomatin.10 Although the specific defects are not known, the pathogenesis of stomatocytes formation in schnauzers and Pomeranians is attributed to an increased monovalent cation (the erythrocyte Na+ and K+ content were increased). The stomatocytosis appeared to be due to erythrocytes overhydration due to a defective cation exchange.7
Hereditary Elliptocytosis (HE)
Elliptocytosis is a physiologic shape in camelids, but is either a hereditary (congenital) or acquired pathologic shape in other domestic mammals. HE results from qualitative or quantitative defects in erythrocyte membrane proteins (i.e., α-spectrin, β-spectrin, protein 4.1, or glycophorin C) in humans.1 These erythrocyte membrane proteins provide horizontal interactions of erythrocyte membrane components and cytoskeletal elements, and defects in the horizontal interactions make the cells prone to elliptocytic shapes.
Hereditary Elliptocytosis of Dogs Due to Protein 4.1 Deficiency
This disorder was discovered by Smith et al.11 Elliptocytosis was due to qualitative and quantitative defects of erythrocytes membrane protein 4.1. This protein is important to stabilize the cytoskeleton of the erythrocyte membrane by providing an anchor for the spectrin/ankrin complex. The elliptocytosis in this case was marked, and the patient had slight anemia with moderate reticulocytosis (appropriate regeneration). Generally these cases are discovered accidentally with no evident clinical signs.12
Congenital Elliptocytosis in a Dog with Qualitative Spectrin Defect13
A case of congenital elliptocytosis with a qualitative spectrin defect was recently discovered accidentally in a dog presented with an orthopedic problem. This case of canine HE was due to a mutant β spectrin. Unfortunately, the unavailability of genetically related dogs prevented additional testing to confirm a genetic pathogenesis.
The proband's disorder was characterized by persistent elliptocytosis, decreased erythrocyte membrane mechanical stability, decreased erythrocyte mechanical deformability, and an increased spectrin dimer to total spectrin ratio in erythrocyte membranes. The dog was not anemic, and there were not an increased percentage of polychromatophilic erythrocytes in the dog's blood, suggesting the proband did not have a compensated anemia. As frequently occurs in human hereditary elliptocytosis cases, this case of canine hereditary elliptocytosis occurred as a heterozygous, asymptomatic state that was detected during the investigation of an unrelated illness. Because the parentage of the mixed-breed dog was not known, it was not possible to determine if the dog inherited the β-spectrin defect or if it resulted from a de novo mutation. The proband's persistent, marked elliptocytosis included type I, II, and III elliptocytes. In people with similar elliptocytoses, the erythrocyte precursors are round cells and the elliptocytes form and become progressively more elliptical during aging in vivo. 14
The membrane mechanical stability of the proband's ghost erythrocytes was decreased compared to the stability of erythrocytes of a control dog. This feature is consistent with human elliptocytosis due to a spectrin structure abnormality (either a defect in α or β chains).15 The deformability of the proband's erythrocytes was reduced compared to a control dog's erythrocytes; a finding also consistent with a spectrin defect in human elliptocytosis. The molecular study on the proband's erythrocyte spectrin revealed that the elliptocytosis was most likely due to a β-spectrin defect that caused reduced self-association of spectrin dimers; either decreased formation of tetramers or decreased stability of tetramers. DNA analysis revealed that the proband was heterozygous for a mutation of β spectrin in which methionine replaced threonine in codon 2110; a region of spectrin that is critical for normal function and structure. Studies demonstrate that threonine at position 2110 in this region of canine β-spectrin is highly conserved in mammals, indicating its importance in normal spectrin structure and function.
Even though the elliptocytosis was not associated with a hemolytic anemia, the dog's erythrocytes did have decreased deformability and decreased stability when subjected to shear stress. Therefore, the dog's erythrocytes may be more susceptible to destruction if the dog develops a vascular or cardiac disorder that creates increased shear forces in its blood.13
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