Vet. Pathol. 21: 370-376 (1984)

Primary Hyperparathyroidism in German Shepherd Dogs: A Disorder of Probable Genetic Origin K. G. THOMPSON, L. P. JONES,W. A. SMYLIE,C. B. QUICK,G. V. SEGRE,D. J. MEUTEN,and M. B. PETRITES-MURPHY Department of Veterinary Pathology, The Texas Agricultural Experiment Station and the Texas Veterinary Medical Diagnostic Laboratory, Texas A&M University, College Station, Tex., and The Endocrine Unit, Massachusetts General Hospital, Boston, Mass.

Abstract. Primary hyperparathyroidism was diagnosed in two German shepherd pups from a litter of four females. Clinical signs were apparent by two weeks of age and included stunted growth, muscular weakness, and polydipsia/polyuria. Radiography revealed diffuse reduction in bone density. Both pups had marked hypercalcemia, hypophosphatemia, increased plasma immunoreactive parathyroid hormone concentrations and increased fractional clearance of inorganic phosphate in the urine. Intravenous infusion of one affected pup with calcium gluconate failed to suppress the plasma concentration of immunoreactive parathyroid hormone, suggesting autonomous secretion of parathyroid hormone. Necropsy of the other pup at eight weeks of age revealed diffuse hyperplasia of parathyroid chief cells, nodular hyperplasia of thyroid C-cells, skeletal alterations consistent with fibrous osteodystrophy, hypercalcemic nephropathy, and extensive mineralization of the lungs and gastric mucosa. The dam and sire were half sibs. One male pup from a previous litter of six had developed similar clinical signs and radiographic lesions, suggesting autosomal recessive inheritance. This is the first report of hereditary primary hyperparathyroidism in domestic animals, a disease which may be analogous to hereditary neonatal primary hyperparathyroidism in children.

Primary hyperparathyroidism in man occurs most often in adults and may be associated with either parathyroid neoplasia, usually solitary adenomas, or diffuse hyperplasia of all four parathyroid gland^.^*^,'*.*^ Some cases have a genetic etiology and may occur in conjunction with neoplasms in other endocrine organs.4, IS. 17.21.30.32 A rare form of hereditary primary hyperparathyroidism developing in the early neonatal period has been described in ~hildren.'.~.~. The disease in children is characterized by failure to thrive, muscular weakness, extreme hypercalcemia, hypophosphatemia, fibrous osteodystrophy, and nephrocalcinosis. Soft tissue calcification involving the lung, stomach, myocardium, and blood vessels occasionally is observed. Affected children die in early infancy if subtotal parathyroidectomy is not d ~ n e . ~Autosomal .~' rece~sive'~ and d ~ m i n a n t * ~ modes . ~ ' of inheritance have been reported. In dogs, primary hyperparathyroidism is rare and usually occurs in aged animals with functional parathyroid adenomas2 Hereditary primary hyperparathyroidism has not been reported previously in dogs or other '3*22324.25.28*31

domestic animals. We report the occurrence of primary hyperparathyroidism, probably with genetic etiology, in two German shepherd pups. Case History At two weeks of age, two German shepherd pups ( 1 and 2) from a litter of four females had difficulty suckling and were dominated by their littermates. By five weeks, both affected pups had stunted growth, lameness, muscular weakness, and polydipsia/polyuria. Radiographic examination of both pups revealed diffuse reduction in bone density with transverse sclerotic lines in long bones and narrowing of the cortices. Similar radiographic findings were present in pup 2 at eight weeks of age in addition to a diffuse increase in pulmonary density consistent with mineralization. At eight weeks of age, pup 1 was unable to support weight on its hind legs and was killed painlessly. Pup 2 was treated with ethane- 1 hydroxy- I , 1-diphosphonate (Procter and Gamble, Cincinnati, Ohio) in an attempt to prevent bone resorption; the results will be reported elsewhere. All four pups were suckled until five weeks of age and then were fed on commercial dog rations. No vitamin or mineral supplements were provided. The sire and dam were half sibs. In a previous litter of six from the same mating, one male pup developed similar

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clinical signs and radiographic lesions to those described above and subsequently was killed at five months of age without being necropsied.

Materials and Methods Serum alkaline phosphatase activity and concentrations of calcium, inorganic phosphorus, total protein, urea nitrogen and creatinine were measured using an SMA 12/60 micro apparatus (Technicon Instruments Corp., Tanytown, N.Y.). Concentrations of calcium, inorganic phosphorus, and creatinine in the urine were measured using the same apparatus. In order to test the responsivenesqof the parathyroid glands in pup 2 to increased plasma calcium concentration, hypercalcemia was induced by intravenous infusion of calcium gluconate (1 mg/ml in 5% dextrose) at 15 mg elemental Ca/ kg/hour then maintained at 7 mg Ca/kg/hour. This study was done after the original hypercalcemia had been treated successfully with ethane- I , hydroxy- 1, 1-diphosphonate. During this study plasma calcium concentrations were measured using a Centrifichem 400 apparatus (Baker Instruments, Bethlehem, Pa.). Immunoreactive parathyroid hormone was measured in heparinized plasma by a modification of methods previously described,26using antiserum GP- 1 (final dilution 1 :450,000). Either partially purified human parathyroid h o r m ~ n e or '~ pooled plasma from human patients with hyperparathyroidism were used as standards. The former was expressed in pg/ ml and the latter in pIEq/ml, an arbitrary unit. Antiserum GP- 1 contains antibodies that recognize two major determinants within the parathyroid hormone molecule, one requiring all or part of the 14-34 region and the other requiring all or part of the 53-84 region.26 It has been previously shown that when used in radioimmunoassay of canine parathyroid hormone, this antiserum readily recognizes both intact hormone and the large carboxy-terminal fragment (G. V. Segre, unpublished data). Moreover, samples of canine plasma with increased concentrations of immunoreactive parathyroid hormone were diluted and found to have displacement curves which were parallel to those given by standard, partially purified human parathyroid hormone. Tissues for light microscopy were fixed in 10% formalin immediately after euthanasia of pup I . Following routine embedding in paraffin, 6 pm sections were cut and stained with hematoxylin and eosin (HE). Selected sections were stained with either von Kossa's or Masson's trichrome method. Bones were demineralized in saturated solutions of ethylene diamine tetracetate before embedding.

37 1

Table I. Biochemical data from two German shepherd pups with primary hyperparathyroidism ( 1 and 2) and their unaffected littermates (3 and 4) at six weeks of age Serum calcium (mg/dl) Serum phosphorus (mg/ dl) Serum alkaline phosphatase (IU/l) Serum creatinine (mg/ dl) Serum urea nitrogen (mg/dl) Fractional clearance of urinary phosphorus

PUD 1 19.8 4.0

PUD2 PUD3 PUP4 15.4 10.4 10.9 4.9 7.8 6.8

880

880

320

356

0.8

1.0

0.3

0.4

22

26

-

-

59%

75%

-

-

values for pups3 Fractional clearance of inorganic phosphorus in the urine was increased in pups 1 and 2 but was not determined in pups 3 and 4. Subsequent samples from both affected pups revealed similar abnormalities. Serum calcium concentrations ranged from 17.9 to 23.7 mg/dl for pup 1 and from 14.4 to 20.6 mg/dl for pup 2. Serum alkaline phosphatase activities in pups 3 and 4 were considered to be normal for rapidly growing pups of a large breed, but the levels in affected pups were increased. Immunoreactive parathyroid hormone concentrations in the plasma of pups 1 and 2 at eight weeks of age were greater than 1000 plEq/ml. Most clinically normal dogs previously assayed by the same method had immunoreactive parathyroid hormone concentrations less than 60 plEq/ml. Intravenous infusion of calcium gluconate into pup 2 over a period of 150 minutes increased the plasma calcium concentration from 10.9 mg/dl to 12.7 mg/dl, but failed to suppress the concentration of immunoreactive parathyroid hormone (fig. 1). Necropsy examination of pup 1 revealed flabby, moderately atrophic limb muscles. The thyroid glands were pale tan. All four parathyroid glands were enlarged slightly. Bones were fragile and had narrow cortices, although there were segmental regions of sclerosis in Results the metaphysis of some limb bones. Firm, pale yellow, The biochemical data obtained from all four pups at thickened regions in the metaphysis of each rib exsix weeks of age are presented in table I. The most tended approximately 2 to 3 cm along the shaft from remarkable biochemical abnormality was extreme hy- near the costochondral junction. Similar swollen segpercalcemia in pups 1 and 2. Serum inorganic phos- ments sometimes were present in the diaphysis of ribs. phorus concentrations in these pups were low in com- The capsular surface of both kidneys was pitted finely. parison to their clinically normal littermates (pups 2 The renal cortex was pale tan and in the outer medulla and 4) and when compared with published reference there was a narrow, incomplete white band interpreted

Thompson et al.

312 c5

a a v

2ooo1

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.-c0

E

c C

:1000Q,

0

0

I

!i m

5a

E

9

Calcium Infusion I

I

l

-30 0

l

I

1

I

1

1

I

30 60 90 120 150 180 210

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h

Time (minutes)

Fig. 1: Effect of intravenous infusion of calcium gluconate on the plasma concentrations of calcium (A)and immunoreactive parathyroid hormone (0)in pup 2 after hypercalcemia had been corrected by treatment with ethane-1 hydroxy-1, 1-diphosphonate.

to be mineralization. The lungs were diffusely firm and failed to collapse when the thoracic cavity was opened. Microscopically, parathyroid glands contained uniform populations of chief cells with an increased amount of lightly basophilic cytoplasm (fig. 2). Perivascular spaces were indistinct and chief cells sometimes formed acinar structures. Occasional chief cells were undergoing mitosis. These changes indicated diffuse hyperplasia and hypertrophy of chief cells. C-cells in thyroid glands also were hyperplastic. Focal aggregates of plump C-cells with abundant cytoplasm separated thyroid follicles (fig. 3). Mitoses were common and occasional cells contained eosinophilic intranuclear inclusions that probably represented cytoplasmic invaginations. The skeletal alterations were consistent with fibrous osteodystrophy and were most severe in the ribs. The metaphyseal and diaphyseal swellings observed macroscopically in the ribs consisted of thickened trabeculae of woven bone separated by loose fibrous connective tissue. Osteoblastic and osteoclastic activities were prominent. Trabeculae sometimes contained resorption cavities lined by osteoclasts. Similar alterations were observed in all limb bones examined. The primary spongiosa contained irregular trabeculae of woven bone lined by either osteoclasts or active osteoblasts, and separated by loose connective tissue (fig. 4). There was excessive thickening of trabeculae in the distal metaphysis of some bones. Resorption cavities were common in the cortices. Renal alterations were mild and consisted of tubular

dilatation, especially in medullary rays, focal interstitial fibrosis,focal mineralization of tubular basement membranes and epithelial cells in the cortex, and more severe focal mineralization in the outer medulla. In the lungs there was diffuse mineralization of alveolar septa, bronchi, and bronchioles (fig. 5). Bronchiolar mineralization was most severe in the superficial lamina propria and muscularis. Alveolar septa were thickened with dense mineral deposits and sometimes contained moderate numbers of mononuclear and polymorphonuclear inflammatory cells. Occasional mineral deposits were in pulmonary arteries. The lamina propria and muscularis mucosa of the stomach also were diffusely mineralized but submucosal arteries usually were spared.

Discussion The presence of elevated plasma immunoreactive parathyroid hormone and diffuseparathyroid hyperplasia in two German shepherd pups with marked hypercalcemia, in addition to increased fractional clearance of inorganic phosphate in the urine and fibrous osteodystrophy, suggests a diagnosis of primary hyperparathyroidism. A genetic etiology is suspected because the disease occurred in only two pups from a litter of four. Parental consanguinity, and the possible occurrence of a similarly affected pup in a previous litter add further support to this hypothesis and suggest autosomal recessive inheritance. The clinical and pathological alterations observed in our pups closely resemble those reported in young children with severe hereditary primary hyperparathyr~idism.~**9, 13- 22*24*25,27*28,31 The disease in children is associated with diffuse hyperplasia of parathyroid chief cells rather than a functional parathyroid a d e n ~ m aAlthough .~~ it is not possible to differentiate grossly or microscopically between adenomatous and hyperplastic parathyroid glands,6*I* the enlargement of all four glands in pup 1 and the presence of apparently active chief cells throughout each gland suggests hyperplasia. Parathyroid hyperplasia and fibrous osteodystrophy may be associated with advanced renal failure, and familial renal disease was considered in the differential diagnosis. Hypercalcemia would not be expected in a dog with renal failure although it has been reported as a possible sequela: presumably secondary to prolonged stimulation of parathyroid chief cells by low plasma ionized calcium concentrations. However, serum biochemical alterations and results of urinalysis did not

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Fig. 2: Parathyroid gland; chief cells have an increased quantity of lightly basophilic cytoplasm, occasional mitotic figures (arrows),and indistinct penvascular spaces. HE. Bar = 30 pm. Fig. 3: Multiple aggregates of plump C-cells in thyroid gland separating thyroid follicles. HE. Bar = 50 pm.

Other possible causes of hypercalcemia such as hyindicate impaired renal function in either of our pups, and the renal lesions observed in pup 1 at necropsy pervitaminosis D, hypervitaminosis A, thyrotoxicosis, were considered to be secondary to hypercalcemia. The and localized bone resorption secondary to skeletal inability of both pups to concentrate urine was presum- neoplasia or osteomyelitis could be excluded on the ably due to inhibition of antidiuretic hormone by cal- basis of clinical history, pathological findings in pup 1, hypophosphatemia and elevated plasma concentrations cium. Pseudohyperparathyroidism is the most common of immunoreactive parathyroid hormone. Hypercalcause of symptomatic hypercalcemia in dogs.3 This cemia and hypophosphatemia, in association with condition is associated with the production by neoplas- slightly elevated plasma immunoreactive parathyroid tic tissue of substances capable of promoting bone hormone concentration, recently was described in an resorption. In dogs, malignant lymphoma and apocrine aged dog with parathyroid chief cell hyperplasia, medadenocarcinoma of the anal sac have been implicated ullary thyroid carcinoma and pheochromocytoma, a most frequently.3In contrast to the disease in our pups, syndrome resembling multiple endocrine neoplasia skeletal alterations in pseudohyperparathyroidism are type IIA in man.” No endocrine neoplasms were demild and parathyroid glands usually are atrophic.” tected at necropsy in our pup and the C-cell hyperplasia Furthermore, there was no evidence of neoplasia in was considered to be secondary to persistent hypercaleither pup. cemia.

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Fig. 4: Prominent osteoblastic and osteoclastic activity in metaphysis of distal ulna. Bone trabeculae separated by loose connective tissue. HE. Bar = 70 pm. Fig. 5: Diffuse mineralization of pulmonary alveolar septa, bronchiolar lamina propria, and muscularis. Von Kossa. Bar = 200 um.

A common cause of fibrous osteodystrophy in young pups is nutritional secondary hyperparathyroidism associated with the feeding of all-meat diets. This is an unlikely possibility in our cases since all four pups were fed similar diets and clinical signs first developed in pups 1 and 2 while they were still suckling. Furthermore, nutritional secondary hyperparathyroidism should not be associated with hypercalcemia. The synthesis and secretion of parathyroid hormone is regulated closely by the extracellular concentration of ionized calcium," although parathyroid hormone secretion is not suppressed completely during hypercalcemia.16 Induced hypercalcemia in pup 2 failed to suppress plasma immunoreactive parathyroid hormone concentration (fig. I), suggesting autonomous secretion of parathyroid hormone in this pup. This is an unusual

finding even in human hyperparathyroidism. Most human patients have elevated immunoreactive parathyroid hormone concentrations in the face of hypercalcemia, suggesting an abnormality in immunoreactive parathyroid hormone regulation. l9 However, several studies", ' . l 9 have demonstrated that in patients with primary hyperparathyroidism, due to either parathyroid hyperplasia or neoplasia, immunoreactive parathyroid hormone is suppressed by increased concentrations of plasma calcium. Greater than normal concentrations of ionized calcium may be required to suppress the synthesis and release of parathyroid hormone from such glands, but failure to suppress immunoreactive parathyroid hormone at least partially, with increased plasma calcium concentration is rare. It has been suggested that a combination of both increased mass of

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roid hormone. N Engl J Med 299:635-644, 1978 12 HABENER, J.F.; POTTS,J.T. JR.: Parathyroid physiology and primary hyperparathyroidism In: Metabolic Bone Disease, ed. Avioli and Krane, pp. 1-147, vol. 2. Academic Press, New York, 1978 I3 HILLMAN, D.A.; SCRIVER, C.R.; PEDVIS, S.; SHRAGOVITCH, I.: Neonatal familial primary hyperparathyroidism. N Engl J Med 270:483-490, 1964 14 KEUTMANN, H.T.; BARLING, P.M.; HENDY, G.N.; SEGRE, G.V.; NIALL,H.D.; AURBACH, G.D.; POTTS,J.T. JR.: Isolation of human parathyroid hormone. Biochemistry (Washington) 13: 1646- 1652, 1974 15 MARX,S.J.; ATTIE, M.F.; SPIEGEL, A.M.; LEVINE, M.A.; LASKER, R.D.; Fox, M.: An association between neonatal severe-primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N Engl J Med 306:257-264, 1982 16 MAYER, G.P.; HABENER, J.F.; POTTS,J.T. JR.: Parathyroid hormone secretion in vivo. Demonstration of a calciumAcknowledgements independent, non-suppressible component of secretion. J This study was supported by Organized Research Reserve Clin Invest 57:678-683, 1976 Grant 1-82 and Biomedical Research Support Grant 2-82. D.J.; COOPER, B.J.; CAPEN,C.C.; CHEW,D.J.; The authors are grateful for the technical assistance of Celia 17 MEUTEN, KOCIBA, G.J.: Hypercalcemia associated with an adenoR. Seiglie. carcinoma derived from the apocrine glands of the anal sac. Vet Pathol 18:454-471, 1981 References M.; EARLL,J.M.: 18 MONCHIK, J.M.; WRAY,H.L.; SCHAAF, 1 BRADFORD, W.D.; WILSON, J.W.; GAEDE,T.T.: Primary Nonautonomy of parathyroid hormone and urinary cyclic neonatal hyperparathyroidism-an unusual cause of failAMP in primary hyperparathyroidism. Am J Surg ure to thrive. Am J Clin Pathol59:267-275, 1973 133:498-505, 1977 19 MURRAY, 2 CAPEN,C.C.; MARTIN,S.L.: Calcium metabolism and T.M.; PEACOCK, M.; POWELL, D.; MONCHIK, disorders of parathyroid glands. Vet Clin North Am J.M.; POTTS,J.T. JR.: Non-autonomy of hormone secretion in primary hyperparathyroidism. Clin Endocrinol 7~513-548,1977 D.J.: Clinical disorders in serum (Oxford) 1:235-246, 1972 3 CHEW,D.J.; MEUTEN, calcium and/or phosphorus metabolism. Vet Clin North 20 PETERSON, M.E.; RANDOLPH, J.F.; ZAKI,F.A.; HEATH, Am 12:411-438, 1982 H.: Multiple endocrine neoplasia in a dog. J Am Vet Med 4 CUTLER, R.E.; REISS,E.; ACKERMAN, L.V.: Familial hy180: 1476-1478, 1982 ASSOC perparathyroidism. A kindred involving eleven cases with 2 1 PONT,A.: Multiple endocrine neoplasia syndromes. West a discussion of primary chief cell hyperplasia. N Engl J J Med 132:301-312, 1980 Med 270:859-865, 1964 22 PRATT,E.L.; GREEN,B.B.; NEUHAUSER, E.B.D.: Hypercalcemia and idiopathic hyperplasia of the parathyroid 5 ESSELSTYN, C.B.; LEVIN,H.S.; EVERSMAN, J.J.; SCHUglands in an infant. J Pediatr 30:388-399, 1947 MACHER, O.P.; SKILLERN, P.G.: Reappraisal of parathyroid pathology in hyperparathyroidism. Surg Clin North 23 PURNELL, D.C.; SCHOLZ, D.A.; BEAHRS, O.H.: HyperparaAm 54:443-447, 1974 thyroidism due to single gland enlargement. Arch Surg 6 FIALKOW, P.M.; JACKSON, C.E.; BLOCK,M.A.; GREEN122:369-372, 1977 WALD,K.A.: Multicellular origin of parathyroid “adeno- 24 RANDAL, C.; LAUCHLAN, S.C.: Parathyroid hyperplasia in mas.” N Engl J Med 297:696-698, 1977 an infant. Am J Dis Child 105:364-367, 1963 7 FINCO,D.R.; ROWLAND, G.H.: Hypercalcemia secondary 25 RHONE, D.P.: Primary neonatal hyperparathyroidism. Reto chronic renal failure in the dog: a report of four cases. port of a case and review of the literature. Am J Clin J Am Vet Med Assoc 173:990-994, 1978 Pathol64:488-499, 1975 8 GARCIA-BUNUEL, R.; KUTCHEMESHGI, A.; BRANDES, D.: 26 SEGRE,G.V.; HABENER, J.F.; POWELL,D.; TRAGEAR, G.W.; POTTS,J.T. JR.: Parathyroid hormone in human Hereditary hyperparathyroidism. Arch Pathol 97:399403, 1974 plasma. Immunochemical characteristics and biological implications. J Clin Invest 51:3 163-3 172, 1972 R.B.; GILLIS, D.A.; PRASAD, M.: Hereditary 9 GOLDBLOOM, parathyroid hyperplasia: a surgical emergency of early 27 SIPPLE, J.H.: The association of pheochromocytoma with carcinoma of the thyroid gland. Am J Med 31: 163-166, infancy. Pediatrics 49:5 14-523, 1972 J.F.: Responsiveness of neoplastic and hyper1961 10 HABENER, plastic parathyroid tissues to calcium in vitro. J Clin Invest 28 SPIEGEL, A.M.; HARRISON, H.E.; MARX,S.J.; BROWN, 62~436-450,1978 E.M.; AURBACH, G.D.: Neonatal primary hyperparathyJ.F.; POTTS,J.T. JR.: Biosynthesis of parathy1 1 HABENER, roidism with autosomal dominant inheritance. J Pediatr glandular tissue and abnormal regulation of parathyroid hormone secretion contribute to the mechanism of hyperparathyroidism.” In our pups, however, the glands were enlarged only slightly, and it appeared that the parathyroid glands failed to respond to elevations in plasma calcium concentration. Although a genetic etiology appears likely for these cases of primary hyperparathyroidism in German shepherd pups, confirmation will require additional breeding studies. If a breeding colony could be established, this canine disease would provide an excellent model for studying the pathogenesis and treatment of human diseases associated with hypercalcemia and hyperparathyroidism.

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90~269-272, 1977 29 STEENDIJK, R.: Metabolic bone disease in children. In: Metabolic Bone Disease, ed. Avioli and Krane, pp. 633702, vol. 2. Academic Press, New York, 1978 30 STEINER, A.L.; GOODMAN, A.D.; POWERS, S.R.: Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing’s disease:

multiple endocrine neoplasia type 2. Medicine (Baltimore) 47:371-409, 1968 31 THOMPSON, N.W.; CARPENTER, L.C.; KESSLER, D.L.; NISHIYAMA, R.D.: Hereditary neonatal hyperparathyroidism. Arch Surg 113:lOO-103, 1978 32 WERMER, P.: Genetic aspects of adenomatosis of endocrine glands. Am J Med 16:363-371, 1954

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