GH Deficiency in a Dalmatian Puppy with Megaesophagus

Introduction

Pituitary dwarfism is a rare endocrine disease that is characterized by a congenital deficiency of growth hormone (GH) in dogs. GH deficiency can occur as either an isolated deficiency or in combination with deficiencies of other hormones that are secreted from the anterior lobe of the pituitary gland.^1,2 In German shepherd dogs, pituitary dwarfism is known to be an autosomal recessive inherited disorder.³ Pituitary dwarfism was originally ascribed to pressure atrophy of the anterior lobe of the pituitary gland due to cyst formation in Rathke’s pouch of German shepherd dogs, but it also appears to be caused by a primary failure of craniopharyngeal ectoderm differentiation into normal tropic hormone-secretory pituitary cells.^4,5 A diagnosis of pituitary dwarfism is based on a reduction in plasma basal insulin-like growth factor 1 (IGF-1) and GH concentrations, but a definitive diagnosis may depend on the results of a GH stimulation test using GH-releasing hormone, clonidine, or xylazine.^2,6

Pituitary dwarfism occurs primarily in the German shepherd dog, as described above, but it has been also reported in other dog breeds including miniature pinschers, Weimaraners, Spitz-type dogs, and Karelian bear dogs.⁷ To the authors’ knowledge, there are no previous reports of GH deficiency in Dalmatians. The case reported herein describes a diagnosis of pituitary dwarfism in a Dalmatian puppy with growth failure.

Case Report

An 8 wk old female Dalmatian weighing .56 kg was referred to the authors’ institution due to growth retardation. The dog had been healthy with no previous medical history. On presentation, there were no abnormal findings during the physical examination other than the significant growth failure (Figure 1). Serum biochemical analyses revealed only a mild elevation of the alanine aminotransferase concentration. The results of a complete blood count were normal, and urine specific gravity was 1.010. Canine trypsin-like immunoreactivity and a bile acid stimulation test were performed and those levels were within the reference ranges. Abnormal findings were not detected either on survey radiographs or abdominal ultrasonography. Endocrine testing revealed the plasma levels of GH and IGF-1 were considerably lower than those of the four littermates (Table 1). After 7 days, the dog returned for a definitive evaluation of GH deficiency, and a GH stimulation test was performed. Plasma GH concentrations were measured before and 20 min after an IV injection of xylazine^a (100 μg/kg). As shown Table 1, there was no elevation in the GH concentration of the sample collected from the patient compared with four 1 yr old healthy beagles. Those results supported a diagnosis of pituitary dwarfism. The concentrations of cortisol and endogenous adrenocorticotropic hormone were normal compared with adult reference ranges; however, the plasma level of total thyroxine (T₄) was lower than the other four littermates, but the level of thyroid-stimulating hormone (TSH) was within the reference range.⁸ Those findings suggested the possibility of hypothyroidism concurrent with pituitary dwarfism.

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TABLE 1 Results of Endocrine Assays of a Dalmatian Puppy Exhibiting Growth Failure, Four Littermates, and Four Healthy Beagles

*

GH levels were determined by a sandwich enzyme-linked immunosorbent assay using a commercial kit^b designed to measure canine growth hormone levels. The assays were performed according to the manufacturer’s protocol. All samples, standards, and controls were assayed in duplicate. Because the concentrations of samples from healthy beagles were predicted to be outside the range of the standard curve, the samples were diluted. The optical density at 450 nm was determined using an automated microplate reader^c. GH was quantified from eight titration points using standard curves generated with purified canine GH. Lower and upper detection limits were .156 μg/L and 10 μg/L, respectively. The intra-assay and interassay coefficients of variation were < 10% and < 12%, respectively.

†

Mean GH concentration in eight adult healthy beagles²

§

IGF-1 levels were measured by a commercial laboratory^d that used an IGF-1 immunoradiometric assay kit^e. The radioactivity was counted by using an immunoradiometric assay-based γ-counter^f.

**

Mean IGF-1 concentration in immature German shepherd dogs²

††

Measurements of T₄ and TSH levels were determined using a chemiluminescent immunoassay-based autoanalyzer^g. All samples were assayed in duplicate. The intra-assay coefficients of variation were 13.8% and 8.2% at T₄ levels between .62 μg/dL and 1.94 μg/dL, respectively. The sensitivity of the T₄ assay was .12 μg/dL. The intra-assay coefficients of variation were 5%, 4%, and 3.8% at TSH levels of .20 μg/L, .50 μg/L, and 2.6 μg/L, respectively. The interassay coefficients of variation were 6.3% and 8.2% at TSH levels of .16 μg/L and 2.8 μg/L, respectively. The sensitivity of the TSH assay was .01 μg/L .

§§

Normal T₄ concentration in adult dogs¹²

***

Normal TSH concentration in adult dogs⁸

GH, growth hormone; IGF-1, insulin-like growth factor 1; NA, not available; ND, not determined, T₄, total thyroxine; TSH, thyroid-stimulating hormone.

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Seven days later, the patient returned to the authors’ institution in a lateral recumbent position after an abrupt collapse. A complete blood count and serum biochemical analysis indicated a very low number of red blood cells (2.61 × 10¹²/L; reference range, 3.7–6.1 × 10¹²/L), a packed cell volume of 12% (reference range, 25–41%), and increases in blood urea nitrogen (BUN; 52.12 mmol/L; reference range, 3.5–13.3 mmol/L), creatinine (176.8 μmol/L; reference range, 34.5–43.3 μmol/L), and alanine aminotransferase (3.842 μkat/L; reference range, ≤ 0.493 μkat/L). Total protein (53 g/L; reference range, 43–58 g/L) and albumin (29 g/L; reference range, 26–37 g/L) were normal. The reference ranges were obtained from dogs between 2 mo and 3 mo.⁹

Unexpectedly, thoracic radiographs showed an air-filled esophagus, indicating the onset of megaesophagus (Figure 2). The puppy was hospitalized with supportive care before the initiation of GH replacement therapy, but the puppy unfortunately died the next morning. The owner did not consent to necropsy, so a postmortem examination was not performed to identify histologic lesions in either the pituitary gland or other organs.

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Discussion

In the case report described herein, the dog exhibited significant growth retardation compared with her littermates. Growth retardation in young dogs can result from pediatric endocrine disorders, including pituitary dwarfism, juvenile diabetes mellitus, juvenile-onset hypothyroidism, and juvenile-onset hyperadrenocorticism.^7,10 Nonendocrine disorders can also cause growth retardation, such as malnutrition, gastrointestinal disorders, liver disease, renal disease, heart failure, and skeletal disorders.⁷ The physical features and the endocrine results of the Dalmatian puppy in this report were compatible with a diagnosis of pituitary dwarfism. The clinical manifestations of pituitary dwarfism result from a deficiency of GH and a secondary deficiency of circulating IGF-1.⁶ There are no reference intervals for IGF-1 and GH concentrations in Dalmatian dogs, so the IGF-1 and GH levels of this patient were compared with her four littermates. The concentration of GH and IGF-1 were considerably lower than those of her four littermates. A GH stimulation test using xylazine was performed to definitely evaluate the GH deficiency because GH and IGF-1 levels may also be low in healthy animals and other juvenile endocrinopathies. An injection of xylazine increases the circulating GH concentrations in normal dogs.¹¹ In the present case, there was no significant increase in the plasma GH concentration 20 min after xylazine administration. A combined anterior pituitary function test is an important dynamic test for assessing complete pituitary function in dogs suspected of pituitary dysfunction.² However, a combined anterior pituitary function test could not be performed in this case due to the risk of taking blood samples from a low body weight animal. That prevented the assessment of a combined deficiency in other pituitary hormones.

A GH stimulation test is often used to diagnose GH deficiency because baseline values of GH levels are low in most dogs. GH deficiency is usually excluded if the GH concentration after stimulation is > 5 μg/L.^7,11 The dose range of xylazine used for the GH stimulation test ranges from 100 μg/kg to 300 μg/kg; however, a higher dose of 300 μg/kg can cause maximal stimulation, and samples are typically taken at five time points over a period of 2 hr.¹¹ Alternatively, a single injection of 100 μg/kg of xylazine and a single time point (20 min after xylazine administration) were used with the puppy in this case, and no control puppy was used. That was because of the side effects of xylazine administration and the risks associated with taking blood samples from an animal with a low body weight. A previous report showed that 100 μg/kg of xylazine can significantly increase the GH value 20 min after administration.¹¹ Three of the littermates included in this study had basal GH concentrations > 5 μg/L, and one littermate had a concentration of 4.96 μg/L. Moreover, xylazine (100 μg/kg) was found to be an effective stimulant when the measured GH value was compared with the GH values of four healthy beagles. The postxylazine GH concentration in four healthy beagles was > 5 μg/L (range, 5.304–26.618 μg/L) at 20 min. Nevertheless, there existed the possibility that the lack of GH secretion in this case was likely due to the protocol used, as described above. A comparison with other littermates would be worth performing.

Pituitary dwarfism may be an inherited condition, which is characterized by a group of well-documented clinical signs in German shepherd dogs.⁷ The pituitary lesion may cause reproductive, thyroid, and adrenocortical dysfunctions, as well as GH deficiency.² The case described herein exhibited GH deficiency, and there was also evidence of decreased thyroid function. In the endocrine assays, the T₄ concentration of the sample collected from the patient was 2.02 μg/dL. Normal puppies ranging from 5 wk to 6 wk of age have T₄ concentrations that are 2–3× higher than normal adult dogs (adult range, 1.2–5.2 μg/dL).¹² Thus, the patient’s T₄ value of 2.02 μg/dL was low and indicative of thyroid dysfunction. The T₄ concentration of the puppy’s sample was also considerably lower than that of her four littermates. However, the TSH level was within the reference range of adult dogs and higher than her littermates, indicating that this case was similar to cases of primary hypothyroidism because the results differed from that expected in a dog with secondary hypothyroidism related to pituitary dwarfism. Therefore, the authors could not definitively diagnose the concurrent onset of hypothyroidism with dwarfism due to GH deficiency. However, the puppy returned with an abrupt collapse, and the diagnostic images unexpectedly indicated the onset of megaesophagus since the radiographs obtained 2 wk previously. A clear association between hypothyroidism and megaesophagus has not yet been proven in dogs, and the authors lacked data on the thyroid function when the puppy presented with megaesophagus.¹³ Thus, there was only a possibility that the megaesophagus was associated with hypothyroidism. However, three human cases of GH deficiency associated with megaesophagus were recently reported, indicating a possible association between GH deficiency and megaesophagus.^14–16 That may be an incidental association, a new syndrome, or possibly an adverse effect of recombinant GH.¹⁶ It remains to be investigated whether that association was purely coincidental.

In the current case, the observation of clinical signs was very early compared with previously reported cases of dwarfism. The early development of clinical signs has not been documented in German shepherd dogs. Dogs with pituitary dwarfism are typically normal in size during the first 1–2 mo of life, following which the rate of growth was slower than littermates.⁷ By 3–4 mo of age, the affected dogs were obviously stunted in growth and they typically never attained full adult dimensions.⁷ Dogs with pituitary dwarfism also developed inappetence and became less active.¹⁰ That situation is usually reached at 2–3 yr of age and has been ascribed to secondary hypothyroidism and impaired renal function.¹⁰ However, the Dalmatian described in this report exhibited significant growth retardation at 8 wk and developed clinical signs, including anemia and abrupt megaesophagus, at 10 wk. That discrepancy in the times of clinical manifestations could be due to reasons other than the general cause of dwarfism. The most likely explanation for the development of pituitary dwarfism was a congenital malformation of the pituitary gland caused by a mutation in a developmental transcription factor. Other explanations could be possible for the sudden-onset azotemia, megaesophagus, and anemia in this puppy. The puppy was 9 wk old when GH stimulation was performed with xylazine. At 9 wk, renal function tends to be immature even in healthy puppies. Moreover, urine specific gravity at presentation was 1.010, suggesting that nonazotemic renal deficiency was present. Therefore, the administration of xylazine to this puppy with GH deficiency may have exacerbated the impairment of renal function by decreasing glomerular clearance. Because xylazine may also cause stretching of the muscular wall of the esophagus, the administration of xylazine could have also resulted in megaesophagus. Moreover, at the time of abrupt collapse, the puppy showed severe anemia, which is a potentially life-threatening condition. Shock caused by gastrointestinal hemorrhage could also have developed; however, the concentrations of plasma total protein and albumin were within normal ranges. A postmortem examination was not performed, which prevented the identification of histologic lesions in the pituitary gland or other organs; thus, the true cause of death could not be definitively determined.

In humans and mice, a combined deficiency of GH, TSH, and prolactin is related to mutations in genes encoding the DNA transcription factors PIT-1, PROP-1, LHX4, and LHX3.¹⁷ In veterinary medicine, a genetic study of pituitary dwarfism has only been performed in the German shepherd dog. The search for the causal gene defect has excluded the transcription factors PIT-1, PROP-1, and LHX4, as well as the LIF-receptor gene as candidates.^18–21 A recent report demonstrated that a contracted DNA repeat in intron 5 of LHX3 is responsible for pituitary dwarfism in German shepherd dogs.²² Further study in different dog breeds will be necessary to identify the mutation in LHX3.

Conclusion

The case reported here describes pituitary dwarfism in a Dalmatian puppy with megaesophagus and a low T₄ level. Further investigations are required to clarify the different modalities of clinical signs that are concurrent with growth failure caused by GH deficiency.