Clinical and Laboratory Findings in Border Collies with Presumed Hereditary Juvenile Cobalamin Deficiency
Juvenile cobalamin deficiency is a rare disease in border collies and its diagnosis requires a high level of clinical suspicion. The goal of this study was to increase awareness of this disease by describing the clinical and laboratory findings in four young border collies with inherited cobalamin deficiency. The median age of the dogs was 11.5 mo (range, 8–42 mo), and two of the four dogs were full siblings. Clinical signs included intermittent lethargy (n = 4), poor body condition (n = 4), odynophagia (n = 2), glossitis (n = 1), and bradyarrhythmia (n = 1). Pertinent laboratory abnormalities were mild to moderate normocytic nonregenerative anemia (n = 3), increased aspartate aminotransferase (AST) activity (n = 3), and mild proteinuria (n = 3). All of the dogs had serum cobalamin levels below the detection limit of the assay, marked methylmalonic aciduria, and hyperhomocysteinemia. Full clinical recovery was achieved in all dogs with regular parenteral cobalamin supplementation, and laboratory abnormalities resolved, except the proteinuria and elevated AST activity persisted. This case series demonstrates the diverse clinical picture of primary cobalamin deficiency in border collies. Young border collies presenting with ambiguous clinical signs should be screened for cobalamin deficiency.
Introduction
Cobalamin (vitamin B12) is a water-soluble vitamin and essential cofactor for mammalian enzyme systems. Adequate amounts are required for nucleic acid synthesis and hematopoiesis1 Once ingested, dietary protein is partially digested by pepsin and hydrochloric acid, and cobalamin is released. Next, cobalamin immediately binds to a transporter protein called haptocorrin, which is mostly synthesized and secreted by the gastric mucosa. Haptocorrin is, in turn, digested by pancreatic proteases in the small intestine, and free cobalamin binds to intrinsic factor, a glycoprotein produced by the stomach and pancreas in dogs.2 The cobalamin-intrinsic factor complex is then internalized into the ileal enterocyte by endocytosis, which is mediated by a specific membrane protein receptor on the cell surface termed the cubam receptor.3,4 In dogs, the most common causes of cobalamin deficiency are chronic, severe small intestinal disease and exocrine pancreatic disease.5 Hereditary forms of cobalamin deficiency have been reported in giant schnauzers, Australian shepherds, and Chinese shar peis.6–9 There have been two case reports of cobalamin deficiency in border collies in which hereditary ileal malabsorption of cobalamin was thought to be the cause (although substantiation of this requires further investigation).10,11
The two most important reactions involving cobalamin are the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A and the remethylation of homocysteine. Cobalamin deficiency leads to reduced activity of those enzyme systems, resulting in increased concentrations of urinary methylmalonic acid (uMMA) and total plasma homocysteine (tHcy). Therefore, measurements of those metabolites allow the assessment of cellular cobalamin availability and are the tests of choice to detect either early or mild cobalamin deficiency in humans.1,12 In one prospective study, serum cobalamin, uMMA, and tHcy concentrations were evaluated in 35 healthy dogs and 113 border collies from Switzerland, and 4 cobalamin-deficient border collies were identified.13 The goal of the current study was to describe the diverse clinical and laboratory findings in those four cases because of the scarcity of information on juvenile cobalamin deficiency in border collies.
Case Report
Measurement of Cobalamin, uMMA, and tHcy
Serum cobalamin was measured using an automated chemiluminescence assaya as described previously.13 The lower detection limit of the assay was 150 ng/L. uMMA was determined by gas chromatography/mass spectrometryb with a lower limit of detection of 0.15 mmol.13 Results of the uMMA assay were expressed as a ratio/mol of urinary creatinine (uMMA/Cr). Cr was measured by the Jaffe methodc. tHcy concentration was measured using high performance liquid chromatography and fluorimetric detection.13
Controls
Thirty-five healthy dogs were used as controls and to establish reference ranges for cobalamin, uMMA, and tHcy. More details can be found in a previous study.13
Case 1
A 12 mo old border collie presented with a history of poor weight gain since the age of 4 wk, chronic diarrhea, and intermittent dysphagia. Physical examination revealed a body condition score (BCS) of 3 out of 9 (body weight was 11.6 kg), pain on manipulation of the tongue (which had no gross abnormalities), and bradyarrhythmia (44 beats/min). Electrocardiography showed a second-degree atrioventricular block, which resolved after administration of atropine. The only abnormality on the complete blood cell count (CBC) and serum biochemical profile was hypoproteinemia (Table 1). A fecal parasite screen was negative. A lateral radiograph of the thorax revealed no evidence of megaesophagus. Gastroduodenoscopy, as well as cobalamin and folated measurements were performed because of chronic diarrhea. All biopsy samples collected from the stomach and duodenum were evaluated according to the World Small Animal Association guidelines and were considered adequate and normal. Intermittent dysphagia was investigated by a videofluoroscopic barium swallow study, determination of an acetylcholine receptor antibody titer, and electromyography. All of those tests were normal. Postprandial serum bile acid concentratione, the results of a thyroid function panelf, the concentrations of insulin growth factor 1g, and fructosamineh were also measured. All values except serum cobalamin were within the reference ranges. Because of severe hypocobalaminemia, uMMA/Cr and the tHcy concentration were measured and both were elevated. Cobalamin supplementationi (500 μg subcutaneously [SC] q 7 days [43 μg/kg]) was initiated. Upon recheck at 8 wk, the diarrhea had resolved without additional treatment, the dog had gained 2 kg, and was significantly more active. Swallowing behavior and the results of an electrocardiogram were normal. A telephone follow-up 11 mo later revealed that the dog was still doing very well, but the owners had decreased cobalamin administration to monthly injections because they appeared to be painful for the dog. On day 325 after discharge (4 wk after the last injection), the serum cobalamin concentration was 311 ng/L (reference range, 261–1,001 ng/L).
Parameters from the CBC and serum biochemical profiles that were abnormal in at least one border collie are shown. All other results were within normal limits. Additional values in parentheses are from later time points.
AST, aspartate aminotransferase; cTLI, canine trypsinogen-like immunoreactivity; N/A, not available; tHcy, total plasma homocysteine; uMMA, urinary methylmalonic acid
Case 2
An 8 mo old border collie presented with a 2 wk history of recurrent fever, poor weight gain, and occasional dropping of food while eating. Physical examination revealed a BCS of 3 out of 9 (body weight was 11.7 kg); swollen palatine tonsils; multifocal, painful red erosions on the tongue; and a grade II/VI systolic murmur on the left. Two episodes of fever (> 40°C) were documented by the referring veterinarian. Radiographs of the abdomen and the thorax were unremarkable. Abnormal results of a CBC, serum biochemical profile, and urinalysis have been summarized in Table 1. Hematologic findings included a moderate normochromic normocytic nonregenerative anemia with mild anisocytosis, polychromasia, and Howell-Jolly bodies. The aspartate aminotransferase (AST) activity was higher than normal, and serum trypsin-like immunoreactivityj was normal, which ruled out exocrine pancreatic insufficiency. Urinalysis revealed mild proteinuria and mild pyuria, and bacteriologic culture was negative. Echocardiography showed a normal heart with adequate function and an increase in left aortic outflow velocity, which explained the murmur. Serum cobalamin, tHcy concentrations, and uMMA/Cr were determined because similar clinical findings including intermittent dysphagia had recently been observed in another young, thin border collie (case 1) by one of the authors (P.K.). Severe cobalamin deficiency was confirmed. Cobalamin supplementation (500 μg SC q 7 days [73 μg/kg]) was initiated, and 8 wk later the dog had gained 3 kg, was bright, alert, and showed normal swallowing behavior. Examination of the oral cavity and the results of a CBC at that time were normal, but the AST was still elevated (Table 1). The serum cobalamin concentration was 955 ng/L (1 wk after last injection), and 98 days after the start of treatment, it was 1,497 ng/L (1 wk after last injection).
Case 3
A 10 mo old border collie weighing 11 kg, a full sibling of the dog in case 2, was perceived by the owners as healthy and was only presented to our clinic when the owners became aware of the sibling’s diagnosis. The body condition of the dog was poor (BCS was 3 out of 9), and hematologic abnormalities included a normochromic microcytic nonregenerative anemia with 25 mature normoblasts/100 leukocytes, moderate anisocytosis, polychromasia, poikilocytosis, and mild neutropenia. Serum AST was higher than normal. The abnormal results of a CBC, serum biochemical analysis and urinalysis have been shown in Table 1. Serum cobalamin, tHcy concentrations, and uMMA/Cr were determined because of the findings in the dog’s sibling. Cobalamin deficiency was confirmed. Cobalamin supplementation (500 μg SC q 7 days [45.45 μg/kg]) was initiated, and at the time of the first re-evaluation 6 wk later, the dog was bright, alert, and had gained 2.5 kg. A CBC was normal, but the AST activity remained elevated. The serum cobalamin concentration was 454 ng/L. The supplementation regimen was decreased to injections q 2 wk. The dog remained bright and active, and serum cobalamin concentration was 561 ng/L on day 136, and 181 ng/L on day 271 after discharge (both values were 2 wk after the previous injection).
Case 4
A 41 mo old border collie that presented as a healthy dog in the course of the screening study of border collies previously mentioned.13 According to the owners, the dog was quiet, but alert, and very active during herding work; however, the dog had high sleep requirements compared with three other border collies in the same household. The dog’s BCS was 3 out of 9 (body weight was 11.8 kg), and hematologic abnormalities included a normochromic normocytic nonregenerative anemia with mild anisocytosis and poikilocytosis. Abnormal results of a CBC, serum biochemical analysis and urinalysis are shown in Table 1. Cobalamin supplementation (500 μg SC q 7 days [41.3 μg/kg]) was initiated, and the dog became more energetic, slept less, and gained 1 kg over 6 wk. The dog was maintained on this regimen for 3 mo after which time 500 μg of cobalamin was administered monthly. On day 183 after discharge, and after the start of treatment, the results of a CBC were normal and the serum cobalamin concentration was 187 ng/L (3 wk after the last injection).
Summary of the Four Cases
Median age of the four border collies with cobalamin deficiency was 11.5 mo (range, 8–41 mo), and median body weight was 11.6 kg (range, 11–12.1 kg). All dogs were female. Serum cobalamin concentrations were below the detection limit of 150 ng/L (reference range, 261–1,001 ng/L) in all dogs. Median uMMA/Cr was 4,064 mmol/mol (upper reference limit, < 4.2 mmol/mol), and median tHcy concentration was 51.5 μmol/L (reference range, 4.3–18.4 μmol/L). All four dogs were fed different commercial dog foods. The cases were described above in chronological order with regard to admission to the Clinic for Small Animal Medicine, University of Zurich.
Discussion
This case report describes four border collies with cobalamin deficiency that was diagnosed based on hypocobalaminemia, methylmalonic aciduria, hyperhomocysteinemia, and unequivocal response to cobalamin supplementation. The cobalamin deficiency was presumed to be hereditary because of the relatively early onset of clinical signs, the close relationship in two patients (siblings), and because of a reported predilection of this condition in border collies.10,11
The concentrations of tHcy and uMMA represent the cobalamin concentration at a cellular level and are considered more sensitive indicators of cobalamin deficiency than serum cobalamin.1 Increases in both variables in eucobalaminemic individuals are only seen with hemoconcentration and renal disease.14,15 The authors recently measured tHcy and uMMA in 113 border collies, and in addition to the four cases described here, there were five clinically normal border collies (four males, one female) with mild hypocobalaminemia (median, 251 ng/L; range, 150–259 ng/L; reference range, 261–1,001 ng/L), but normal tHcy concentration and an undetectable uMMA/Cr. None of the five dogs had any clinical or laboratory abnormality. Although tHcy and uMMA were beneficial in differentiating those five border collies from the border collies with presumed hereditary cobalamin deficiency, further investigation of the diagnostic usefulness of both markers is required.
The current study describes the first cases of presumed hereditary cobalamin deficiency in the border collie breed in continental Europe. There was one case reported in the USA and one case in the UK. The authors’ goal was to increase the understanding of this rare disease because clinical signs vary greatly and there is the potential for misdiagnosis because of a lack of awareness among veterinarians.
Although all four cases described herein were intact females, the aforementioned case from the UK was an 8 mo old male border collie.11 In affected giant schnauzers, family studies and breeding experiments demonstrated an autosomal recessive inheritance.6,7 The same holds true for humans suffering from hereditary selective cobalamin malabsorption (Imerslund-Gräsbeck syndrome).16 The authors are currently working on mapping the causative genetic defect in border collies.
The historical and clinicopathologic findings of the dogs described in this report differed from the two previously published case reports on two severely diseased border collies.10,11 Cases 1 and 2 described above were indeed presented because of illness; however, cases 3 and 4 were perceived by their owners to be healthy and were only identified because they were subjects in a previous study on tHcy and uMMA/Cr involving a larger number of border collies.13 Nevertheless, the predominant findings shared by the four cases included in this report were poor body condition and satisfactory weight gain after cobalamin administration. This is in agreement with reports from the veterinary literature and is also a typical finding in humans suffering from selective intestinal vitamin B12 malabsorption.7,10,11,17–19 Impaired DNA replication associated with hypocobalaminemia predominantly affects rapidly dividing cells, such as intestinal mucosal cells, and results in malabsorption of nutrients.18 In addition, reduced food intake due to cobalamin deficiency is thought to contribute to poor body condition.
Chronic fatigue was another major clinical sign in the four patients described herein, which is in agreement with other studies of cobalamin-deficient dogs and humans.10,17,20 The owners of cases 3 and 4 had not been alarmed by the chronic fatigue, but were amazed at the increased liveliness of their dogs after administration of parenteral cobalamin. Further, intermittent, impaired swallowing was a clinical feature in two of the dogs in this report, a finding that has not been described before. A fluoroscopic swallowing study was unremarkable in case 1 with glossodynia, and case 2 had gross evidence of glossitis. Those findings point to primary stomatodynia as the cause of dysphagia, rather than a neuromuscular disorder. Glossitis, mucosal ulcerations, glossodynia, stomatodynia, and peripheral paresthesia have all been reported in cobalamin-deficient human infants and adults.20,21
Reversible bradyarrhythmia caused by a second-degree atrioventricular block was documented in case 1. Unspecified bradycardia has previously been reported in a juvenile beagle with cobalamin deficiency.17 Dysfunction of the autonomic nervous system leads to reversible bradyarrhythmia in cobalamin-deficient humans, but the underlying mechanism is not known.22
At the time of admission, case 2 had fever, swollen palatine tonsils, and erosive lesions on the tongue. This combination of signs has been described in humans with cobalamin deficiency, and more recent investigations have shown that defective neutrophil chemotaxis is responsible for recurrent aphthous stomatitis in cobalamin-deficient infants.19,23 Administration of cobalamin in case 2 resulted in resolution of both the oral lesions and fever.
The results of gastroduodenoscopy and biopsy evaluation carried out according to World Small Animal Association guidelines in case 1 with chronic diarrhea were unremarkable. This is in contrast to the findings in three cobalamin-deficient giant schnauzers. Biopsies of the gastrointestinal tract showed diffuse atrophy of the gastric mucosa in one dog, diffuse atrophy of the duodenal and proximal jejunal mucosa, and mucosal edema and lymphangiectasia of the entire intestinal tract in all three giant schnauzers.7 Typical changes in the jejunal mucosa in humans with cobalamin deficiency include mild abnormalities of villous architecture (villus blunting) and increased lymphocytic and plasma cell infiltration of the lamina propria.24 The authors of this report are aware that it is not possible to draw conclusions from a single case, but it is conceivable that cobalamin deficiency in border collies is not routinely associated with histologic changes in the gastrointestinal tract.
Anemia, especially megaloblastic anemia, is a typical laboratory finding in cobalamin-deficient humans.18,19 Normochromic normocytic anemia has been reported in cobalamin-deficient dogs and occurred in two of the four cases presented above.7,10,11,17 Evaluation of blood smears from case 3 with microcytic nonregenerative anemia revealed 25 normoblasts/100 leukocytes, which is indicative of dyserythropoesis. In contrast, bone marrow cytology of the first reported case of cobalamin deficiency in a border collie with very mild normocytic anemia revealed erythroid hyperplasia with erythrophagocytosis.10 Hematologic abnormalities of cobalamin-deficient giant schnauzers included occasional macrocytic normoblasts with immature nuclei together with reduced erythroid precursors in a hypocellular bone marrow.7 Those differences may reflect either individual or breed-specific variations of cobalamin-deficient states. Subtle hematologic abnormalities, which were seen in case 3 in this report, may go undetected when only an automated blood cell count is done.10 Interestingly, the complete hematologic evaluation in case 1 was unremarkable, which has been occasionally documented in cobalamin-deficient humans, but has never been reported in dogs with cobalamin deficiency.1 That finding suggests that juvenile cobalamin deficiency in border collies may not be routinely associated with hematologic changes. Normal hematologic results in a border collie should therefore not preclude cobalamin assessment.
In three of the four cases described above, there was increased AST activity with normal alanine aminotransferase (ALT) activity, and AST activity remained elevated in two dogs even after full clinical recovery. It is not likely that malnutrition was the main reason for the increase, because AST activity remained higher than normal even after weight gain. Although increased AST activity in the absence of abnormal ALT activity may be due to muscle injury, there may be clinical exceptions relating to the zonal location of hepatic damage. It has been shown that AST achieves higher concentrations in periacinar hepatocytes.25 Although centrilobular hepatocytes are more susceptible to oxidative stress, relative centrilobular hypoxia secondary to anemia still seems an unlikely explanation because AST activity remained elevated in two of three patients even after resolution of the anemia. Another explanation relates to the finding of hyperhomocysteinemia in the present cases. Specifically, serum AST levels have been shown to correlate with tHcy concentrations in hyperhomocysteinemic humans and are thought to reflect increased metabolic activity via transamination pathways.26 In this context, it is interesting to note that knockout mice deficient in the enzyme regulating homocysteine metabolism, suffered from hepatic steatosis.27 The underlying mechanisms for hyperhomocysteinemia-associated liver steatosis were shown to be oxidative stress and decreases in hepatic antioxidant enzymes.28,29 To elucidate, if the same mechanism holds true for the elevated AST activity without an increase in ALT activity in the border collie, liver biopsies before and after cobalamin supplementation, as well as serial tHcy determinations, would have been required, but this could not be justified ethically. Furthermore, serial measurement of tHcy concentration was not practical because of cumbersome preanalytical blood handling.
Mild proteinuria was found in three of the four border collies. Proteinuria has also been reported in previous cases of cobalamin deficiency.11,17 It is believed that absence of the cubam receptor complex in proximal renal tubular cells results in reduced reabsorption of albumin.4 In the human literature, it is reported that mild proteinuria often persists, even after successful treatment with cobalamin is initiated.16 Similarly, persistent proteinuria was documented in two of the cases presented in this report.
Not all of the four patients included in this report were screened for exocrine pancreatic insufficiency or intestinal disease; however, the authors are confident that a congenital deficiency/defect was the cause of disease in all cases. Exocrine pancreatic insufficiency or ileal disease seem highly unlikely given the age, presenting clinical signs, and quick response to cobalamin administration as the sole treatment. Dogs suffering from hypoadrenocorticism could also present with vague clinical symptoms and gastrointestinal signs, and its diagnosis usually requires an adrenocorticotropin hormone stimulation test. However, considering the clinical courses of all four cases, Addison’s disease can confidently be ruled out.
Treatment was monitored via serum cobalamin measurement, and subnormal values occurred in two of the four cases. Subnormal values have been reported previously in cobalamin-deficient dogs on long-term cobalamin supplementation.11,17,30 In dogs, cobalamin is mostly bound to two proteins taken up by tissues and bound to enzymes with a long half-life. Thus, it is possible that cobalamin remains active in tissues even when serum concentrations are low.17,31,32 However, inadequate cobalamin supplementation could not be ruled out in the four patients in this report because the optimal cobalamin supplementation regimen, monitored via concurrent uMMA/Cr and tHcy measurements, is not known in dogs. Unfortunately, owner compliance with regular monitoring and follow-up examinations was difficult. Once the disease had been discussed and cobalamin administration had been initiated, the owners tended to think that the vitamin deficiency could be managed at home without veterinary guidance and further blood tests.
In humans suffering from Imerslund-Gräsbeck syndrome, successful oral treatment has also been described. It is based on the finding that when large doses of cobalamin are administered orally, sufficient amounts are absorbed, even in the absence of intrinsic factor.33 That treatment modality has not been examined in dogs so far.
Conclusion
The results of the present case report suggest that cobalamin deficiency is a familial problem in border collies associated with a variety of diverse historical and clinicopathologic abnormalities differing in severity. Furthermore, results of routine laboratory evaluation may be in the normal ranges, even if the dog presents quite ill. A potential cobalamin deficiency could be missed. Juvenile border collies with nonspecific clinical signs, such as anorexia, lethargy, and failure to gain weight, should be screened for cobalamin deficiency before more advanced tests are carried out.
The authors would like to thank Professor Ralph Gräsbeck for his helpful discussions. Parts of the study were supported by a grant from the Albert-Heim Foundation of the Swiss Cynologic Society, Bern, Switzerland.
Contributor Notes
S. Lutz’s updated credentials since article acceptance are Dr.med.vet.
