Hereditary Chondrodystrophy in the Rabbit

Transcription

1 The Journal of Heredity 66: Hereditary Chondrodystrophy in the Rabbit Genetics and pathology of a new mutant, a model for metatropic dwarfism R. R. Fox AND D. D. CRARY M 16 AROTEAUX et a/. delineated a new syndrome called metatropic dwarfism based on 5 personal cases and 12 others gathered from the literature. This condition has been confused at birth with achondroplasia because of the short limbs and relatively long trunk, and as growth progresses with Morquio's disease 18 because of the marked kyphoscoliosis of the axial skeleton and relatively short trunk. Kniest's syndrome 22 also has been confused with metatropic dwarfism 24 but is now considered a separate entity. Subsequent to the initial report by Maroteaux et al., nine additional cases have been reported 2>9 - l2>13>24>25. Bailey 1, in his review article, reported that limited data suggested metatropic dwarfism is inherited as an autosomal recessive condition in man. In addition to these original articles, a source of easy identification and comparison of the metatropic dwarfism syndrome with many other syndromes may be found in the review by Felson 6 in which a brief description of 1 syndromes of dwarfs and other little people is given. Further descriptions with excellent radiographs of the metatropic dwarf are given by Saldino 2, Bailey 1, and Silverman 23. In brief, however, at birth a metatropic dwarf has short limbs, normal length trunk, and a narrow chest features that are common to other bone dysplasias. Of greater significance are the swollen joints that, on radiological examination, show metaphyseal enlargement giving a trumpet-like expansion, hyperlucency, and a thin cortical rim. The bones of all the limbs are shortened with a very noticeable effect on the femur. Retardation of maturation of the epiphyseal centers of the knee is often observed. The Dr.' Fox is a staff scientist, and Mrs. Crary a senior professional research assistant at the Jackson Laboratory, Bar Harbor, Maine 469. This investigation was supported initially by NIH Research Grant HD-1496 from the National Institute of Child Health and Human Development, followed by NIH Research Grant EY-148 from the National Eye Institutes and an allocation from the NIH General Research Support Grant RR-5545 from the Division of Research Resources to the Jackson Laboratory and, in part, by NIH Research Grant RR-251 from the Division of Research Resources. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care. 271 vertebral bodies, or centra, are generally irregular and immature compared with the posterior elements of the vertebrae 2. Neonatal death may result from respiratory problems or pulmonary aspiration secondary to a cleft palate'. In 197 a mutation more closely resembling the metatropic dwarf than any other syndrome in man was observed in the III/J strain of rabbits at the Jackson Laboratory. A brief description of the first four cases and the provisional gene symbol, cd, were reported in Subsequently, 27 additional cases have been observed in closely related rabbits. In this communication we report the mode of inheritance and describe the pathology of this new model in the rabbit for study of the metatropic dwarf in man. Materials and Methods Our data were obtained from matings within the III/J strain (formerly called III or IHmo) of rabbits maintained at the Jackson Laboratory. Crosses also were made between rabbits transmitting chondrodystrophy and those of another strain where this condition has not been observed. In addition to gross observations and radiographs, skeletal measurements of length and width were made on 25 of the 31 chondrodystrophics (ranging in gestational age from 32 to 36 days). Of the other six, three were partly resorbed, therefore not suitable for valid growth comparisons, and three were used either for sectioning or for whole mounts. Normal controls comprised 5 strain III/J rabbits of comparable weight and gestational age, some of which were siblings of affected chondrodystrophics. Measurements were made in centimeters to the nearest half millimeter. Overall length was taken from the base of the skull (or from the anterior cervical border after the head had been removed) to the base of the tail, skull length was from the base of the incisors to the external occipital protuberance, and skull width was measured at the widest part. Nasal length was the length of the midline between the two nasal bones, and width was taken at the broadest part including both nasal bones. Mandibular length was measured from the base of the

2 The Journal of Heredity 272 FIGURE 1 Newborn chondrodystrophic (cdlcd) rabbits: A ventral view of alizarin stained specimen showing aberrant centra, ribs, sternebrae and long bones. B and C external ventral and dorsal views showing general features; note in particular the forelimbs clasped over the chest and talipes varus. D dorsal view.of specimen in A showing relatively normal cranium and neural arches but abnormal scapula, long bones, and ribs. normal newborn rabbit shown for comparison. incisors to the pterygoid tuberosity, and mandibular width was the distance between the pterygoid tuberosities. Lengths of basioccipital and basisphenoid were taken at midline, with widths at the posterior borders. Lengths of long bones were taken from metaphysis to metaphysis, and widths at the narrowest point (approximately mid shaft), except for the scapula that was measured at the posterior border. Student's " t " statistic for unpaired data and chisquare were used for data analyses. Standard genetic ratios were tested for analysis of progeny data. The condition is neither sex linked nor sex limited. There were 17 female, 13 male, and one of undetermined sex in the 31 chondrodystrophic animals. We propose the symbol cd for the gene responsible for this new chondrodystrophic rabbit in accordance with both our original provisional symbol8 and the symbol reported in the Biology of the Laboratory Rabbit7. There is a reduction in litter size from transmitting does of slightly more than one rabbit per litter. This is observed whether the transmitting does are mated to transmitting males or to males of another strain, and suggests a pleiotropic effect of a single dose of the gene. Since the ratio of normal to affected progeny does not deviate appreciably from the expected, the effect on litter size is a general maternal effect on all fetuses and not a genotype-dependent fetal effect. If the latter were true, we should have observed a marked reduction in the number of affected progeny resulting in an aberrant progeny ratio. Results Genetics The first chondrodystrophic rabbits were progeny from a brother-sister mating in the III/J strain. All subsequent specimens were from offspring of this initial pair. A separate substrain, comprising the descendants of this pair, is being established and designated Wiled} (the coefficient of inbreeding at the time of Pathology separation was about.97 or the equivalent of between animals are short, plump, and have 16 and 17 generations of brother-sister mating). Matings firm muscles. Their heads are large and rounded with within this substrain have been primarily sib or parent short broad faces and small features. Their tongues offspring, but some cousin matings were made. The protrude slightly and the incisors are pointed (normally progeny data are strongly indicative of autosomal re- the incisors are square tipped by 32 days of gestation). cessive inheritance. Matings of transmitter by trans- The limbs are short with large joints, the fore feet mitter produced 15 normal and 31 affected progeny, are often clasped over the chest and the hind feet which is in accord with a 3:1 ratio (x2 =.2451) are clubbed (talipes varus) (Figure \B and C). Suband matings of transmitters within the WVcdi sub- dermal hemorrhages, indicative of difficult parturition, strain with nontransmitters within the substrain and with are sometimes present. Most cdlcd rabbits lived to term nontransmitters of the IIFVO/J strain produced 61 and in utero; only three were partly resorbed. Those we 7 progeny, respectively, all of which were normal. observed at the time of parturition gasped only a few

3 Fox and Crary: Chondrodystrophy in Rabbits 273 times and then expired. The cause of death is uncertain, but is probably due to respiratory insufficiency associated with the inability of the lungs to expand in the cramped chest. Birth weights were compared in litters containing both normal and chondrodystrophic progeny with the following results: 24 normal males 68.5 ± 1.6g; 21 normal females 64.7 ± 1.94g; 11 chondrodystrophic males 6.5 ± 2.4g; and 14 chondrodystrophic females 62.5 ± l.53g. The only significant effect was on the depression of birth weight of the chondrodystrophic males when compared with the normal males (P <.1). Internally the thyroid cartilage is enlarged as are the cartilagenous ends of the long bones. The tracheal cartilage is compressed. The palate is cleft in all but one (97 percent) of the animals. The tooth rows are arched laterally and development of molars and premolars is somewhat erratic. Normally the upper molars and premolars appear to develop slightly in advance of the lower ones, i.e., there are four uppers more frequently than four lowers. Among 49 normals only one animal had four lowers (2 percent) whereas all but three (94 percent) had four uppers at least on one side. In 25 cdlcd animals there were only three upper and three lower molars in 36 percent of the animals as compared to 6 percent of normals. However, 44 percent had four lowers at least on one side compared with 2 percent of normals. Normals, on the other hand, had four uppers more frequently than did cdlcd animals (94 percent vs. 4 percent). Affected rabbits had significantly more cardiovascular anomalies (11/31) and bifurcated appendices (7/31) than their normal sibs (2/13 and 6/13 respectively; P <.1 in both cases). All other internal organs appeared normal except for the thyroid, which was large. Examination of the skeleton of cdlcd animals showed a well ossified skull with a small normal fontanelle (Figure ID). The shape of the foramen magnum was variable. In 67 percent of the rabbits it was longer than broad, being either pear-shaped or diamond-shaped, sometimes with both ventral and dorsal notches. It was broader than long 29 percent and round 4 percent of the time. In contrast, the foramen magnum in normal animals is predominantly broader than long (86 percent of the time), being oval or round with an occasional dorsal notch. It was longer than broad only 2 percent and round 12 percent of the time. Small extra centers of ossification lateral to and articulating with the exoccipitals either bilaterally or unilaterally were found in 84 percent of cdlcd animals and none in normals. The body of the hyoid was bipartite or tripartite in 6 percent of cdlcd rabbits and the arches were broadly flaring. Only 12 percent of normal animals had bipartite hyoid bodies. Neural arches of chondrodystrophic rabbits were essentially normal except for a few small ones in cervical and lumbosacra! regions. Centra, on the other hand, were highly variable throughout the vertebral column. They were absent, underdeveloped, bipartite, or asymmetrical (Figure LA). They were most nearly normal in the posterior thoracic and anterior lumbar regions. Most of the defective centra were clustered around six ventral depressions occurring from vertebra 2-4 (anteriorcervical), 7-9 (cervicothoracic), 2-21 (thoracolumbar), (posterior lumbar), (sacrocaudal) and (caudal). The exception was the peak of poor development on vertebra 12 with no depression. However, centra in the thoracic area from vertebra 1-18 were almost without exception present but merely abnormally developed. Ribs were short, bowed, and had flared ends (Figure \A and D). There was no difference in numbers of presacral vertebrae and pairs of ribs between normal and cdlcd animals. Sternebrae normally are somewhat variable in expression in the rabbit, usually 6 in number, but occasionally a small seventh sternebra inserted between 5 and 6 can be observed in some genetic backgrounds 17. In the present population, only one normal animal had a small bipartite 7th sternebra and none of the cdlcd animals did. In the normal population 2 percent of the animals had small or bipartite 5th sternebrae and 16 percent had small, asymmetrical or bipartite 6th sternebrae. Only minimal reduction was seen on sternebrae 2, 3, and 4 occurring 6, 4, and 6 percent of the time, respectively. The cdlcd rabbits, on the other hand, showed much more variation, with 88 percent having small, absent, or bipartite 5th sternebrae, 35 percent having a similar effect on sternebra 6, 27 percent on sternebra 4, and 19 percent on sternebra 2. The effect on sternebra 3 was minimal (4 percent). In neither normal nor chondrodystrophic rabbits was there any reduction of sternebra 1. The appendicular skeleton of cdlcd animals is well ossified. The scapulae are broad with wavy, flared distal ends. The clavicles appear high. Long bones of the limbs are short, the diaphyses somewhat bowed. The epiphyseal cartilages are greatly enlarged and the metaphyses are broadly flared with a thin hyperlucent cortical rim (Figure \A and D). The ossification centers of the epiphyses of the long bones were present in varying degrees in both normal and cdlcd animals. Table I shows the order in which the centers appeared inferred from the relative incidence in the present population. This incidence is similar to that found in Table I. The relative incidence in percent of the ossification centers of the epiphyses in newborn normal and chondrodystrophic rabbits Epiphyseal center Proximal humerus Distal humerus Proximal ulna Proximal radius Distal radius Distal ulna Distal femur Proximal tibia Proximal femur Distal tibia Distal fibula Proximal fibula Normal Bilateral Unilateral Bilateral Unilateral

4 274 The Journal of Heredity strain III rabbits 4. The incidence in cdlcd animals was much less than for normals except for proximal humerus in which 96 percent of cdlcd animals had an epiphyseal center at least on one side. The order of appearance was the same for chondrodystrophic and normal rabbits except that the distal ulna developed before the distal radius in cdlcd animals and in no cdlcd rabbits were there centers for proximal radius, distal tibia, or distal fibula. The cd gene, therefore, not only retards the ossification of the epiphyseal centers, but also changes the order of their appearance to some extent, at least in the forelimb. Results of measurements of skull and long bones of normal and cdlcd animals showed that, except for length of mandible and width of basisphenoid, all lengths of cdlcd animals were significantly reduced and all widths were significantly increased (Table II). The decrease in limb length is greater than the effect on skull length, with the effect in the limbs being greater in the proximal portion. The effect on width is also greater in the limbs than in the skull, but the effect on limb width is greater distally. Discussion Genetic analysis of the data clearly indicates autosomal recessive inheritance for chondrodystrophy {cdlcd) in the rabbit. This is in accord with the review by Bailey 1 on the metatropic dwarf in man. The data in man, however, are too limited to be conclusive. Direct comparison of the phenotype of the metatropic dwarf in man with the chondrodystrophic dwarf {cdlcd) in the rabbit can best be seen in Table III. This table shows clearly that while there is not perfect agreement of the anatomical differences, the major diagnostic criteria are in excellent accord. In both the chondrodystrophic rabbits and the examples of the metatropic dwarf, the bodies, or centra, of the vertebrae are undeveloped, irregular, and immature compared with the vertebral arches and costal processes, which are essentially normal. In the developing vertebrae differentiation occurs so that the cells of each protovertebra are more closely packed together in the caudal half (posterior portion of sclerotome) and more loosely arranged in the cephalic half (anterior portion of sclerotome) 1. From the caudal portion dorsal and lateral extensions arise to form the vertebral arches and the costal processes. The cephalic portion of one segment plus a small part of the preceding caudal segment give rise to the vertebral centra. The similarity of the abnormal centra in both species and the differential effect on two separate portions of the vertebrae of differing embryological origin suggests that the gene effect in the rabbit, and possibly in man, acts on the cephalic but not the caudal half. This would result in the centra, primarily from the more loosely packed cephalic half, being retarded and defective, whereas the vertebral arches from the denser caudal portion would be essentially normal. Appropriate embryological studies will be necessary to determine the validity of this hypothesis and the time and location of gene action. Comparison of the effects of the cd gene on the skeleton with those of the dachs {Da) 19 and the achondroplasia (ac) 3 ' 5 ' 11 genes reveals some interesting differences. The cd gene decreases lengths and increases widths in contrast to the ac and Da genes both of which decrease lengths and widths at birth 3>1I>19. In the DA strain at 31 days of. gestation normal animals had ossified epiphyseal centers of proximal and distal humerus, distal femur and proximal tibia in 1 percent of the animals. Fewer Da/Da animals had these centers present, but in addition a small percentage had centers of distal radius, distal ulna, and proximal femur 19. In strain AC/J (previously called AC) at 31 days of gestation 5 normals had centers of proximal and distal humerus, distal femur, and proximal tibia in 1 percent of animals and proximal femur in 62 percent. By 32 days of gestation all except the proximal fibula may be present. In aclac rabbits no epiphyseal centers are ossified prior to 32 days of gestation and then ossification occurs in only 2 percent Table II. Skeletal measurements of length and width of bones of newborn normal and chondrodystrophic rabbits Length* Width* Measurements Normal P Normal P Overall length r.785 ±.58 Hyoid Skull ;..968 ±.18 Nasal ().822 : t.9 Mandible.95 : t.13 Basioccipital I).511 : t.6 Basisphenoid Scapula Humerus Radius Ulna Femur Tibia-fibula.419 ii it : t : t : t it dt ± : t : t : t : t di.8.71 : t : t : t : t Ht it.18 <.1 <.1 <.1 >.5 <.1 <.1 <.1 <.1 <.1 <.1 <.1 < ± ± ± ± ± ±.3.81 ± ± ±.3.12 ± ±.3.29 ± ± ±.2.92 ± ± ± ± ± ±.6.26 ± ±.4.36 ±.7.37 ±.7 <.1 <.1 <.1 <.1 <.1 >.5 <.1 <.1 <.1 <.1 <.1 <.1 Mean ± SE (in centimeters)

5 of the animals. Crary and Sawin 4 showed that the first appearance of ossification centers of the epiphyses is due to a slowing of growth of the long bones in question. Thus it may be inferred that growth in cd/cd and Da/Da rabbits is slower and does not reach the critical reduction in rate quite as soon as in normals and that the order in which the bones slow in growth is, to some extent, different. That any aclac animals have any epiphyses ossified is surprising in light of their extreme immaturity. Both Da and ac have been put on different strain backgrounds with the effects of the genes remaining very similar. All three of the genes either now are {cd or Da) or have been (ac) on strain III background where their individual effects remain as stated. The effects of these three genes on the skeleton, therefore, are markedly different and not the result of a strain background effect. An interesting observation is that the "depressions" found in the vertebral column of cd/cd animals correspond closely with the regional borders, or areas of lesser growth, described by Sawin and Crary 21. These regional borders define areas of greater growth that appear in a wave-like pattern along the length of the vertebral column. That a gene affecting the development of the skeleton, as cd does, should have a more pronounced effect in regions that are normally retarded during development is not at all surprising. Summary Thirty-one cases of a new hereditary chondrodystrophy in the rabbit have been shown to be associated Fox and Crary: Chondrodystrophy in Rabbits 275 with a fully penetrant autosomal recessive gene symbolized cd. The mutant is viable prenatally but does not survive after birth. It differs from the two other inherited chondrodystrophies, dachs and achondroplasia, but is very similar to the metatropic dwarf reported in man. Literature Cited 1. BAILEY, J.A., II. Forms of dwarfism recognizable at birth. Clin. Orthopaed. Relat. Res. 76: , J.P. DORST, and R.W. SAUNDERSON, JR. Metatropic dwarfism, recognized retrospectively from the roentgenographic features. Proc. 1st Conf. on Clin. Delineation of Birth Defects. Part IV. Bone dysplasias. D. Bergsma, Ed. In Birth Defects: Orig. Art. Series, The Natl. Fdn. N.Y. 5: COVALT, D. A quantitative and qualitative study of the effects of the ac gene on the newborn rabbit skeleton. Summer Student Report. The Jackson Laboratory, Bar Harbor, Maine CRARY, D.D. and P.B. SAWIN. Morphogenetic studies of the rabbit. VI. Genetic factors influencing the ossification pattern of the limbs. Genetics 34: and. Morphogenetic studies of the rabbit. XXXII. Qualitative skeletal variations induced by the ac gene (achondroplasia). Amer. J. Anat. 113: FELSON, B., Ed. Dwarfs and other little people. Seminars in Roentgenol. 8: Fox, R.R. Taxonomy and genetics. Chapter 1. In Biology of the Laboratory Rabbit. S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, Eds. Academic Press, New York and D.D. CRARY. A new recessive chondrodystrophy in the rabbit. Teratology 4:2< Table ID. A comparison of the pathological findings of the metatropic dwarf in man with the chondrodystrophic dwarf (cd/cd) in the rabbit 1. External a. Short limbs with swollen joints b. Relatively normal length trunk Pathology c. Narrow chest d. Long slender fingers & toes e. Tail-like appendage in region of coccyx f. Joint movement is restricted 2. Radiographic findings a. Metaphyseal enlargement of long bones (the metaphyses show trumpet-like expansion, hyperlucency and a thin cortical rim) b. Shortened limbs have dumbbell-like appearance (particularly the femurs) c. Delayed maturation of the epiphyseal centers of the knee d. Vertebral bodies (centra) are generally irregular and immature e. Shortened and flared ribs f. Well ossified sternum g. Cleft palate h. Cranium normal 3. Viable postnatally Metatropic dwarf in man at birth but shorter with age slight but subject to early respiratory death dwarf in rabbit shorter at birth present normally severe no no

6 276 The Journal of Heredity 9. GEFFERTH, K. BeitragezurDiagnostik des metatropischen Zwergwuchses. Z. Kinderheilk. 13: GRAY, H. Osteology. In Anatomy of the Human Body. C. M. Goss, Ed. 27th edition. Lea and Febiger, Philadelphia, p HOOF, D. The effects of the achondroplasia (ac) gene on the skull development in the rabbit. Summer Student Report. The Jackson Laboratory, Bar Harbor, Maine JENKINS, P., M.B. SMITH, and J.S. MCKINNELL. Metatropic dwarfism. Brit. J. Radiol. 43: LAROSE, J.H. and B.B. GAY, JR. Metatropic dwarfism. Am.J. Roentgenol. 16: MAROTEAUX, P. The chondrodystrophies detectable at birth. In Congenital Malformations. F. C. Fraser and V. A. McKusick, Eds. Excerpta Medica, Amsterdam, p Spondyloepiphyseal dysplasias and metatropic dwarfism. In Birth Defects. D. Bergsma, Ed. Proc. 1st Conf. on Clin. Delineation of Birth Defects. Part IV. Bone Dysplasias. Orig. Art. Series, The Natl. Fnd. N.Y. 5: , J. SPRANGER, and H-R. WEIDEMANN. Der metatropische Zwergwuchs. Arch. Kinderheilk. 173: PECK, E.D. and P.B. SAWIN. Morphogenetic studies of the rabbit. VIII. Genetic variations in the sternum as determined by the interaction of general and of regionally specific growth factors. J. Exp. Zool. 114: POHL, J.F. Chondro-osteodystrophy (Morquio's disease). Progressive kyphosis from congenital wedge-shaped vertebrae. J. Bone Joint Surg. 21: RAFFERTY, S. The effects of the dachs gene on the skeleton of rabbit newborn. Summer Student Report. The Jackson Laboratory, Bar Harbor, Maine SALDINO, R.M. Radiographic diagnosis of neonatal shortlimbed dwarfism. Med. Radiog. Photog. (Eastman Kodak) 49: SAWIN, P.B. and D.D. CRARY. Genetics of skeletal deformities in the domestic rabbit (Oryctolagus cuniculus). Clin. Orthopaed. Relat. Res. 33: SIGGERS, D.C., D.L. RIMOIN, J.P. DORST, S.B. DOTY, B.R. WILLIAMS, D.W. HOLLISTER, R. SILBERBERG, R.E. CRANLEY, R.L. KAUFMAN, and V.A. MCKUSICK. The Kniest syndrome. In Birth Defects. D. Bergsma, Ed. Skeletal Dysplasias-Boston Conf. Orig. Art. Series, The Natl. Fnd. N.Y. 1: SILVERMAN, F.N. A differential diagnosis of achondroplasia. Radiol. Clin. of N. Am. 6: Discussion on spondyloepihyseal dysplasia and metatropic dwarfism. In Birth Defects. D. Bergsma, Ed. Proc. 1st Conf. on Clin. Delineation of Birth Defects. Part IV. Bone Dysplasias. Orig. Art. Series, The Natl. Fnd. N.Y. 5: SUGIURA, Y., Y. TERASHIMA, K. WATANABE, and K. IWASE. Metatropic dwarfism. Ann. Genet. 17: