A Syndrome of Hereditary Tyrosinemia in Mink (Mustela vison Schreb.) K. Christensen, P. Fischer, K. E. B. Knudsen, S. Larsen, H. Sorensen and 0. Venge* ABSTRACT A hereditary disease in mink (Mustela vison Schreb.) leading to death when the affected kits are about six weeks old has been investigated. The disorder is inherited as a simple autosomal recessive character. Strongly elevated plasma tyrosine concentration is an outstanding feature of the disease. An enzyme defect in tyrosine aminotransferase (EC 2.6.1.5) or 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) is considered together with the possibility of a parallel between the disease in mink and the disease tyrosinosis or hereditary tyrosinemia in man.
RESUME Cette etude portait sur une maladie hMr& ditaire du vison, Mustela vison Schreb., qui entraine la mort des sujets atteints, vers l'age de six semaines. La transmission de cette maladie est attribuable a un gene autosome recessif. Une elevation marquee de la teneur du plasma en tyrosine caract6rise cette condition. On pense donc a un defaut enzymatique de la fyrosine aminotransferase (EC 2.6.1.5) ou de la 4-hydroxyphenylpyruvate dioxygenase (EC 1. 13.11.27), ainsi qu'a la possibilite d'un parallele entre cette maladie du vison et la tyrosinose, i.e. la tyrosinemie her6ditaire de l'homme.
During the last few years some Danish mink farmers have observed a rather high *Department of Animal Genetics (Christensen and Venge), Chemistry Department (Fischer, Knudsen and Sorensen) and Department of Pathology Larsen), Royal Veterinary and Agricultural University, Copenhagen V. Denmark. Correspondence to: H. Sorensen, Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Copenhagen V, Denmark. Submitted August 3, 1978.
Volume 43
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July, 1979
mortality among kits of the Standard type mink. Death usually occurred in the course of two to three days after the first symptoms had been observed when the kits were about weaning age. The general view among the farmers has been that it is a genetic disorder. The object of the present investigation was to confirm or invalidate, respectively, a hypothesis of a simple recessive inheritance and attempt to reveal the cause and pathogenesis of the disease. The disease has occurred on several farms but the genetic investigation was restricted to only two where pedigree information was available. It was further supplemented by a few test matings at an experimental farm. The studies were based on a total of 150 kits from 25 litters. Forty-six of these animals eventually showed clinical signs of the disease while the rest remained normal. Pedigrees for affected animals with regard to relationship between the litters could be traced back to 1969. The results are shown in Fig. 1 from which it is seen that in litters with affected kits the sire and the dam have common ancestors. In a few cases, however, the ancestry could only be traced back to animals which were purchased from the same farm from which the common ancestor was bought. A closer examination of the pedigrees revealed that the litters were slightly inbred with an inbreeding coefficient between 0.5-2.0 percent. A test of the hypothesis of a simple recessive inheritance, corrected to a 3:1 segregation according to previously described principles (1) is presented in Table I. The X2-test supports the assumption of an autosomal recessive inheritance. The assumption of a 3:1 segregation was additionally based on the following observations: 1) All the affected animals were slightly inbred. 2) A normal sex ratio.
333
Generation
Farm No. I
69
70 71
72
73 74
75
76
Farm No. 11 72 73 74
75 76
77
o0
a0
Fig. 1. Genealogical diagram of litters with diseased animals from farm No. I and from farm No. II. The sign X means: purchased from the same stock. A square is the symbol for a male and a circle for the female. The diseased animals are marked by a filled symbol.
334
Can. J. comp. Med.
Table I. The Table Shows the Segregation Ratio for the Litters from Fig. 1. The Calculations are Based on a Corrected 3:1 Segregation, as the Expected Number is Corrected for Litters, Where the Number of Diseased is Zero, Even When Both Parents of the Litter Were Carriers of the Gene Which Caused the Disease Normal
Diseased
Farm No. I Observed number Expected number
42 39.1
14 16.9 x2 = 0.8; P > 0.3
Farm No. II Observed number Expected number
62 65.1
32 28.9 x2 = 0.5. P > 0.5
3) No affected kits have been observed when one of the parents was test mated to nonrelated partners. 4) No chromosomal aberrations were detected when chromosome studies by ordinary staining (8) were performed on cultured cells from the diaphragm of affected animals. The kits appeared clinically healthy until the age of about six weeks. As a rule, the disease then developed rapidly and in most cases the kits died within two or three days. In a few cases affected kits have survived for a little over a week. The first clinical signs were watery eyes with subsequent sticking together of the eyelids by a tenacious mucoid material which left a yellowish incrustation along the margins of the lids. A common finding, particularly in male kits, was a soaked abdominal haircoat apparently due to frequent urination. Matting of the hairs dorsally on the toes by a small amount of greyish branlike material occurred in some cases. The disease has only been observed among mink of the Standard colour type, although the farms included in the investigation also produced mink of other colour phases. Necropsies were performed on ten diseased and two normal kits killed by intraperitoneal injection of pentobarbital sodium. Gross changes, apart from those of the eyes and toes already mentioned, were mild, inconsistent and confined to the urinary system. They consisted of slight enlargement and abnormal paleness of the kidneys and/or varying degrees of dilatation of the renal pelvis and proximal part of the ureters but without demonstrable obstruction. Lung, myocardium, liver, kidneys, urinary bladder, stomach, intestine,
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pancreas, pituitary, thyroid and adrenal glands, ovaries, testicles, thymus, spleen, bone marrow, brain, skeletal muscle, eyelids, conjunctiva and skin from the toes were examined histopathologically. The changes of the eyelids and skin from the toes were identical. The initial lesion appeared as patchy acantholysis or acute epidermal necrosis, total or subtotal, leaving the basal layer intact. In some cases the hair follicles were similarly affected. The subsequent development of the lesions consisted of migration of neutrophilic granulocytes into the damaged epithelium, formation of micropustules, epithelial sloughing, erosion or ulceration with exudative inflammatory changes in the subjacent corium and superficial crusting. Acute but mild and superficial neutrophilic and fibrinous conjunctivitis accompanied the lesions of the eyelids. In the kidneys of all but one of the diseased kits small foci of necrosis, usually superficial, were present in the renal papillae and associated with a mild acute inflammatory reaction with interstitial edema, neutrophilic granulocytes and sometimes small amounts of fibrinous exudate. In some animals similar inflammatory changes were seen in the renal pelvis and urinary bladder. The kidneys also contained discrete, interstitial or perivascular focal accumulations, 50-300 jxm in greatest dimension, of a homogeneous, faintly PASpositive material with the appearance of amyloid but not convincingly metachromatic in methylviolet stains. Compared with the normal controls variable depletion of glycogen was the only change observed in the livers of the diseased kits. No other microscopic lesions were found. Chemical analyses were carried out on plasma samples from ten diseased and six clinically healthy kits. Blood samples taken from the kits before the first symptoms of the disease could be observed were obtained by cutting the tail and collecting blood in heparinized capillary tubes. At the time of killing heparinized blood samples were obtained by cardiac puncture. The red blood cells were separated from the plasma by centrifugation at 14,000 g, 4°C, 10 min. The supernatant plasma was pipetted into vials which were kept at -20°C until the analyses were performed. Amino acids and carboxylic acids of analytical grade used as reference compounds were purchased from Sigma, St. Louis. Sephadex G-25 (fine)
335
was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Proteins were separated from amino acids and carboxylic acids by gel filtration on Sephadex G-25 (fine) columns (0.9 x 50 cm) using water as an eluent, flow rate 20 ml/hr, 3 ml/fraction and continuously measuring absorption at 280 nm of the effluent. Amino acid analyses were performed on an automatic amino acid analyzer (Beckman Model 120 C). Absorption spectra were recorded on a Perkin-Elmer 402 UltravioletVisible Spectrophotometer. Paper chromatography (PC) was performed in n-butanolacetic acid-water (12:3:5) (solvent 1), phenol-water-concentrated aqueous ammonia (120:30:1) (w/v/v) (solvent 2), isopropanol-concentrated aqueous ammoniawater (8:1:1 (solvent 3) by the descending technique on Whatman No. 1 paper. High voltage electrophoresis (HVE) was carried out on Whatman No. 3 MM paper using a flat-plate unit (Shandon Model L. 24) in the following systems: (a) Buffer pH 3.6 (Pyridine-acetic acid-water) (1 :10:200), 30 min at 3 kV, and 90 mA; (b) Buffer pH 6.5 (Pyridine-acetic acid-water) (25:1: 500), 30 min at 5 kV, and 90 mA. Chromatograms and electrophoresis papers were dipped in ninhydrin (0.2% in acetone) or in aniline-xylose (1 g xylose in 3 ml water + 1 ml aniline in 100 ml methanol) to reveal carboxylic acids or Pauly's reagent to
detect phenols (7,24), followed by heating at 100°C for 2-5 min. The identity of commercial and isolated amino acids was established by the elution volume from the gel filtration columns, the Rf in PC, the mobility in HVE, the peak retention time from the amino acid analyzer and for the aromatic amino acids by UV spectral comparison. The amount of the amino acids in the samples was established by quantitative ninhydrin determination (22) and peak size from the amino acid analyzer and, in addition, for tyrosine from the 294 nm absorption peak when the compound was dissolved in 1 N NaOH. Plasma concentrations of urea (Berthelot reaction), creatinine (kinetic picric acid method), Na and K (IL 143 flame photometer) Ca, and Mg (IL 153 atomic absorption spectrophotometer) were determined at the Central Laboratory of the Royal Veterinary and Agricultural University. Results from investigations of plasma samples from four normal and three affected kits born in 1976 are presented in Table II. No appreciable differences be-
336
tween the concentrations of Na, K, Mg and Ca in plasma from normal and affected kits were observed. However, the concentrations of urea and creatinine in plasma from affected kits were strongly elevated compared to the concentrations found in plasma from normal kits. Plasma samples from some kits of the litters born in 1977 (Fig. 1) were further investigated for free amino acids. After gel filtration, the proteins appeared in fractions 5-7, phenylalanine and tyrosine in fractions 10-11 and the other free amino acids in fractions 12-20. The fractions containing phenylalanine and tyrosine also contained other aromatic low molecular weight compounds (vide infra) which were extracted with ether after dilution of the fractions with 1 N HCl. Urea, creatinine and tyrosine concentrations were strongly elevated in plasma from affected kits (Table III). Two dimensional PC of free amino acids from plasma samples of these mink kits confirmed the observation concerning tyrosine and showed furthermore that the disease results in significantly lower concentration of all other ninhydrin reacting compounds except for the basic amino acids and phenylalanine. The results obtained by use of HVE and amino acid analyzer showed that about 80% of the basic amino acid pool was ornithine. In addition, results from amino acid analyses of plasma from kit No. 7 (Table III) and a normal kit from the same litter are presented in Table IV. These plasma samples (200 IAI) were obtained five days before killing the animals and they showed that the disease at this age had drastically changed the content of free amino acids in plasma similar to that found in later stages of the disease (Table III) though clinical symptoms were not observable at that time. Further investigations concerning metabolites accumulated in plasma of affected kits were hampered due to the small amount of plasma available. Therefore, it was not possible to make a final identification of the aromatic compounds in the ether extract from acidified solutions of fractions 10-11 (see above). However, preliminary investigations using PC and HVE using 2- and 4-
hydroxyphenylacetate, 4-hydroxyphenylpyruvate, imidazoleacetate and other related carboxylic acids as reference compounds, as well as colour reactions with Pauly's reagent and the reactions on the paper with the aniline-xylose reagent for carboxylic acids indicated that 4-hydroxyphenyl sub-
Can. J. comp. Med.
Table II. Results from Chemical Analyses of Plasma Samples from Mink Kits Born in 1976 (see Fig. 1)
Mink No 1 affected ........................ " ........................ 2 " ........................ 3
Urea 26.7 27.0 28.8
4 normal " 5 " 6 " 7
12.7 12.2 10.5 12.2
........................
........................ ........................ ........................
Concentration in Plasma mM K Creatinine Na 0.08 163 5.1 0.10 6.2 163 148 5.6
0.04 0.04 0.02 0.04
151 155 145 152
4.9 8.3 8.4 5.7
of Ca 2.88 2.80 2.93
Mg 1.37 1.40 1.06
2.80 3.33 3.13 3.16
0.93 1.59 1.39 1.14
Table III. Data for Nine Mink Kits born in 1977 (see Fig. 1) and Results from Analyses of Plasma Samples
Mink No. 1 affected .......... 2 " .
.
.
.
4 5
",,.
6
"
.
.
7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Date at Killing Birth 13.6 1.5 13.6 26.4 13.6 1.5 14.6 30.4 1.5 14.6 14.6 1.5 15.6 27.4
8 normal .......... "p.......... 9
Sex
9 9 9Q 9 9 9
15.6 15.6
Table IV. Amino Acid Analyses of Plasma Samples Obtained from Mink Kits Five Days Before Killing the Animals
Compound Lysine Ornithine Arginine Aspartic acid Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine
T,yrosine Phienylalanine
Concentration in Plasma mM Kit No. 7 Normal Kit (Table III) 0.113 0.009 0.023 0.018 0.110 0.300 0.054 0.270 0.158 0.078 0.048 0.076 0.025 0.032
0.018 0.143 0.012 0.005 0.062 0.198 0.009 0.160 0.063 0.032 0.021 0.037 0.487 0.047
stituted carboxylic acids, but not imidazole derivatives, were present in the plasma of affected kits. The same indication was obtained from measuring the UV-absorption spectra in 1 N HCl and in 1 N NaOH after preparative HVE isolations of the compounds.
In conclusion, the disorder is inherited
Volume 43
July, 1979
as
Concentration in Plasma mM of Creatinine Tyrosine Urea 75.3 1.7 0.14 27.3 0.09 2.8 18.8 2.2 0.05 41.8 2.2 0.09 56.5 1.1 0.17 56.0 2.2 0.13 14.0 1.8 0.08 7.5 6.8
0.02 0.02
0.03 0.03
recessive character, due to homozygosity at a single locus. No indication of pleiotropic effect, coupling between genes, etc. has been observed. The recessive inheritance implies that both the sire as well as the dam of the diseased kits are heterozygous for the gene in question. Such results are also described for hereditary tyrosinemia in man (2,9). Environmental causes (feeding, management, etc.) of the disease can be excluded. No indication of infection as a releasing cause was found. These statements are confirmed by the results of controlled matings at an experimental farm. A mink disease has been described in the USA where the clinical observations were very similar to ours (18). Without presenting definite proof a genetic background was assumed. The disease occurs about one week after the kits have started eating their normal diet which contains at least double the amount of protein per energy unit compared to a normal human diet. Thus, a defect in an enzyme in the tyrosine catabolism will result in accumulation of this amino acid. However, the disease in mink seems to be comparable to the disease in man dea
337
OH
-ICOO*1
+1
2fl NH3
3. NH3
if
~CO-2-b/'
COO-
kCOO~~
2a.
VCOOOH
OH
OH COO~
5.
02
w
ii 1 7.4 coo
-OOC-, OFumarate
+
Acetoacetate
Fig. 2. Catabolic pathways of phenylalanine and tyrosine in animals. The heavy arrows indicate the normal degradation of phenylalanine and tyrosine. The broken arrows indicate degradative pathways with a limited function in the normal catabolism of these amino acids.
338
Can. J. comp. Med.
scribed as hereditary tyrosinemia or hered- are dominating compounds in the urine toitary tyrosinosis. The normal catabolic gether with the citric acid cycle carboxylic pathways of phenylalanine and tyrosine in acids and the aromatic carboxylic acids animals are presented in Fig. 2. The oxida- expected from tyrosine catabolism when the tions 1, 4 and 5 are irreversible reactions enzyme at reaction 4 (Fig. 2) is defect. The catalyzed by the enzymes phenylalanine-4- possibility of a defect in the enzyme fumahydroxylase, 4-hydroxyphenylpyruvate di- rylacetoacetase, reaction 7 in Fig. 2, has oxygenase and homogentisate oxidase, re- therefore been proposed as the primary spectively. The decarboxylations 2b and 3b enzyme defect in hereditary tyrosinemia are irreversible too, whereas the transamin- in man (13). In the diseased mink, the acase catalyzed reactions 2 and 3 as well as cumulation in the urine of succinylacetothe redox reactions 2a and 3a are reversible. acetate and the decarboxylation product The enzymes tyrosine aminotransferase thereof, succinylacetone, has not yet been (EC 2.6.1.5) and 4-hydroxyphenylpyruvate investigated. However, the affected kits dioxygenase (EC 1.13.11.27) are found in showed an increase in the plasma urea conliver and kidneys (3). A low activity of centrations of 2-2.5 times that found for one or both of these enzymes thus leads to the normal kits and a corresponding inaccumulation in these tissues, in blood and crease in the plasma creatinine concentrain urine of 4-hydroxyphenylpyruvate and tions was also observed (Table II and III). products thereof, e.g. tyrosine, 4-hydroxy- The increase in the plasma tyrosine conphenyllactate, and 4-hydroxyphenylacetate, centration and for kit No. 7 also the plasma as shown in Fig. 2 reactions 3, 3a and 3b. ornithine concentration (Table IV) was Relatively high concentrations of these much more pronounced. Thus, it seems compounds are toxic leading to tissue dam- likely that liver enzymes of both the tyroage via binding to the macromolecules sine catabolism and, secondarily, the urea (6,23) and inhibition of enzymes (12,13, cycle are involved in the disease. It is tempting to assume that the lesions 15). In hereditary tyrosinemia in man damage of liver and kidneys occurs (14,16,23) in the urinary system and possibly also as well as accumulation of 4-hydroxyphenyl- those. of the conjunctiva and skin are elicpyruvate, 4-hydroxyphenyllactate and 4-hy- ited by excretion or deposition of qualdroxyphenylacetate in urine (4,13) and ty- itatively or quantitatively abnormal merosine and products thereof in blood (5, 17, tabolites in the affected tissues. 19,21). The inheritance, and the accumulation of tyrosine, together with 4-hydroxyphenyl REFERENCES substituted carboxylic acids in plasma indicate that the described hereditary mink 1. ANDRESEN, E. The effect of ascertainment by trundisease is similar to human hereditary tyrocate selection on segregation ratios. - 1. World Congr. Genetics Applied to Livestock Production, sinemia. Although the accumulation of 01.3, Madrid. pp. 111-114. 1974. DALLAIRE, L. Genetic aspects of tyrosinemia. Can. tyrosine is most commonly believed to occur 2. Med. Ass. J. 97: 1098-1099. 1967. from a deficiency or reduced activity of 3. FELLMAN J. H., T. S. FUJITA and E. S. ROTH. properties and tissue distribution of p-hydroAssay, 4-hydroxyphenylpyruvate dioxygenase (5, xyphenylpyruvate hydroxylase. Biochim. biophys. 10), considerable evidence against a geneActa 284: 90-100. 1972. J., B. LINDBLAD, S. LINDSTEDT. L. tically-determined primary deficiency of 4. GENTZ, LEVY, W. SHASTEEN and R. ZETTERSTROM. Dietary treatment of tyrosinemia (tyrosinosis). Am. this enzyme has appeared in the literature J. Dis. Child. 113: 31-37. 1967. (4,11,15,20). 5. GENTZ, J., B. LINDBLAD, S. LINDSTEDT and R. ZETTERSTROM. Studies on the metabolism of The chemical observations do not allow the phenolic acids in hereditary tyrosinemia by a gas-liquid chromatographic method. J. Lab. elin. a distinction between a defect in the en74: 185-202. 1969. zymes tyrosine aminotransferase and/or 6. Med. GILETTE, J. R. A perspective on the role of chemically reactive metabolites of foreign compounds in 4-hydroxyphenylpyruvate dioxygenase, but toxicity - I Correlation of changes in covalent from the established inheritance of the binding of reactivity metabolites with changes in the incidence and severity of toxicity. Biochem. Phardisease we conclude that only one of these mac. 23: 2785-2794. 1974. GRIMMET. M. R. and E. L. RICHARDS. Separation enzymes is involved. As the accumulation 7. of imidazoles by cellulose thin-layer chromatography. of tyrosine far exceeds that of 4-hydroxyJ. Chromat. 20: 171-173. 1965. I. Cytogenetic, distribution and phe8. GUSTAVSSON, phenyllactate and 4-hydroxyphenylacetate notypic effect of a translocation in Swedish cattle. 63: 68-169. 1969. it is most likely a defect in tyrosine amino- 9. Hereditas LABERGE, C. and L. DALLAIRE. Genetic aspects of transferase. tyrosinemia in the Chicoutimi Region. Can. Med. Ass. 97: 1099-1100. 1967. In tyrosinemia in man the compounds 10. J.LaDU, B. N. The enzymatic deficiency in tyrosinemia. Am. J. Dis. Child. 113: 54-57. 1967. succinylacetoacetate and succinylacetone
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339
11. LaDU, B. N. and L. R. GJESSING. Tyrosinosis and tyrosinemia. In The Metabolic Basis of Inherited Disease. J. B. Stanberry, J. B. Wyngaarden and D. S. Fr edeirickson, Editors. pp. 296-307. New York: McGraw-Hill Book Co. 1972. 12. LINDBLAD, B., G. LINDSTEDT, S. LINDSTEDT and M. RUNDGREN. Pur-ification and some properties of human 4-hydroxyphenylpyruvate dioxygenase (I). J. biol. Chem. 252: 5075-5084. 1977. 13. LINDBLAD, B., S. LINDSTEDT and G. STEEN. On the enzymic defects in hereditary tyrosinemia. Proc. Natn. Acad. Sci. 74: 4641-4645. 1977. 14. PARTINGTON, M. W. and M. D. HAUST. A patient with tyrosinemia and hypermethioninemia. Can. Med. Ass. .J. 97: 1059-1067. 1967. 15. PERRY, T. L. Tyrosinemia associated with hypermethioninemia and islet cell hyperplasia. Can Med. Ass. J. 97: 1067-1072. 1967. 16. PRIVE, L. Pathological findings in patients with tyrosinemia. Can. Med. Ass. J. 97: 1054-1056. 1967. 17. SASS-KORTSAK, A., S. FICICI, L. RAIJNIER, L. W. KOOH, D. FRASER and S. H. JACKSON. Secondar-y metabolic derangements in patients with tyr-osyluria. Can. Med. Ass. J. 97: 1079-1089. 1967.
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18. SCHWARTZ, T. M. and R. M. SHACKELFORD. Pseudodistemper an apparently new ailment of mink. U.S. Fur Rancher 52. No. 8, p. 6, 1973. 19. SCRIVER, C. R. The phenotypic manifestations of hereditary tyrosinemia and tyrosyluria: A hypothesis. Can. Med. Ass. J. 97: 1073-1075. 1967. 20. SCRIVER, C. R. and E. DAVIES. Investigation in vivo of the biochemical defect in hereditary tyrosinemia and tyrosyluria. Can. Med. Ass. J. 97: 10761078. 1967. 21. SCRIVER, C. R., M. SILVERBERG and C. L. CLOW. Hereditar-y tyrosinemia and tyrosyluria: Clinical repoirt of four patients. Can. Med. Ass. J. 97: 10471050. 1967. 22. S0RENSEN, H. Saccharopine and 2-aminoadipic acid in Reseda odorata. Phytochem. 15: 1527-1529. 1976. 23. WEINBERG, A. G., C. E. MIZE and H. G. WORTHEN. The occurrence of hepatoma in the chr onic form of hereditary tyrosinemia. J. Pediatr. 88: 434-438. 1976. 24. WHITFIELD, A. E. Diazotised sulphanilic acid reagent as an aid to the identification of some oxo acid 2.4-dinitirophenyl-hydrozones. J. Chromat. 20: 401-402. 1965.
Can. J. comp. Med.
RESUME Cette etude portait sur une maladie hMr& ditaire du vison, Mustela vison Schreb., qui entraine la mort des sujets atteints, vers l'age de six semaines. La transmission de cette maladie est attribuable a un gene autosome recessif. Une elevation marquee de la teneur du plasma en tyrosine caract6rise cette condition. On pense donc a un defaut enzymatique de la fyrosine aminotransferase (EC 2.6.1.5) ou de la 4-hydroxyphenylpyruvate dioxygenase (EC 1. 13.11.27), ainsi qu'a la possibilite d'un parallele entre cette maladie du vison et la tyrosinose, i.e. la tyrosinemie her6ditaire de l'homme.
During the last few years some Danish mink farmers have observed a rather high *Department of Animal Genetics (Christensen and Venge), Chemistry Department (Fischer, Knudsen and Sorensen) and Department of Pathology Larsen), Royal Veterinary and Agricultural University, Copenhagen V. Denmark. Correspondence to: H. Sorensen, Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Copenhagen V, Denmark. Submitted August 3, 1978.
Volume 43
-
July, 1979
mortality among kits of the Standard type mink. Death usually occurred in the course of two to three days after the first symptoms had been observed when the kits were about weaning age. The general view among the farmers has been that it is a genetic disorder. The object of the present investigation was to confirm or invalidate, respectively, a hypothesis of a simple recessive inheritance and attempt to reveal the cause and pathogenesis of the disease. The disease has occurred on several farms but the genetic investigation was restricted to only two where pedigree information was available. It was further supplemented by a few test matings at an experimental farm. The studies were based on a total of 150 kits from 25 litters. Forty-six of these animals eventually showed clinical signs of the disease while the rest remained normal. Pedigrees for affected animals with regard to relationship between the litters could be traced back to 1969. The results are shown in Fig. 1 from which it is seen that in litters with affected kits the sire and the dam have common ancestors. In a few cases, however, the ancestry could only be traced back to animals which were purchased from the same farm from which the common ancestor was bought. A closer examination of the pedigrees revealed that the litters were slightly inbred with an inbreeding coefficient between 0.5-2.0 percent. A test of the hypothesis of a simple recessive inheritance, corrected to a 3:1 segregation according to previously described principles (1) is presented in Table I. The X2-test supports the assumption of an autosomal recessive inheritance. The assumption of a 3:1 segregation was additionally based on the following observations: 1) All the affected animals were slightly inbred. 2) A normal sex ratio.
333
Generation
Farm No. I
69
70 71
72
73 74
75
76
Farm No. 11 72 73 74
75 76
77
o0
a0
Fig. 1. Genealogical diagram of litters with diseased animals from farm No. I and from farm No. II. The sign X means: purchased from the same stock. A square is the symbol for a male and a circle for the female. The diseased animals are marked by a filled symbol.
334
Can. J. comp. Med.
Table I. The Table Shows the Segregation Ratio for the Litters from Fig. 1. The Calculations are Based on a Corrected 3:1 Segregation, as the Expected Number is Corrected for Litters, Where the Number of Diseased is Zero, Even When Both Parents of the Litter Were Carriers of the Gene Which Caused the Disease Normal
Diseased
Farm No. I Observed number Expected number
42 39.1
14 16.9 x2 = 0.8; P > 0.3
Farm No. II Observed number Expected number
62 65.1
32 28.9 x2 = 0.5. P > 0.5
3) No affected kits have been observed when one of the parents was test mated to nonrelated partners. 4) No chromosomal aberrations were detected when chromosome studies by ordinary staining (8) were performed on cultured cells from the diaphragm of affected animals. The kits appeared clinically healthy until the age of about six weeks. As a rule, the disease then developed rapidly and in most cases the kits died within two or three days. In a few cases affected kits have survived for a little over a week. The first clinical signs were watery eyes with subsequent sticking together of the eyelids by a tenacious mucoid material which left a yellowish incrustation along the margins of the lids. A common finding, particularly in male kits, was a soaked abdominal haircoat apparently due to frequent urination. Matting of the hairs dorsally on the toes by a small amount of greyish branlike material occurred in some cases. The disease has only been observed among mink of the Standard colour type, although the farms included in the investigation also produced mink of other colour phases. Necropsies were performed on ten diseased and two normal kits killed by intraperitoneal injection of pentobarbital sodium. Gross changes, apart from those of the eyes and toes already mentioned, were mild, inconsistent and confined to the urinary system. They consisted of slight enlargement and abnormal paleness of the kidneys and/or varying degrees of dilatation of the renal pelvis and proximal part of the ureters but without demonstrable obstruction. Lung, myocardium, liver, kidneys, urinary bladder, stomach, intestine,
Volume 43
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July, 1979
pancreas, pituitary, thyroid and adrenal glands, ovaries, testicles, thymus, spleen, bone marrow, brain, skeletal muscle, eyelids, conjunctiva and skin from the toes were examined histopathologically. The changes of the eyelids and skin from the toes were identical. The initial lesion appeared as patchy acantholysis or acute epidermal necrosis, total or subtotal, leaving the basal layer intact. In some cases the hair follicles were similarly affected. The subsequent development of the lesions consisted of migration of neutrophilic granulocytes into the damaged epithelium, formation of micropustules, epithelial sloughing, erosion or ulceration with exudative inflammatory changes in the subjacent corium and superficial crusting. Acute but mild and superficial neutrophilic and fibrinous conjunctivitis accompanied the lesions of the eyelids. In the kidneys of all but one of the diseased kits small foci of necrosis, usually superficial, were present in the renal papillae and associated with a mild acute inflammatory reaction with interstitial edema, neutrophilic granulocytes and sometimes small amounts of fibrinous exudate. In some animals similar inflammatory changes were seen in the renal pelvis and urinary bladder. The kidneys also contained discrete, interstitial or perivascular focal accumulations, 50-300 jxm in greatest dimension, of a homogeneous, faintly PASpositive material with the appearance of amyloid but not convincingly metachromatic in methylviolet stains. Compared with the normal controls variable depletion of glycogen was the only change observed in the livers of the diseased kits. No other microscopic lesions were found. Chemical analyses were carried out on plasma samples from ten diseased and six clinically healthy kits. Blood samples taken from the kits before the first symptoms of the disease could be observed were obtained by cutting the tail and collecting blood in heparinized capillary tubes. At the time of killing heparinized blood samples were obtained by cardiac puncture. The red blood cells were separated from the plasma by centrifugation at 14,000 g, 4°C, 10 min. The supernatant plasma was pipetted into vials which were kept at -20°C until the analyses were performed. Amino acids and carboxylic acids of analytical grade used as reference compounds were purchased from Sigma, St. Louis. Sephadex G-25 (fine)
335
was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Proteins were separated from amino acids and carboxylic acids by gel filtration on Sephadex G-25 (fine) columns (0.9 x 50 cm) using water as an eluent, flow rate 20 ml/hr, 3 ml/fraction and continuously measuring absorption at 280 nm of the effluent. Amino acid analyses were performed on an automatic amino acid analyzer (Beckman Model 120 C). Absorption spectra were recorded on a Perkin-Elmer 402 UltravioletVisible Spectrophotometer. Paper chromatography (PC) was performed in n-butanolacetic acid-water (12:3:5) (solvent 1), phenol-water-concentrated aqueous ammonia (120:30:1) (w/v/v) (solvent 2), isopropanol-concentrated aqueous ammoniawater (8:1:1 (solvent 3) by the descending technique on Whatman No. 1 paper. High voltage electrophoresis (HVE) was carried out on Whatman No. 3 MM paper using a flat-plate unit (Shandon Model L. 24) in the following systems: (a) Buffer pH 3.6 (Pyridine-acetic acid-water) (1 :10:200), 30 min at 3 kV, and 90 mA; (b) Buffer pH 6.5 (Pyridine-acetic acid-water) (25:1: 500), 30 min at 5 kV, and 90 mA. Chromatograms and electrophoresis papers were dipped in ninhydrin (0.2% in acetone) or in aniline-xylose (1 g xylose in 3 ml water + 1 ml aniline in 100 ml methanol) to reveal carboxylic acids or Pauly's reagent to
detect phenols (7,24), followed by heating at 100°C for 2-5 min. The identity of commercial and isolated amino acids was established by the elution volume from the gel filtration columns, the Rf in PC, the mobility in HVE, the peak retention time from the amino acid analyzer and for the aromatic amino acids by UV spectral comparison. The amount of the amino acids in the samples was established by quantitative ninhydrin determination (22) and peak size from the amino acid analyzer and, in addition, for tyrosine from the 294 nm absorption peak when the compound was dissolved in 1 N NaOH. Plasma concentrations of urea (Berthelot reaction), creatinine (kinetic picric acid method), Na and K (IL 143 flame photometer) Ca, and Mg (IL 153 atomic absorption spectrophotometer) were determined at the Central Laboratory of the Royal Veterinary and Agricultural University. Results from investigations of plasma samples from four normal and three affected kits born in 1976 are presented in Table II. No appreciable differences be-
336
tween the concentrations of Na, K, Mg and Ca in plasma from normal and affected kits were observed. However, the concentrations of urea and creatinine in plasma from affected kits were strongly elevated compared to the concentrations found in plasma from normal kits. Plasma samples from some kits of the litters born in 1977 (Fig. 1) were further investigated for free amino acids. After gel filtration, the proteins appeared in fractions 5-7, phenylalanine and tyrosine in fractions 10-11 and the other free amino acids in fractions 12-20. The fractions containing phenylalanine and tyrosine also contained other aromatic low molecular weight compounds (vide infra) which were extracted with ether after dilution of the fractions with 1 N HCl. Urea, creatinine and tyrosine concentrations were strongly elevated in plasma from affected kits (Table III). Two dimensional PC of free amino acids from plasma samples of these mink kits confirmed the observation concerning tyrosine and showed furthermore that the disease results in significantly lower concentration of all other ninhydrin reacting compounds except for the basic amino acids and phenylalanine. The results obtained by use of HVE and amino acid analyzer showed that about 80% of the basic amino acid pool was ornithine. In addition, results from amino acid analyses of plasma from kit No. 7 (Table III) and a normal kit from the same litter are presented in Table IV. These plasma samples (200 IAI) were obtained five days before killing the animals and they showed that the disease at this age had drastically changed the content of free amino acids in plasma similar to that found in later stages of the disease (Table III) though clinical symptoms were not observable at that time. Further investigations concerning metabolites accumulated in plasma of affected kits were hampered due to the small amount of plasma available. Therefore, it was not possible to make a final identification of the aromatic compounds in the ether extract from acidified solutions of fractions 10-11 (see above). However, preliminary investigations using PC and HVE using 2- and 4-
hydroxyphenylacetate, 4-hydroxyphenylpyruvate, imidazoleacetate and other related carboxylic acids as reference compounds, as well as colour reactions with Pauly's reagent and the reactions on the paper with the aniline-xylose reagent for carboxylic acids indicated that 4-hydroxyphenyl sub-
Can. J. comp. Med.
Table II. Results from Chemical Analyses of Plasma Samples from Mink Kits Born in 1976 (see Fig. 1)
Mink No 1 affected ........................ " ........................ 2 " ........................ 3
Urea 26.7 27.0 28.8
4 normal " 5 " 6 " 7
12.7 12.2 10.5 12.2
........................
........................ ........................ ........................
Concentration in Plasma mM K Creatinine Na 0.08 163 5.1 0.10 6.2 163 148 5.6
0.04 0.04 0.02 0.04
151 155 145 152
4.9 8.3 8.4 5.7
of Ca 2.88 2.80 2.93
Mg 1.37 1.40 1.06
2.80 3.33 3.13 3.16
0.93 1.59 1.39 1.14
Table III. Data for Nine Mink Kits born in 1977 (see Fig. 1) and Results from Analyses of Plasma Samples
Mink No. 1 affected .......... 2 " .
.
.
.
4 5
",,.
6
"
.
.
7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Date at Killing Birth 13.6 1.5 13.6 26.4 13.6 1.5 14.6 30.4 1.5 14.6 14.6 1.5 15.6 27.4
8 normal .......... "p.......... 9
Sex
9 9 9Q 9 9 9
15.6 15.6
Table IV. Amino Acid Analyses of Plasma Samples Obtained from Mink Kits Five Days Before Killing the Animals
Compound Lysine Ornithine Arginine Aspartic acid Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine
T,yrosine Phienylalanine
Concentration in Plasma mM Kit No. 7 Normal Kit (Table III) 0.113 0.009 0.023 0.018 0.110 0.300 0.054 0.270 0.158 0.078 0.048 0.076 0.025 0.032
0.018 0.143 0.012 0.005 0.062 0.198 0.009 0.160 0.063 0.032 0.021 0.037 0.487 0.047
stituted carboxylic acids, but not imidazole derivatives, were present in the plasma of affected kits. The same indication was obtained from measuring the UV-absorption spectra in 1 N HCl and in 1 N NaOH after preparative HVE isolations of the compounds.
In conclusion, the disorder is inherited
Volume 43
July, 1979
as
Concentration in Plasma mM of Creatinine Tyrosine Urea 75.3 1.7 0.14 27.3 0.09 2.8 18.8 2.2 0.05 41.8 2.2 0.09 56.5 1.1 0.17 56.0 2.2 0.13 14.0 1.8 0.08 7.5 6.8
0.02 0.02
0.03 0.03
recessive character, due to homozygosity at a single locus. No indication of pleiotropic effect, coupling between genes, etc. has been observed. The recessive inheritance implies that both the sire as well as the dam of the diseased kits are heterozygous for the gene in question. Such results are also described for hereditary tyrosinemia in man (2,9). Environmental causes (feeding, management, etc.) of the disease can be excluded. No indication of infection as a releasing cause was found. These statements are confirmed by the results of controlled matings at an experimental farm. A mink disease has been described in the USA where the clinical observations were very similar to ours (18). Without presenting definite proof a genetic background was assumed. The disease occurs about one week after the kits have started eating their normal diet which contains at least double the amount of protein per energy unit compared to a normal human diet. Thus, a defect in an enzyme in the tyrosine catabolism will result in accumulation of this amino acid. However, the disease in mink seems to be comparable to the disease in man dea
337
OH
-ICOO*1
+1
2fl NH3
3. NH3
if
~CO-2-b/'
COO-
kCOO~~
2a.
VCOOOH
OH
OH COO~
5.
02
w
ii 1 7.4 coo
-OOC-, OFumarate
+
Acetoacetate
Fig. 2. Catabolic pathways of phenylalanine and tyrosine in animals. The heavy arrows indicate the normal degradation of phenylalanine and tyrosine. The broken arrows indicate degradative pathways with a limited function in the normal catabolism of these amino acids.
338
Can. J. comp. Med.
scribed as hereditary tyrosinemia or hered- are dominating compounds in the urine toitary tyrosinosis. The normal catabolic gether with the citric acid cycle carboxylic pathways of phenylalanine and tyrosine in acids and the aromatic carboxylic acids animals are presented in Fig. 2. The oxida- expected from tyrosine catabolism when the tions 1, 4 and 5 are irreversible reactions enzyme at reaction 4 (Fig. 2) is defect. The catalyzed by the enzymes phenylalanine-4- possibility of a defect in the enzyme fumahydroxylase, 4-hydroxyphenylpyruvate di- rylacetoacetase, reaction 7 in Fig. 2, has oxygenase and homogentisate oxidase, re- therefore been proposed as the primary spectively. The decarboxylations 2b and 3b enzyme defect in hereditary tyrosinemia are irreversible too, whereas the transamin- in man (13). In the diseased mink, the acase catalyzed reactions 2 and 3 as well as cumulation in the urine of succinylacetothe redox reactions 2a and 3a are reversible. acetate and the decarboxylation product The enzymes tyrosine aminotransferase thereof, succinylacetone, has not yet been (EC 2.6.1.5) and 4-hydroxyphenylpyruvate investigated. However, the affected kits dioxygenase (EC 1.13.11.27) are found in showed an increase in the plasma urea conliver and kidneys (3). A low activity of centrations of 2-2.5 times that found for one or both of these enzymes thus leads to the normal kits and a corresponding inaccumulation in these tissues, in blood and crease in the plasma creatinine concentrain urine of 4-hydroxyphenylpyruvate and tions was also observed (Table II and III). products thereof, e.g. tyrosine, 4-hydroxy- The increase in the plasma tyrosine conphenyllactate, and 4-hydroxyphenylacetate, centration and for kit No. 7 also the plasma as shown in Fig. 2 reactions 3, 3a and 3b. ornithine concentration (Table IV) was Relatively high concentrations of these much more pronounced. Thus, it seems compounds are toxic leading to tissue dam- likely that liver enzymes of both the tyroage via binding to the macromolecules sine catabolism and, secondarily, the urea (6,23) and inhibition of enzymes (12,13, cycle are involved in the disease. It is tempting to assume that the lesions 15). In hereditary tyrosinemia in man damage of liver and kidneys occurs (14,16,23) in the urinary system and possibly also as well as accumulation of 4-hydroxyphenyl- those. of the conjunctiva and skin are elicpyruvate, 4-hydroxyphenyllactate and 4-hy- ited by excretion or deposition of qualdroxyphenylacetate in urine (4,13) and ty- itatively or quantitatively abnormal merosine and products thereof in blood (5, 17, tabolites in the affected tissues. 19,21). The inheritance, and the accumulation of tyrosine, together with 4-hydroxyphenyl REFERENCES substituted carboxylic acids in plasma indicate that the described hereditary mink 1. ANDRESEN, E. The effect of ascertainment by trundisease is similar to human hereditary tyrocate selection on segregation ratios. - 1. World Congr. Genetics Applied to Livestock Production, sinemia. Although the accumulation of 01.3, Madrid. pp. 111-114. 1974. DALLAIRE, L. Genetic aspects of tyrosinemia. Can. tyrosine is most commonly believed to occur 2. Med. Ass. J. 97: 1098-1099. 1967. from a deficiency or reduced activity of 3. FELLMAN J. H., T. S. FUJITA and E. S. ROTH. properties and tissue distribution of p-hydroAssay, 4-hydroxyphenylpyruvate dioxygenase (5, xyphenylpyruvate hydroxylase. Biochim. biophys. 10), considerable evidence against a geneActa 284: 90-100. 1972. J., B. LINDBLAD, S. LINDSTEDT. L. tically-determined primary deficiency of 4. GENTZ, LEVY, W. SHASTEEN and R. ZETTERSTROM. Dietary treatment of tyrosinemia (tyrosinosis). Am. this enzyme has appeared in the literature J. Dis. Child. 113: 31-37. 1967. (4,11,15,20). 5. GENTZ, J., B. LINDBLAD, S. LINDSTEDT and R. ZETTERSTROM. Studies on the metabolism of The chemical observations do not allow the phenolic acids in hereditary tyrosinemia by a gas-liquid chromatographic method. J. Lab. elin. a distinction between a defect in the en74: 185-202. 1969. zymes tyrosine aminotransferase and/or 6. Med. GILETTE, J. R. A perspective on the role of chemically reactive metabolites of foreign compounds in 4-hydroxyphenylpyruvate dioxygenase, but toxicity - I Correlation of changes in covalent from the established inheritance of the binding of reactivity metabolites with changes in the incidence and severity of toxicity. Biochem. Phardisease we conclude that only one of these mac. 23: 2785-2794. 1974. GRIMMET. M. R. and E. L. RICHARDS. Separation enzymes is involved. As the accumulation 7. of imidazoles by cellulose thin-layer chromatography. of tyrosine far exceeds that of 4-hydroxyJ. Chromat. 20: 171-173. 1965. I. Cytogenetic, distribution and phe8. GUSTAVSSON, phenyllactate and 4-hydroxyphenylacetate notypic effect of a translocation in Swedish cattle. 63: 68-169. 1969. it is most likely a defect in tyrosine amino- 9. Hereditas LABERGE, C. and L. DALLAIRE. Genetic aspects of transferase. tyrosinemia in the Chicoutimi Region. Can. Med. Ass. 97: 1099-1100. 1967. In tyrosinemia in man the compounds 10. J.LaDU, B. N. The enzymatic deficiency in tyrosinemia. Am. J. Dis. Child. 113: 54-57. 1967. succinylacetoacetate and succinylacetone
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11. LaDU, B. N. and L. R. GJESSING. Tyrosinosis and tyrosinemia. In The Metabolic Basis of Inherited Disease. J. B. Stanberry, J. B. Wyngaarden and D. S. Fr edeirickson, Editors. pp. 296-307. New York: McGraw-Hill Book Co. 1972. 12. LINDBLAD, B., G. LINDSTEDT, S. LINDSTEDT and M. RUNDGREN. Pur-ification and some properties of human 4-hydroxyphenylpyruvate dioxygenase (I). J. biol. Chem. 252: 5075-5084. 1977. 13. LINDBLAD, B., S. LINDSTEDT and G. STEEN. On the enzymic defects in hereditary tyrosinemia. Proc. Natn. Acad. Sci. 74: 4641-4645. 1977. 14. PARTINGTON, M. W. and M. D. HAUST. A patient with tyrosinemia and hypermethioninemia. Can. Med. Ass. .J. 97: 1059-1067. 1967. 15. PERRY, T. L. Tyrosinemia associated with hypermethioninemia and islet cell hyperplasia. Can Med. Ass. J. 97: 1067-1072. 1967. 16. PRIVE, L. Pathological findings in patients with tyrosinemia. Can. Med. Ass. J. 97: 1054-1056. 1967. 17. SASS-KORTSAK, A., S. FICICI, L. RAIJNIER, L. W. KOOH, D. FRASER and S. H. JACKSON. Secondar-y metabolic derangements in patients with tyr-osyluria. Can. Med. Ass. J. 97: 1079-1089. 1967.
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18. SCHWARTZ, T. M. and R. M. SHACKELFORD. Pseudodistemper an apparently new ailment of mink. U.S. Fur Rancher 52. No. 8, p. 6, 1973. 19. SCRIVER, C. R. The phenotypic manifestations of hereditary tyrosinemia and tyrosyluria: A hypothesis. Can. Med. Ass. J. 97: 1073-1075. 1967. 20. SCRIVER, C. R. and E. DAVIES. Investigation in vivo of the biochemical defect in hereditary tyrosinemia and tyrosyluria. Can. Med. Ass. J. 97: 10761078. 1967. 21. SCRIVER, C. R., M. SILVERBERG and C. L. CLOW. Hereditar-y tyrosinemia and tyrosyluria: Clinical repoirt of four patients. Can. Med. Ass. J. 97: 10471050. 1967. 22. S0RENSEN, H. Saccharopine and 2-aminoadipic acid in Reseda odorata. Phytochem. 15: 1527-1529. 1976. 23. WEINBERG, A. G., C. E. MIZE and H. G. WORTHEN. The occurrence of hepatoma in the chr onic form of hereditary tyrosinemia. J. Pediatr. 88: 434-438. 1976. 24. WHITFIELD, A. E. Diazotised sulphanilic acid reagent as an aid to the identification of some oxo acid 2.4-dinitirophenyl-hydrozones. J. Chromat. 20: 401-402. 1965.
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