Hereditary red blood cell disorders in the dog and cat

The main hereditary red blood cell disorders in the dog and cat include enzyme deficiencies, membrane alterations or erythropoiesis abnormalities.Haemoglobinopathies are instead rare, while in humans they are responsible for relatively widespread diseases (e.g. sickle cell anaemia).

RED CELL ENZYMOPATHIES

These are hereditary, usually autosomal recessive, disorders in which affected individuals show reduced function of intraerythrocytic enzymes involved in energy metabolism and in its accessory pathways. The metabolism of erythrocytes is relatively simple when compared to that of other cells (Fig. 1). Indeed, the lack of a nucleus and of many organelles prevents the erythrocyte from developing complex metabolic pathways. Basically, the only constantly active metabolic pathway is anaerobic glycolysis (Embden-Meyerhof pathway), which supplies energy to the erythrocyte, and which can give rise to accessory pathways like the pentose phosphate pathway (or hexose monophosphate shunt), important because it produces reducing substances that the erythrocyte uses to defend itself from oxidative damage, the polyol pathway and a few others.The most common enzyme deficiencies that can affect these metabolic pathways are listed below.

Phosphofructokinase (PFK) deficiency
PFK is the key enzyme of anaerobic glycolysis and if it is inhibited (e.g. by an excess of ATP) or deficient, the complete process of glycolysis is less active. Canine erythrocytes contain two PFK isoenzymes: of the two the muscle isoform (M-PFK) is the most abundant, however the platelet isoform (P-PFK) is also present. M-PFK is absent in hereditary deficiencies, similarly to the corresponding human disease. In humans, however, symptoms linked to myopathy predominate, as M-PFK and the “hepatic” isoenzyme (L-PFK) are present in equal measure in human erythrocytes. Even in the absence of M-PFK, therefore, L-PFK activity makes it possible to maintain erythrocyte energy requirements.

In the dog, PFK deficiency is an autosomal recessive disorder reported in Springer Spaniels, as well as in American Cocker Spaniels and Whippets: the abnormality resides in a mutation of the gene encoding for M-PFK, which is truncated and rapidly degraded. Consequently, muscle phosphofructokinase activity is completely absent and erythrocyte activity is considerably reduced. Thus, on the one hand less glucose is consumed and an accumulation of glycogen in the muscles is present (the disease is also called glycogen storage disease type VII) and, on the other hand, there is a decrease in ATP production. The production of 2,3-diphosphoglycerate (2,3-DPG), a glycolytic intermediate capable of affecting haemoglobin’s affinity for oxygen, within the erythrocytes is also reduced. Given that the erythrocytes contain small amounts of isoforms other than M-PFK under normal conditions the erythrocyte energy deficiency is compensated, however the average life span of erythrocytes is reduced (around 2 weeks instead of 3-4 weeks).

In the dog, haemolytic crises occur after hyperventilation (e.g. after hunting or in case of high environmental temperatures). In fact, during hyperventilation a tendency toward alkalosis is noticed, and PFK deficient erythrocytes are more fragile in an alkaline environment. During the crises, which usually begin at a young age, dogs appear prostrate and without appetite and exhibit fever, haemoglobinuria and/or bilirubinuria (the latter also during convalescence) and pale mucous membranes. Muscular symptoms, which are marked in humans, are less evident in dogs (cramps, exercise intolerance), except for in Whippets, in which a form of myocardial disease associated with PFK deficiency is reported. During the crises, the anaemia can at times be very severe and acquire the typical characteristics of haemolytic forms (anisocytosis, polychromasia, reticulocytosis, possible presence of nucleated erythrocytes). The haemolytic crises last a few days and are usually compensated by active regeneration, stimulated by the fact that, because of the persistent hypoxia caused by the “chronic” 2,3 DPG deficiency, affected dogs produce more erythropoietin.

Diagnosis should be based on signalment and  the clinical history: the presence of anaemia and haemoglobinuria associated with stress or exercise induced hyperventilation in dogs belonging to the above-mentioned breeds is very suggestive of PFK deficiency. Diagnostic confirmation can be obtained by determining the intraerythrocytic PFK activity or by looking for the mutation with molecular techniques (PCR). Determination of the activity is complex and laborious, not always available in laboratories and, in the final analysis, can give false negative results in the young or in subjects in which the activity of the isoforms present compensates the lack of M-PFK. It is therefore preferable to use PCR, which makes it possible to identify not only symptomatic animals (recessive homozygotes) but also healthy carriers.

In affected animals haemolytic crises must be prevented, avoiding behaviours potentially associated with hyperventilation. Treatment for haemolytic crises is symptomatic (rehydration, possible transfusions in case of severe anaemia, antipyretics, ACE inhibitors to counteract alkalemia).

Pyruvate kinase (PK) deficiency
PK is the last enzyme in the glycolytic pathway. The erythroid precursors primarily express one of the two muscle isoenzymes (M2-PK), while mature erythrocytes express the  erythrocyte isoenzyme (R-PK), which is encoded, like the liver isoenzyme (L-PK), from a gene different than the preceding one.

PK deficiency has been reported as an autosomal recessive disease in Basenjis, Beagles, Poodles, Dachshunds, West Highland White Terriers and Cairn Terriers and occasionally in other canine breeds, as well as in Abyssinian, Somali and European cats.R-PK mutations differ in the different breeds and have been identified in Basenjis, in Terriers, in Beagles and in cats. Regardless of the type of mutation, the abnormal R-PK is not functional and is partially compensated, in dogs, by the increase in M2-PK expression which, however, in vivo, appears inactive. The determination of enzyme activity is therefore not useful for diagnostic purposes in the dog, as the total activity can remain unvaried in vitro. The erythrocyte encounters an energy deficiency, not being able to complete the glycolysis, and its average life span is reduced to less than one week. On the other hand, the accumulation of intermediates of glycolysis lead to increased production of 2,3 DPG and thus to a decrease in haemoglobin’s affinity for oxygen, guaranteeing a good level of oxygenation of the tissues in spite of the anaemia.

Symptoms differ in dogs and cats. In the dog, affected subjects show exercise intolerance at a young age and a very regenerative, persistent and progressive enzymopaenic haemolytic anaemia of medium-high severity (anisocytosis, polychromasia, reticulocytosis) associated with hepatosplenomegaly due to both haemosiderosis and extramedullary haematopoiesis, as well as to myelofibrosis and medullary osteosclerosis, which can be detected radiographically. Osteosclerosis, more severe in Basenjis than in other breeds, leads to an unfavourable prognosis and death already in the early years of life. In the cat, instead, osteosclerosis is never present, and anaemia, detectable in around 12% of Somali or Abyssinian cats (approximately 24% are asymptomatic carriers), is chronic and intermittent, usually moderate, slightly macrocytic and hypochromic and strongly regenerative, besides being associated with splenomegaly.

Diagnosis is based on signalment, on the detection of anaemia with the above characteristics and, in the dog, on the presence of osteosclerosis. Confirmation is obtained by highlighting the mutation with PCR, in breeds in which the mutation is known or, in other breeds, highlighting the abnormal protein with special techniques (Western blotting, immunofixation, etc.). Treatment is basically symptomatic. Experimentally, both marrow transplant, in the dog, and splenectomy have proven effective in slowing down the progression of the disease.

NADPH-dependent methaemoglobin reductase deficiency (Cytochrome b5 reductase or Cb5R)
Cb5R is involved in bringing the haemoglobin molecules that oxidise daily, through spontaneous mechanisms or due to exposure to oxidising agents, back to the reduced state. In its absence, the iron contained in haemoglobin is oxidised to Fe_³⁺. The thus oxidised haemoglobin (methaemoglobin) is no longer capable of binding oxygen. Methaemoglobinaemia can be detected in all subjects following particularly severe oxidative insults but it is especially severe if it occurs in the absence of obvious oxidative insults in subjects with Cb5R deficiency.

Cb5R deficiency is an autosomal recessive disorder found in many canine breeds (Borzois, Chihuahuas, English Setters, Pomeranians and other less popular breeds) and in some cats. The mutation responsible for the disease has not been identified. Affected dogs appear exercise intolerant but generally show few symptoms. A constant finding is the presence of cyanosis in spite of normal blood pO₂ levels, a cyanosis which persists even if oxygen is administered. An empirical method for evaluating the possible presence of methaemoglobinaemia is to put a drop of blood on a paper napkin: in the presence of methaemoglobinaemia the drop colours brown instead of the normal bright red colour. If the methaemoglobinaemia is pronounced, the brownish colour may already be perceptible in the test tube. Affected subjects can be moderately anaemic but the modest degree of hypoxia resulting from the methaemoglobinaemia induces a regenerative response such that, in the majority of cases, the haematocrit appears normal, if not actually moderately increased, as reported in the majority of cats in which this abnormality has been detected. Consequently, positive animals do not require treatment.

Glucose-6-phosphate dehydrogenasedeficiency (G6PD)
This is one of the most common red cell enzymopathies in humans (responsible for favism). Given that this enzyme takes part in the pentose phosphate pathway, which produces reducing equivalents used by the erythrocyte to defend itself from oxidative damage, to which it is particularly exposed, when it is deficient, haemolytic crises caused by oxidising agents develop (characterised by eccentrocytosis and/or methaemoglobinaemia). Affected subjects are thus more exposed to oxidative insults, which in healthy subjects are rendered harmless by the antioxidant defences. In humans, for example, favism gets its name precisely because the haemolytic crises manifest themselves after eating fava beans, which contain oxidising compounds. In pets, clinically evident G6PD deficiency has never been reported, except in one horse, but reduced G6PD activity has been reported in a clinically healthy Weimaraner dog.

Flavin adenine dinucleotide deficiency (FAD)
FAD is a cofactor for antioxidant enzymes (methaemoglobin reductase, glutathione reductase). In its absence, these enzymes are no longer able to protect the haemoglobin and the membranes, respectively, from oxidation. As in G6PD deficiency, in FAD deficiency anaemia appears after exposure to oxidising agents and, in this case also, there are no reports of this type of deficiency in the dog and cat, while the disease has been reported in the horse.

MEMBRANE ALTERATIONS

The erythrocyte membrane is composed of a protein network underneath the classic lipid bilayer (spectrin, actin and myosin, protein 4.1), which forms the erythrocyte cytoskeleton. Transmembrane proteins are found in the membrane (integral proteins, e.g. band 3, ankyrin, glycophorin, stomatin, etc.), involved in the exchanges of substances between erythrocyte and surrounding environment and in the maintenance of the relationships between lipid layer and cytoskeleton. On the whole, these structures guarantee the cell the deformability that is indispensable for it to adapt itself to the changes in size of the capillaries and to resist the friction encountered in the peripheral circulation. Abnormalities of these structures cause alterations in the stability of the membrane itself, leading to morphological alterations of the erythrocytes (the most common are eccentrocytosis, stomatocytosis and elliptocytosis), the average life span of which decreases as the erythrocytes are destroyed in the haemocatheretic organs. Some of these alterations can only be detected in laboratory animals or in bovines. One example is hereditary spherocytosis caused by a deficiency in the band 3 integral protein and in other cytoskeletal proteins, associated with severe haemolytic crises in newborn calves. Spectrin deficiencies not associated with haemolytic diseases have been reported in the dog: however, elliptocytes were found in one dog, while in another report on Golden Retrievers, the erythrocytes appeared morphologically normal. The clinically significant associated membrane abnormalities reported in the dog are described below.

Protein 4.1 deficiency
Protein 4.1 deficiency prevents maintenance of the stability of the bonds between proteins of the erythrocyte cytoskeleton. Because of this instability, the erythrocytes take on an ellipsoid shape, which probably allows them to survive better in the circulation even though they are less stable. The greater fragility of these cells, however, makes it also possible to detect poikilocytosis, macrocytosis and erythrocyte fragments in the circulation. This form has also been reported in the dog, due both to an actual protein 4.1 deficiency and to a structural abnormality of the protein itself, with consequent lesser binding affinity with actin and spectrin. In spite of the morphological abnormalities of the erythrocytes, however, affected subjects did not show signs of anaemia.

Hereditary stomatocytoses
Several hereditary diseases are known in humans and dogs characterised by the presence of stomatocytes in circulation. The two forms of hereditary stomatocytosis known in humans are described below:

1.     Dehydrated stomatocytosis(DHS), characterised by moderate compensated haemolytic anaemiaand by a low percentage ofstomatocytes;

2.     Overhydrated stomatocytosis(OHS) or hydrocytosis, characterised by a high percentage of stomatocytes and by predisposition to thrombosis following splenectomy. Hypochromic macrocytic anaemia is present in this form and the erythrocytes show an especially high corpuscular volume because of a high sodium content and a low potassium content in the erythrocytes. The cause of this abnormality is unknown, but it is believed that a quantitative alteration of integral protein 7.2b or stomatin might play a role.

In the dog, the known forms of stomatocytosis have been reported in some breeds, in particular:

• Miniature Schnauzers: autosomal recessive, asymptomatic form characterised by reduction in the average life span of erythrocytes, which show macrocytosis, hypochromia and greater osmotic fragility.
• Standard Schnauzers (Fig. 2): the heritability is unknown. Affected animals [5] can be asymptomatic or slightly anaemic and show macrocytosis and alterations in the intraerythrocytic concentration of sodium and potassium similar to those detected in OHS. However, stomatin deficiency is not involved in the pathogenesis.
• Alaskan Malamute: affected dogs show chondrodysplasia, dwarfism and hypochromic macrocytic anaemia with greater osmotic fragility and reduction in average erythrocyte life span, followed in its turn by medullary erythroid hyperplasia and reticulocytosis. The pathogenesis is unknown but it is believed that a mechanism similar to that of human OHS might be involved.
• Drentse Patrijshond: affected dogs show hypertrophic gastritis and haemolytic anaemia associated with stomatocytosis. Polyneuropathy also develops over time, leading to the appearance of neurological symptoms. The stomatocytes are not macrocytic and do not have intracellular sodium and potassium content abnormalities. The pathogenesis of this form appears to reside in an altered membrane lipid composition, in its turn dependent on a hereditary abnormality of the metabolism of phosphatidylcholine, the concentration  of which is reduced in membranes and in plasma (the concentration of sphingomyelin instead increases).

ERYTHROPOIESIS ABNORMALITIES

These are hereditary abnormalities of the synthesis of erythrocytes or of their components (e.g. haemoglobin). The most frequent forms found in the dog and cat are listed below.

Congenital erythropoietic porphyria
This is a hereditary abnormality of haemoglobin synthesis reported in the cat as an autosomal recessive disease: the abnormality resides in the deficiency (not absolute) of the enzyme uroporphyrinogen III cosynthetase, involved in haemoglobin synthesis.The precursors of haemoglobin are thus converted into the I isomer of uroporphyrinogen and into coproporphyrinogen I. These compounds are then oxidised to uro- and coproporphyrin. These porphyrins accumulate in the erythroid precursors, making them more fragile. The rupture of these cells releases the porphyrins into the circulation, where they act like photosensitising substances. Depositing themselves in the tissues, they absorb ultraviolet rays, releasing electromagnetic waves of energy lower than that absorbed but in any case sufficient to cause oxidoreductive reactions in the affected cells. These lesions manifest themselves as cutaneous inflammatory processes that can generate bullous or necrotic lesions in the areas exposed to the sun. The deposition of porphyrins in hard tissues is instead responsible for the reddish-brown discolouration of the teeth. Alongside these skin lesions haemolytic crises are also found due to abnormal haemoglobin production or to abnormalities in maturation of the erythroid precursors in which the porphyrins have accumulated.

Treatment of this disease is essentially based on prevention of skin lesions caused by radiation (affected subjects should not be exposed to sunlight), as well as on the control and symptomatic treatment of haemolytic crises.

Macrocytosis of Poodles
This is a genetically determined abnormality, found in both sexes, usually not associated with anaemia, characterised by severe macrocytosis (high MCV) and by the possible presence in the circulation of nucleated erythrocytes (metarubricytes), often dysplastic due to the presence of Howell-Jolly bodies (at times multiple and of different size) and of nucleo-cytoplasmic maturation asynchronies. The erythroid precursors in the marrow appear more voluminous than usual (megaloblasts) and are often bi- or multi-nucleated or characterised by other abnormalities in nucleus shape and colourability. For example, the nuclei often appear fragmented or form the so-called “nuclear bridging”, i.e. a type of chromatin bridge between two adjacent metarubricytes, perhaps the result of incomplete mitosis. The same mitoses are very frequent and often atypical. The nature of the genetic abnormality responsible for this alteration has not been clarified and, all in all, given that affected animals do not show any symptoms whatsoever, it is considered more than anything a morphological alteration that does not require particular diagnostic or therapeutic interventions.

Vitamin B₁₂deficiency anaemia
Vitamin B₁₂ or cobalamin is essential for erythropoiesis since it acts as a cofactor during the synthesis of nucleic acids and is thus essential for cell replication. When this vitamin is deficient, cell replication slows down and the erythroid precursors are more voluminous (megaloblasts). Vitamin B₁₂ deficiency is not common in animals because high amounts of  this vitamin are produced by the intestinal flora. In giant Schnauzers and, to a lesser degree, in Border Collies and Beagles, a hereditary autosomal recessive disease is reported that prevents the intestinal absorption of cobalamin since the receptor that allows enterocyte internalisation of the intrinsic factor-cobalamin complex is missing.

The disease appears in very young animals and is characterised by anorexia, cachexia and by nonregenerative, normocytic-normochromic  anaemia associated with acanthocytosis, anisocytosis (detectable instrumentally as an increase in RDW) and possible circulating erythroid blasts. Neutropenia with hypersegmentation of the neutrophils and macrothrombocytopenia (presence of voluminous platelets) are also present.The medullary megaloblasts also show signs of dysplasia. Diagnosis is based on determination of Vitamin B₁₂ in the blood, as well as on signalment and clinical and haematological data. Treatment consists in administering Vitamin B₁₂ supplements administered parenterally.

Suggested readings

1. Feldman B.F., Zinkl J.G., Jain N.C.: Schalm’s Veterinary Hematology, 5a edizione. Philadelphia: Lippincott Williams & Wilkins, 2000.
2. Stockham S.L., Scott M.A.: Fundamentals of Veterinary Clinical Pathology, 2a edizione. Blackwell Publishing, 2008.
3. Paltrinieri S., Bertazzolo W., Giordano A. Patologia Clinica del Cane e del gatto. Approccio pratico alla diagnostica di laboratorio. ISBN: 978-88-2143-159-3. Elsevier Masson, 2010.
4. Paltrinieri S., Comazzi S., Ceciliani F., Prohaska R., Bonfanti U. Stomatocytosis of Standard Schnauzers does not depend on stomatin deficiency. Vet J. 173:202-205, 2007.
5. Harvey JW. Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses. Vet Clin Pathol. 35:144-56, 2006.
6. Clavero S., Bishop D.F., Giger U., Haskins M.E., Desnick R.J. Feline congenital erythropoietic porphyria: two homozygous UROS missense mutations cause the enzyme deficiency and porphyrin accumulation. Mol Med. 16:381-383, 2010.
7. Fyfe J.C., Giger U., Hall C.A., Jezyk P.F., Klumpp S.A., Levine J.S., Patterson D.F. Inherited selective malabsorption of vitamin B12 in giant schnauzers . J Am Anim Hosp Assoc; 25:533-539, 1989
8. Canfield P.J., Watson A.D.J. Investigations of bone marrow dyscrasia in a poodle with macrocytosis . J Comp Pathol; 101:269-278. 1989