Comparative Medicine Copyright 2013 by the American Association for Laboratory Animal Science
Vol 63, No 4 August 2013 Pages 342–347
Case Report
Multiple Complex Congenital Malformations in a Rabbit Kit (Oryctolagus cuniculi) Jennifer L Booth,1 Xuwen Peng,1 Jennifer Baccon,2,3 and Timothy K Cooper1,2,* Congenital malformations may occur during early embryogenesis in cases of genetic abnormalities or various environmental factors. Affected subjects most often have only one or 2 abnormalities; subjects rarely have several unrelated congenital defects. Here we describe a case of a stillborn New Zealand white rabbit with multiple complex congenital malformations, including synophthalmia, holoprosencephaly, gastroschisis, and a supernumerary hindlimb, among other anomalies. There was no historical exposure to teratogens or other known environmental causes. Although not confirmed, this case was most likely a rare spontaneous genetic event.
Congenital malformations occur when there is derangement of the embryologic developmental process. Neural development and organogenesis is a critical time of development that occurs during early embryogenesis.2,37 Congenital malformations that manifest at this stage of development may occur in association with various genetic abnormalities, such as point mutations and chromosomal abnormalities.25,29 In addition, environmental factors, including maternal health status, nutritional deficiencies, and exposure to teratogenic drugs or chemicals, may play a role in the development of congenital malformations.12,25 However, in 65% to 75% of human cases, the cause is unknown, resulting from a complex set of events such as polygenic or multifactorial genetic disorders, spontaneous genetic errors, and synergistic interactions of teratogens.3,25 Approximately 78% of human cases demonstrate only a single developmental malformation, with cardiovascular defects accounting for approximately 30% to 35% of organ defects. Cases of more than 2 or 3 malformations in a single person are extremely rare.28 Here we describe a New Zealand white rabbit that was stillborn with numerous complex developmental abnormalities, including synophthalmia, a supraoptic proboscis, holoprosencephaly with other associated craniofacial deformities, Chiari malformation type I, gastroschisis, a supernumerary hindlimb, a fused (horseshoe) kidney with a supernumerary kidney, and male pseudohermaphroditism.
Case Report
This breeding colony of New Zealand white rabbits was established in an AAALAC-accredited facility at Penn State University (Hershey, PA). All breeding protocols were reviewed, approved, Received: 21 Dec 2012. Revision requested: 04 Feb 2013. Accepted: 15 Feb 2013. Departments of 1Comparative Medicine, 2Pathology, and 3Neurosurgery, Penn State Hershey College of Medicine, Hershey, Pennsylvania. * Corresponding author. Email: [email protected]
and performed in accordance with the guidelines of the IACUC at Penn State Hershey and the Guide for the Care and Use of Laboratory Animals.15 The animal room was maintained at 19 ± 3 °C with a relative humidity between 30% to 70% and a 12:12-h light:dark cycle (lights on, 0700 to 1900). Rabbits were singly housed in double stainless-steel cages attached via a pass-through doorway, with grated floors. Space requirements were adequate in regard to the standards set in the Guide. Each housing unit contained a plastic nesting box. Dams were fed a commercial pelleted feed (no. 8630, Harlan Teklad, Madison, WI) with access to fresh water ad libitum and supplied by an automatic watering system. Rabbits in this colony are free of Encephalitozoon cuniculi, rotavirus, Treponema spp., cilia-associated respiratory bacillus, Clostridium piliforme, Pasteurella multocida, coccidian, and other intestinal and external parasites. Rabbits in this breeding colony included transgenic long QT syndrome type 1 male rabbits and normal (wildtype) female breeders. The transgenic male breeders came from a line derived from pronuclear injection of the human gene KCNQ1 Y315S (KVLQT1) under the control of the rabbit β-myosin heavy chain.4 Breedings in this colony were performed between transgenic male and wildtype female rabbits only. All wildtype female breeders in this colony were purchased from Millbrook Labs (Amherst, MA) and were replaced every 4 to 5 litters. A normal mating of a 15-mo-old wildtype dam and a 3-y-old transgenic long QT syndrome type 1 sire produced a litter of 6 kits after an unremarkable gestation. Two kits were stillborn, whereas the other 4 were viable and healthy. A necropsy was performed on one of the stillborn rabbit kits. Grossly, the head of the kit was severely malformed and smaller than normal, with no openings for the ears, mouth, or nose (Figure 1 A). There was a single large but otherwise grossly normal eye located in the center of the face. Dorsal to the eye was a tubular bony appendage (proboscis) measuring 1.4 × 0.5 × 0.5 cm, with a small, red, pedunculated structure (diameter, 2 mm) protruding from the rostral tip (Figure 1 B). Ear pinnae were present bilaterally but slightly curled.
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Figure 1. (A) Ventral view of the entire length of the body demonstrating cyclopia with proboscis and absence of oral and nasal orifices and gastroschisis without an omphalocoele. The supernumerary right kidney is visible at the cranial margin of the defect of the ventral abdomen. There are 3 well-formed hindlimbs. (B) Lateral view of head. Cyclopia with proboscis and absence of normally developed lower craniofacial structures. (C) The ventral skin of the cervical region is reflected to reveal the tongue and a rudimentary oral cavity. The esophagus and trachea are not visible and are covered by a large bony structure. (D) Dorsal view of the caudal half of the kit, illustrating supernumerary limb with a laceration or defect through the skin and muscle left of the dorsal midline.
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The anatomy of the ventral cervical structures was abnormal (Figure 1 C). Reflecting the skin of the ventral neck revealed a grossly normal tongue and rostrally truncated oral cavity. The trachea and esophagus were not grossly apparent. There was a large bony structure within the ventral cervical region, extending from the head to the thoracic inlet, measuring approximately 10 × 5 mm. The lungs did not float in formalin. The small intestines, large intestines, cecum, liver, and stomach were displaced outside of the body, anchored by the distal colon, esophagus, and caudal vena cava through a 1-cm midline defect of the abdominal body wall and skin. The organs were not enclosed in a membranous sac. A single pale-pink kidney partially protruded through the cranial margin of the abdominal defect. It had a spherical rather than reniform shape. In addition, there were 2 other retroperitoneal kidneys, normal in color but fused at the cranial poles, resulting a bilobed appearance (horseshoe kidney), and located slightly left of midline. There was a small amount of fecal material (meconium) present within the intestines and cecum. There were 3 hindlimbs present (polymelia, Figure 1 D). The outermost (supernumerary) limb on the fetus’ left side had moderately less muscle mass than did the other 2 hindlimbs. The thigh of the middle hindlimb had a moderately wider circumference than did the other 2 hindlimbs. All limbs appeared to be normal in length, with no arthrogryposis, and each ended in a normally formed foot. There was a 12 × 6 mm laceration or defect through the skin and muscle, extending just left of the dorsal midline from the pelvic region to the lateral thigh of the middle hindlimb and just medial and dorsal to the leftmost limb. A swelling resembling external genitalia was present between the supernumerary limb and the abdominal body wall. The dorsal skin defect was medial to the genitalia. There was a well-formed tail present between the 2 other (normal) legs. Forelimbs were grossly normal. The ribs extended to just cranial of the pelvis, making the actual abdominal cavity severely shorter in length than normal. Tissues were fixed in 10% neutral buffered formalin or Bouin solution (head). Tissues were processed in an automated processor (Tissue-Tek VIP, Sakura Finetek USA, Torrance, CA) and paraffin-embedded (Tissue-Tek TEC embedding station, Sakura Finetek USA). Sections were cut at 6 µm for routine staining with hematoxylin and eosin. All images were obtained by using a microscope (model BX51, Olympus America, Center Valley, PA), digital camera (model DP71, Olympus America), and cellSens Standard 1.6 imaging software (Olympus America). A sagittal section of the head contained a large rostral supraorbital proboscis with a central core of irregular segmented hyaline cartilage surrounded by peripheral trabeculae of lamellar bone (Figure 2 A). There was a mushroom-like process at the tip, with a hyaline cartilage core covered by highly congested haired skin. The olfactory bulb of the brain extended partway into the proboscis, dorsal to the cartilage core. Multiple sinus hairs (vibrissae) were present in the skin of the proboscis. The retina of the eye was longer than normal, with retinal redundancy and rosettes. The internal and external nuclear layers were irregularly thickened and occasionally merged. Retinal outer nuclear layer cells focally infiltrated the rostral optic nerve. The rostral end of the oropharynx and nasopharynx were not patent, coming to a blind end caudoventral to the eye. Anlages of trachea and larynx were present. There was a single large central ventricle of the forebrain that opened to the dorsal surface of the brain in the center of the rostral–caudal axis. There was no cortex overlying the ventricle
Figure 2. (A) Sagittal section of the decalcified head with single midline eye, supraorbital proboscis, holoprosencephaly with hippocampal dysplasia, and herniation of the cerebellum through the foramen magnum. A scanned digital version of this slide can be accessed at the Penn State Zebrafish Atlas (http://zfatlas.psu.edu/view. php?s=35351&z=7&c=48034,28974). (B) Fusion of 2 kidneys at the cranial poles. There are 2 distinct renal pelves. Hematoxylin and eosin stain.
here, and the choroid plexus and pineal gland lay directly ventral to a thin fibrous membrane that formed the dorsal cranial vault at this site. The hippocampus was oriented abnormally and formed the cranial margin of the dorsal cerebral defect. The cerebellum was partially displaced caudally through the foramen magnum and was markedly distorted by compression. The choroid plexus of the fourth ventricle was in the vertebral canal between the first and second vertebral vertebrae. The cranial poles of 2 kidneys were fused, but 2 distinct renal pelves were maintained (Figure 2 B). The renal pelvis of the separate third kidney was mildly dilated. All kidneys had an immature renal cortex, with predominantly fetal glomeruli. An immature (fetal) testis and epididymis was present adjacent to the kidney. Cross sections of the spine at the level of the skin defect showed that the defect was lined by a capsule of multiple layers of plump fibroblasts in a loose and edematous stroma. A ganglion and several myelinated nerve fibers were present within the defect. In a separate section through the same tissue, there was a cross-section of spinal cord near the cauda equina, with multiple cyst-like blood-filled lakes in the neuropil. Cross-sections of the spine cranial
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to the defect revealed multiple blood filled lakes (acute hemorrhage) present in the white and, to a lesser extent, gray matter of the spinal cord. In one section, erythrocytes filled the central canal. The slide containing cross-sections of the spine caudal to the defect included sections of normal vagina and uterus. The lungs were markedly atelectatic, consistent with the diagnosis of stillbirth. The heart was normal, with no defects.
Discussion
Congenital malformations may occur due to abnormalities during the early embryogenesis stage of development. The cause of these congenital malformations is not always well understood. Some of these deformities have been linked to etiologies such as environmental factors, including exposure to teratogens, and maternal nutritional or health status. Genetic aberrations of chromosomes, expression of autosomal recessive conditions linked to inbreeding, and various gene mutations, deletions, and duplications have also been identified as underlying causes of congenital malformations.17,30 Under many circumstances, the cause of the congenital defect cannot be identified. Many pregnancies, in which the fetus has a severe congenital abnormality, will not be carried to full term. According to the World Health Organization, approximately 50% of all human pregnancies result in spontaneous abortion, with chromosomal abnormalities being responsible for those which occur in the first trimester.8 Neonates that are carried to full term with congenital defects most often have only one or 2 abnormalities. It is very rare for a subject to have several malformations. In this particular case, this rabbit kit was stillborn with numerous complex and severe developmental abnormalities. Holoprosencephaly is a condition in which there is impairment of midline cleavage of the forebrain during fetal development. This failure of division often is accompanied by several facial malformations, such as midline facial clefts, cyclopia (synophthalmia), and absence of the nasal cavity with a supraorbital proboscis.16,25 Holoprosencephaly may occur due to exposure to teratogenic drugs or environmental factors during the organogenesis stage of fetal development. Maternal diabetes and alcohol consumption have been identified as environmental factors associated with holoprosencephaly.25 Cyclopia in rabbits has been experimentally documented to occur when exposed to drugs or chemicals such as thalidomide, phthalimidobenzene, methamphetamine, and cyclopamine and jervine, which are derived from the Veratrum plant genus.13,18 The pathogenic mechanism through which V. californicum causes these malformations involves the molecular signaling along the Sonic Hedgehog (SHH) pathway.6,14,25 Genetic deletions or mutations of the SHH gene can result in this developmental defect.7,33 Genetic errors with chromosomal abnormalities have been identified as a cause of holoprosencephaly, with trisomy 13 being the most common.11,25 At least 12 chromosomal regions on 11 chromosomes have been implicated to play a role in holoprosencephaly.11 Cyclopia has previously been documented as an uncommon isolated defect in rabbits and suggested to be caused by a recessive gene, cy.21 The cause of holoprosencephaly in this rabbit kit was most likely due to a genetic error. Confirmation would require genetic testing. Chiari malformation type I of the cerebellum occurs congenitally when there is an abnormally small cerebellar fossa, which causes the growing cerebellum to herniate through the foramen magnum. This type of malformation may be a result of defective
embryogenesis or birth injury;27 in the case we present, defective embryogenesis is more likely. A genetic cause for Chiari malformation type I has not been identified but has been supported through several reports of human familial cases and the association of Chiari malformation type I with other genetic conditions.22 Chiari malformation type I has been surgically induced in rabbits,10 but there are no previous reports of this abnormality occurring spontaneously in rabbits. During fetal development of the limbs, the somatopleuric mesenchyme interacts with the overlying ectoderm to cause proliferation of the mesenchyme and formation of an apical ectodermal ridge.19 This site, known as the zone of polarizing activity, is the position of further limb development. The patterning and ordered arrangement of the skeletal elements of the limbs is dependent on the SHH gene. The mechanism of action is unknown. Moreover, SHH expression has been identified to occur in the zone of polarizing activity at the posterior margin of the limb bud during development. When an ectopic zone of polarizing activity occurs in the anterior limb margin, it results in the development of extra digits. A polydactyly mouse mutant carries a sasquatch (Ssq) mutation located 1 Mb from SHH that disrupts SHH expression. Humans that are heterozygous for the SSQ mutation develop normal forelimbs, but the hindlimbs have preaxial polydactyly.14 Altered expression of SHH in the rabbit we present might provide a plausible link between the development of a supernumerary hindlimb and cyclopia. However, these 2 malformations have not been reported to occur simultaneously in the human or animal populations. Alternatively, these 2 anomalies could have occurred independently. The occurrence of spontaneous conjoined twins in rabbits has been reported5 but is extremely rare. The extent of fusion between the twins can vary in degree. Some conjoined twins have normal development of the body parts that are not fused, whereas other twins have an autosite–parasite relationship.5 The parasitic twin is often underdeveloped, with multiple malformations, and is dependent on the autosite, which has relatively normal development. Monozygotic twins are formed from the incomplete division and separation of a single blastocyst. The mechanism of this phenomenon is not understood, and evidence for genetic or environmental association is sparse.5,26 Caudal duplication anomaly (dipygus) is another abnormality that has been well documented in the human literature but that has never been described to occur in rabbits. Many authors argue whether there is a relationship between caudal duplication and monozygotic twinning.20 An etiologic pathogenesis has not yet been explained. The distal caudal end of the trunk may be affected by a variety of anomalies, including complete agenesis, fusion, partial splitting, and complete duplication of organs. Spina bifida, malformed caudal spinal cords, supernumerary kidneys, and duplicate genitalia are among the abnormalities that can occur.9,35 This syndrome could represent an alternative theory to the monozygotic twinning and may explain the presence of several of the anomalies in this rabbit kit. Gastroschisis is a rare congenital defect of the abdominal wall through which the abdominal organs eviscerate. The pathogenesis of this anomaly is not understood, with many proposed hypotheses. However, specific risk factors for susceptibility have been identified in humans, including young maternal age, primigravidity or primiparity, lower prepregnancy body mass index, poor maternal diet, the use of vasoactive medications, maternal
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infection, and other environmental factors.16 Many of these risk factors do not apply to this dam. She was multiparous, in good body condition, received adequate nutrition, and had no history of medications or exposure to other environmental factors. Gastroschisis seems to be widespread in occurrence and has not been clearly linked to a genetic cause.34 Experimental exposure to teratogens, such as thalidomide, vinblastine, and clomiphene, have been demonstrated to cause gastroschisis in rabbits.13 This rabbit kit had a single fused kidney (horseshoe kidney) and a separate well-formed supernumerary kidney that was grossly and histologically normal, with the exception of mild hydronephrosis. This rare anomaly is often an incidental finding; however in two-thirds of cases, symptoms such as urinary tract infections and renal or ureteral calculi develop. During normal embryologic development of the kidneys, the mesonephric duct bilaterally gives rise to the ureteric bud, which grows cranially into the metanephric blastema. This metanephric mass ultimately becomes the adult kidney. A supernumerary kidney is developed when there is a secondary outpouching of the nephrogenic cord into 2 metanephric blastemas, resulting in a partial or completely duplicated ureteral bud. The lower branch of this division often fuses with the left kidney, giving it a horseshoe appearance.31,36 Renal fusion has been induced experimentally in rabbits that were exposed to teratogens, such as thalidomide and L-asparaginase.13 Male pseudohermaphroditism is defined as the presence of male gonads with female genitalia. This type of reproductive development may occur with any genetic defect that impairs androgen synthesis or activity and with failure of Müllerian duct regression.24,32 The SRY gene on the Y chromosome is responsible for the initiation of testis development.4 Sertoli cells secrete Müllerian inhibiting factor, which causes regression of the Müllerianassociated structures. When Müllerian inhibiting factor is deficient, the affected subject develops reproductive structures of both Müllerian and Wolffian origin.32 The production of testosterone and 5α-dihydrotestosterone are responsible for the development of Wolffian duct structures and external male genitalia. Mutation of the androgen receptor causes complete or incomplete testicular feminization, in which the affected subject develops external female genitalia but internal male reproductive structures and gonads.23,24,29 Disorders of sexual differentiation in rabbits have not been previously described, but the case we present is consistent with persistent Müllerian duct. The defect in the caudodorsal body wall near the supernumerary limb was of undetermined etiology. Although spina bifida was suspected in light of the gross appearance, a simple musculocutaneous defect or postmortem trauma from the dam could not be excluded. Surprisingly, no cardiovascular malformations were present. Neither this breeding pair nor the entire breeding colony have been associated with any other reports of kits with congenital malformations. Given the clinical and breeding history of the rabbits in this colony, the case we present was most likely an isolated and rare spontaneous event that undoubtedly had some genetic influence. Whether the anomalies of the caudal end of the kit were due to monozygotic twinning or another pathologic entity such as caudal duplication anomaly is unclear. Karyotyping for chromosomal abnormalities and gene sequencing for mutations that are known to be associated with the witnessed anomalies in this rabbit would have been ideal. However, we did not have
any unfixed tissues or specimens available for testing nor any gene candidate(s) for sequencing. Genetic tools for testing in rabbits are very limited and only available on a research basis, thus presenting many difficulties for definitive diagnoses. In addition, rabbit gene polymorphisms are poorly documented.
Acknowledgments
We thank Dr Ronald Wilson, Chair of the Department of Comparative Medicine and director of the laboratory animal medicine residency training program, and the Comparative Medicine histology lab staff, Weifang Lin and Ellen Mullady. Jean Copper, Steve Peckins, and Dr Keith Cheng administer the Penn State Zebrafish Atlas, funded by NIH grant R24 OD011152 (formerly RR017441).
References
1. Alam AR, Cho YG, Cho SJ, Lee JI, Lee HB, Tae HJ, Kim IS, Kim NS. 2007. Male pseudohermaphroditism in dogs: 3 case reports. Vet Med (Praha) 52:74–78. 2. Barkovich AJ, Millen KJ, Dobyns WB. 2009. A developmental and genetic classification for midbrain-hindbrain malformations. Brain 132:3199–3230. 3. Brent RL. 2004. Environmental causes of human congenital malformations: the pediatrician’s role in dealing with these complex clinical problems caused by a multiplicity of environmental and genetic factors. Pediatrics 113:957–968. 4. Brunner M, Peng X, Liu GX, Ren XQ, Ziv O, Choi BR, Mathur R, Hajjiri M, Odening KE, Steinberg E, Folco EJ, Pringa E, Centracchio J, Macharzina RR, Donahay T, Schofield L, Rana N, Kirk M, Mitchell GF, Poppas A, Zehender M, Koren G. 2008. Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome. J Clin Invest 118:2246–2259. 5. Chai CK, Crary DD. 1971. Conjoined twinning in rabbits. Teratology 4:433–444. 6. Chan A, Lakshminrusimha S, Heffner R, Gonzalez-Fernandez F. 2007. Histogenesis of retinal dysplasia in trisomy 13. Diagn Pathol 2:48. 7. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. 1996. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene. Nature 3:383. 0 8. Damstra T, Barlow S, Bergman A, Kavlock R, van der Kraak G, editors. 2002. Global assessment of the state-of-the-science of endocrine disruptors. Geneva (Switzerland): World Health Organization– International Programme on Chemical Safety. 9. Dominguez R, Rott J, Castillo M, Pittaluga RR, Corriere JN Jr. 1993. Caudal duplication syndrome. Am J Dis Child 147:1048–1052. 10. Fontecha CG, Peiro JL, Aguirre M, Soldado F, Paz P, Oria M, Toran N, Martinez-Ibanez V. 2007. The effect of prenatal treatment with steroids and preterm delivery in a model of myelomeningocele on the rabbit foetus. Pediatr Surg Int 23:425–429. 11. Garzozi HJ, Barkay S. 1985. Case of true cyclopia. Br J Ophthalmol 69:307–311. 12. Hajduk P, Sato H, Puri P, Murphy P. 2011. Abnormal notochord branching is associated with foregut malformations in the Adriamycin-treated mouse model. PLoS ONE 6:e27635. 13. Hartman HA. 1974. The fetus in experimental teratology, p 104–128. In: Weisbroth SH, Flatt RE, Kraus AL, editors. The biology of the laboratory rabbit. New York (NY): Academic Press. 14. Hill RE, Heaney SJ, Lettice LA. 2003. Sonic hedgehog: restricted expression and limb dysmorphologies. J Anat 202:13–20. 15. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 16. Jones KL, Benirschke K, Chambers CD. 2009. Gastroschisis: etiology and developmental pathogenesis. Clin Genet 75:322–325. 17. Kaur A, Singh JR. 2010. Chromosomal abnormalities: genetic disease burden in India. Int J Hum Genet 10:1–14.
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18. Keeler RF. 1970. Teratogenic compounds of Veratrum californicum (Durand) X. Cyclopia in rabbits produced by cyclopamine. Teratology 3:175–180. 19. Klaassen Z, Shoja MM, Tubbs RS, Loukas M. 2011. Supernumerary and absent limbs and digits of the lower limb: a review of the literature. Clin Anat 24:570–575. 20. Kroes HY, Takahashi M, Zijlstra RJ, Baert JA, Kooi KA, Hofstra RM, van Essen AJ. 2002. Two cases of the caudal duplication anomaly including a discordant monozygotic twin. Am J Med Genet 112:390–393. 21. Lindsey JR, Fox RR. 1994. Inherited diseases and variations. In: Manning PJ, Ringler DH, Newcomer CE, editors. Biology of the laboratory rabbit. San Diego (CA): Academic Press. 22. Mavinkurve GG, Sciubba D, Amundson E, Jallo GI. 2005. Familial Chiari type I malformation with syringomyelia in 2 siblings: case report and review of the literature. Childs Nerv Syst 21:955–959. 23. McPhaul MJ, Griffin JE. 1999. Male pseudohermaphroditism caused by mutations of the human androgen receptor. J Clin Endocrinol Metab 84:3435–3441. 24. Meyers-Wallen VN. 2000. Inherited abnormalities of sexual development in dogs and cats. In: Concannon PW, England G, Verstegen J, editors. Recent advances in small animal reproduction. Ithaca (NY): International Veterinary Information Service. 25. Ming JE, Golden JA. Midline patterning defects. In: Golden JA, Harding BN, editors. Developmental neuropathology. Lawrence (KS): Allen Press. 26. Mutchinick OM, Luna-Munoz L, Amar E, Bakker MK, Clementi M, Cocchi G, da Graca Dutra M, Feldkamp ML, Landau D, Leoncini E, Li Z, Lowry B, Marengo LK, Martinez-Frias ML, Mastroiacovo P, Metneki J, Morgan M, Pierini A, Rissman A, Ritvanen A, Scarano G, Siffel C, Szabova E, Arteaga-Vazquez J. 2011. Conjoined twins: a worldwide collaborative epidemiological study of the International Clearinghouse for Birth Defects Surveillance and Research. Am J Med Genet C Semin Med Genet 157C:274–287. 27. Nash J, Cheng JS, Meyer GA, Remler BF. 2002. Chiari type I malformation: overview of diagnosis and treatment. WMJ 101:35–40.
28. New York State Department of Health. [Internet] 2012. Congenital malformations registry summary report: statistical summary of children born in 2007 and diagnosed through 2009. Available at: http:// www.health.ny.gov/diseases/congenital_malformations/2007/ summary.htm. 29. Nik-Zainal S, Strick R, Storer M, Huang N, Rad R, Willatt L, Fitzgerald T, Martin V, Sandford R, Carter NP, Janecke AR, Renner SP, Oppelt PG, Oppelt P, Schulze C, Brucker S, Hurles M, Beckmann MW, Strissel PL, Shaw-Smith C. 2011. High incidence of recurrent copy number variants in patients with isolated and syndromic Mullerian aplasia. J Med Genet 48:197–204. 30. Pierpont ME, Basson CT, Benson DW Jr, Gelb BD, Giglia TM, Goldmuntz E, McGee G, Sable CA, Srivastava D, Webb CL. 2007. Genetic basis for congenital heart defects: current knowledge. A scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation 115:3015–3038. 31. Ramasamy S, Paramasivam J, Janardhanam K. 2009. Supernumerary kidney presenting as pyonephrosis. Indian J Urol 25:389–391. 32. Renu D, Rao BG, Ranganath K, Namitha. 2010. Persistent mullerian duct syndrome. Indian J Radiol Imaging 20:72–74. 33. Roessler E, Belloni E, Gaudenz K, Jay P, Berta P, Scherer SW, Tsui LC, Muenke M. 1996. Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat Genet 14:357–360. 34. Sawin PB, Crary DD. 1964. Genetics of skeletal deformities in the domestic rabbit (Oryctolagus cuniculus). Clin Orthop Relat Res 33:71–90. 35. Seseke F. 2003. Clinical aspects of pediatric urology. In: Becker W, Meller J, Zappel H, Leenen A, Seseke F, editors. Imaging in pediatric urology. Berlin (Germany): Springer-Verlag. 36. Suresh J, Gnanasekaran N, Dev B. 2011. Fused supernumerary kidney. Radiology Case Reports 6:1–4. 37. Yamada S, Samtani RR, Lee ES, Lockett E, Uwabe C, Shiota K, Anderson SA, Lo CW. 2010. Developmental atlas of the early firsttrimester human embryo. Dev Dyn 239:1585–1595.
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Case Report
Multiple Complex Congenital Malformations in a Rabbit Kit (Oryctolagus cuniculi) Jennifer L Booth,1 Xuwen Peng,1 Jennifer Baccon,2,3 and Timothy K Cooper1,2,* Congenital malformations may occur during early embryogenesis in cases of genetic abnormalities or various environmental factors. Affected subjects most often have only one or 2 abnormalities; subjects rarely have several unrelated congenital defects. Here we describe a case of a stillborn New Zealand white rabbit with multiple complex congenital malformations, including synophthalmia, holoprosencephaly, gastroschisis, and a supernumerary hindlimb, among other anomalies. There was no historical exposure to teratogens or other known environmental causes. Although not confirmed, this case was most likely a rare spontaneous genetic event.
Congenital malformations occur when there is derangement of the embryologic developmental process. Neural development and organogenesis is a critical time of development that occurs during early embryogenesis.2,37 Congenital malformations that manifest at this stage of development may occur in association with various genetic abnormalities, such as point mutations and chromosomal abnormalities.25,29 In addition, environmental factors, including maternal health status, nutritional deficiencies, and exposure to teratogenic drugs or chemicals, may play a role in the development of congenital malformations.12,25 However, in 65% to 75% of human cases, the cause is unknown, resulting from a complex set of events such as polygenic or multifactorial genetic disorders, spontaneous genetic errors, and synergistic interactions of teratogens.3,25 Approximately 78% of human cases demonstrate only a single developmental malformation, with cardiovascular defects accounting for approximately 30% to 35% of organ defects. Cases of more than 2 or 3 malformations in a single person are extremely rare.28 Here we describe a New Zealand white rabbit that was stillborn with numerous complex developmental abnormalities, including synophthalmia, a supraoptic proboscis, holoprosencephaly with other associated craniofacial deformities, Chiari malformation type I, gastroschisis, a supernumerary hindlimb, a fused (horseshoe) kidney with a supernumerary kidney, and male pseudohermaphroditism.
Case Report
This breeding colony of New Zealand white rabbits was established in an AAALAC-accredited facility at Penn State University (Hershey, PA). All breeding protocols were reviewed, approved, Received: 21 Dec 2012. Revision requested: 04 Feb 2013. Accepted: 15 Feb 2013. Departments of 1Comparative Medicine, 2Pathology, and 3Neurosurgery, Penn State Hershey College of Medicine, Hershey, Pennsylvania. * Corresponding author. Email: [email protected]
and performed in accordance with the guidelines of the IACUC at Penn State Hershey and the Guide for the Care and Use of Laboratory Animals.15 The animal room was maintained at 19 ± 3 °C with a relative humidity between 30% to 70% and a 12:12-h light:dark cycle (lights on, 0700 to 1900). Rabbits were singly housed in double stainless-steel cages attached via a pass-through doorway, with grated floors. Space requirements were adequate in regard to the standards set in the Guide. Each housing unit contained a plastic nesting box. Dams were fed a commercial pelleted feed (no. 8630, Harlan Teklad, Madison, WI) with access to fresh water ad libitum and supplied by an automatic watering system. Rabbits in this colony are free of Encephalitozoon cuniculi, rotavirus, Treponema spp., cilia-associated respiratory bacillus, Clostridium piliforme, Pasteurella multocida, coccidian, and other intestinal and external parasites. Rabbits in this breeding colony included transgenic long QT syndrome type 1 male rabbits and normal (wildtype) female breeders. The transgenic male breeders came from a line derived from pronuclear injection of the human gene KCNQ1 Y315S (KVLQT1) under the control of the rabbit β-myosin heavy chain.4 Breedings in this colony were performed between transgenic male and wildtype female rabbits only. All wildtype female breeders in this colony were purchased from Millbrook Labs (Amherst, MA) and were replaced every 4 to 5 litters. A normal mating of a 15-mo-old wildtype dam and a 3-y-old transgenic long QT syndrome type 1 sire produced a litter of 6 kits after an unremarkable gestation. Two kits were stillborn, whereas the other 4 were viable and healthy. A necropsy was performed on one of the stillborn rabbit kits. Grossly, the head of the kit was severely malformed and smaller than normal, with no openings for the ears, mouth, or nose (Figure 1 A). There was a single large but otherwise grossly normal eye located in the center of the face. Dorsal to the eye was a tubular bony appendage (proboscis) measuring 1.4 × 0.5 × 0.5 cm, with a small, red, pedunculated structure (diameter, 2 mm) protruding from the rostral tip (Figure 1 B). Ear pinnae were present bilaterally but slightly curled.
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Figure 1. (A) Ventral view of the entire length of the body demonstrating cyclopia with proboscis and absence of oral and nasal orifices and gastroschisis without an omphalocoele. The supernumerary right kidney is visible at the cranial margin of the defect of the ventral abdomen. There are 3 well-formed hindlimbs. (B) Lateral view of head. Cyclopia with proboscis and absence of normally developed lower craniofacial structures. (C) The ventral skin of the cervical region is reflected to reveal the tongue and a rudimentary oral cavity. The esophagus and trachea are not visible and are covered by a large bony structure. (D) Dorsal view of the caudal half of the kit, illustrating supernumerary limb with a laceration or defect through the skin and muscle left of the dorsal midline.
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The anatomy of the ventral cervical structures was abnormal (Figure 1 C). Reflecting the skin of the ventral neck revealed a grossly normal tongue and rostrally truncated oral cavity. The trachea and esophagus were not grossly apparent. There was a large bony structure within the ventral cervical region, extending from the head to the thoracic inlet, measuring approximately 10 × 5 mm. The lungs did not float in formalin. The small intestines, large intestines, cecum, liver, and stomach were displaced outside of the body, anchored by the distal colon, esophagus, and caudal vena cava through a 1-cm midline defect of the abdominal body wall and skin. The organs were not enclosed in a membranous sac. A single pale-pink kidney partially protruded through the cranial margin of the abdominal defect. It had a spherical rather than reniform shape. In addition, there were 2 other retroperitoneal kidneys, normal in color but fused at the cranial poles, resulting a bilobed appearance (horseshoe kidney), and located slightly left of midline. There was a small amount of fecal material (meconium) present within the intestines and cecum. There were 3 hindlimbs present (polymelia, Figure 1 D). The outermost (supernumerary) limb on the fetus’ left side had moderately less muscle mass than did the other 2 hindlimbs. The thigh of the middle hindlimb had a moderately wider circumference than did the other 2 hindlimbs. All limbs appeared to be normal in length, with no arthrogryposis, and each ended in a normally formed foot. There was a 12 × 6 mm laceration or defect through the skin and muscle, extending just left of the dorsal midline from the pelvic region to the lateral thigh of the middle hindlimb and just medial and dorsal to the leftmost limb. A swelling resembling external genitalia was present between the supernumerary limb and the abdominal body wall. The dorsal skin defect was medial to the genitalia. There was a well-formed tail present between the 2 other (normal) legs. Forelimbs were grossly normal. The ribs extended to just cranial of the pelvis, making the actual abdominal cavity severely shorter in length than normal. Tissues were fixed in 10% neutral buffered formalin or Bouin solution (head). Tissues were processed in an automated processor (Tissue-Tek VIP, Sakura Finetek USA, Torrance, CA) and paraffin-embedded (Tissue-Tek TEC embedding station, Sakura Finetek USA). Sections were cut at 6 µm for routine staining with hematoxylin and eosin. All images were obtained by using a microscope (model BX51, Olympus America, Center Valley, PA), digital camera (model DP71, Olympus America), and cellSens Standard 1.6 imaging software (Olympus America). A sagittal section of the head contained a large rostral supraorbital proboscis with a central core of irregular segmented hyaline cartilage surrounded by peripheral trabeculae of lamellar bone (Figure 2 A). There was a mushroom-like process at the tip, with a hyaline cartilage core covered by highly congested haired skin. The olfactory bulb of the brain extended partway into the proboscis, dorsal to the cartilage core. Multiple sinus hairs (vibrissae) were present in the skin of the proboscis. The retina of the eye was longer than normal, with retinal redundancy and rosettes. The internal and external nuclear layers were irregularly thickened and occasionally merged. Retinal outer nuclear layer cells focally infiltrated the rostral optic nerve. The rostral end of the oropharynx and nasopharynx were not patent, coming to a blind end caudoventral to the eye. Anlages of trachea and larynx were present. There was a single large central ventricle of the forebrain that opened to the dorsal surface of the brain in the center of the rostral–caudal axis. There was no cortex overlying the ventricle
Figure 2. (A) Sagittal section of the decalcified head with single midline eye, supraorbital proboscis, holoprosencephaly with hippocampal dysplasia, and herniation of the cerebellum through the foramen magnum. A scanned digital version of this slide can be accessed at the Penn State Zebrafish Atlas (http://zfatlas.psu.edu/view. php?s=35351&z=7&c=48034,28974). (B) Fusion of 2 kidneys at the cranial poles. There are 2 distinct renal pelves. Hematoxylin and eosin stain.
here, and the choroid plexus and pineal gland lay directly ventral to a thin fibrous membrane that formed the dorsal cranial vault at this site. The hippocampus was oriented abnormally and formed the cranial margin of the dorsal cerebral defect. The cerebellum was partially displaced caudally through the foramen magnum and was markedly distorted by compression. The choroid plexus of the fourth ventricle was in the vertebral canal between the first and second vertebral vertebrae. The cranial poles of 2 kidneys were fused, but 2 distinct renal pelves were maintained (Figure 2 B). The renal pelvis of the separate third kidney was mildly dilated. All kidneys had an immature renal cortex, with predominantly fetal glomeruli. An immature (fetal) testis and epididymis was present adjacent to the kidney. Cross sections of the spine at the level of the skin defect showed that the defect was lined by a capsule of multiple layers of plump fibroblasts in a loose and edematous stroma. A ganglion and several myelinated nerve fibers were present within the defect. In a separate section through the same tissue, there was a cross-section of spinal cord near the cauda equina, with multiple cyst-like blood-filled lakes in the neuropil. Cross-sections of the spine cranial
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to the defect revealed multiple blood filled lakes (acute hemorrhage) present in the white and, to a lesser extent, gray matter of the spinal cord. In one section, erythrocytes filled the central canal. The slide containing cross-sections of the spine caudal to the defect included sections of normal vagina and uterus. The lungs were markedly atelectatic, consistent with the diagnosis of stillbirth. The heart was normal, with no defects.
Discussion
Congenital malformations may occur due to abnormalities during the early embryogenesis stage of development. The cause of these congenital malformations is not always well understood. Some of these deformities have been linked to etiologies such as environmental factors, including exposure to teratogens, and maternal nutritional or health status. Genetic aberrations of chromosomes, expression of autosomal recessive conditions linked to inbreeding, and various gene mutations, deletions, and duplications have also been identified as underlying causes of congenital malformations.17,30 Under many circumstances, the cause of the congenital defect cannot be identified. Many pregnancies, in which the fetus has a severe congenital abnormality, will not be carried to full term. According to the World Health Organization, approximately 50% of all human pregnancies result in spontaneous abortion, with chromosomal abnormalities being responsible for those which occur in the first trimester.8 Neonates that are carried to full term with congenital defects most often have only one or 2 abnormalities. It is very rare for a subject to have several malformations. In this particular case, this rabbit kit was stillborn with numerous complex and severe developmental abnormalities. Holoprosencephaly is a condition in which there is impairment of midline cleavage of the forebrain during fetal development. This failure of division often is accompanied by several facial malformations, such as midline facial clefts, cyclopia (synophthalmia), and absence of the nasal cavity with a supraorbital proboscis.16,25 Holoprosencephaly may occur due to exposure to teratogenic drugs or environmental factors during the organogenesis stage of fetal development. Maternal diabetes and alcohol consumption have been identified as environmental factors associated with holoprosencephaly.25 Cyclopia in rabbits has been experimentally documented to occur when exposed to drugs or chemicals such as thalidomide, phthalimidobenzene, methamphetamine, and cyclopamine and jervine, which are derived from the Veratrum plant genus.13,18 The pathogenic mechanism through which V. californicum causes these malformations involves the molecular signaling along the Sonic Hedgehog (SHH) pathway.6,14,25 Genetic deletions or mutations of the SHH gene can result in this developmental defect.7,33 Genetic errors with chromosomal abnormalities have been identified as a cause of holoprosencephaly, with trisomy 13 being the most common.11,25 At least 12 chromosomal regions on 11 chromosomes have been implicated to play a role in holoprosencephaly.11 Cyclopia has previously been documented as an uncommon isolated defect in rabbits and suggested to be caused by a recessive gene, cy.21 The cause of holoprosencephaly in this rabbit kit was most likely due to a genetic error. Confirmation would require genetic testing. Chiari malformation type I of the cerebellum occurs congenitally when there is an abnormally small cerebellar fossa, which causes the growing cerebellum to herniate through the foramen magnum. This type of malformation may be a result of defective
embryogenesis or birth injury;27 in the case we present, defective embryogenesis is more likely. A genetic cause for Chiari malformation type I has not been identified but has been supported through several reports of human familial cases and the association of Chiari malformation type I with other genetic conditions.22 Chiari malformation type I has been surgically induced in rabbits,10 but there are no previous reports of this abnormality occurring spontaneously in rabbits. During fetal development of the limbs, the somatopleuric mesenchyme interacts with the overlying ectoderm to cause proliferation of the mesenchyme and formation of an apical ectodermal ridge.19 This site, known as the zone of polarizing activity, is the position of further limb development. The patterning and ordered arrangement of the skeletal elements of the limbs is dependent on the SHH gene. The mechanism of action is unknown. Moreover, SHH expression has been identified to occur in the zone of polarizing activity at the posterior margin of the limb bud during development. When an ectopic zone of polarizing activity occurs in the anterior limb margin, it results in the development of extra digits. A polydactyly mouse mutant carries a sasquatch (Ssq) mutation located 1 Mb from SHH that disrupts SHH expression. Humans that are heterozygous for the SSQ mutation develop normal forelimbs, but the hindlimbs have preaxial polydactyly.14 Altered expression of SHH in the rabbit we present might provide a plausible link between the development of a supernumerary hindlimb and cyclopia. However, these 2 malformations have not been reported to occur simultaneously in the human or animal populations. Alternatively, these 2 anomalies could have occurred independently. The occurrence of spontaneous conjoined twins in rabbits has been reported5 but is extremely rare. The extent of fusion between the twins can vary in degree. Some conjoined twins have normal development of the body parts that are not fused, whereas other twins have an autosite–parasite relationship.5 The parasitic twin is often underdeveloped, with multiple malformations, and is dependent on the autosite, which has relatively normal development. Monozygotic twins are formed from the incomplete division and separation of a single blastocyst. The mechanism of this phenomenon is not understood, and evidence for genetic or environmental association is sparse.5,26 Caudal duplication anomaly (dipygus) is another abnormality that has been well documented in the human literature but that has never been described to occur in rabbits. Many authors argue whether there is a relationship between caudal duplication and monozygotic twinning.20 An etiologic pathogenesis has not yet been explained. The distal caudal end of the trunk may be affected by a variety of anomalies, including complete agenesis, fusion, partial splitting, and complete duplication of organs. Spina bifida, malformed caudal spinal cords, supernumerary kidneys, and duplicate genitalia are among the abnormalities that can occur.9,35 This syndrome could represent an alternative theory to the monozygotic twinning and may explain the presence of several of the anomalies in this rabbit kit. Gastroschisis is a rare congenital defect of the abdominal wall through which the abdominal organs eviscerate. The pathogenesis of this anomaly is not understood, with many proposed hypotheses. However, specific risk factors for susceptibility have been identified in humans, including young maternal age, primigravidity or primiparity, lower prepregnancy body mass index, poor maternal diet, the use of vasoactive medications, maternal
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infection, and other environmental factors.16 Many of these risk factors do not apply to this dam. She was multiparous, in good body condition, received adequate nutrition, and had no history of medications or exposure to other environmental factors. Gastroschisis seems to be widespread in occurrence and has not been clearly linked to a genetic cause.34 Experimental exposure to teratogens, such as thalidomide, vinblastine, and clomiphene, have been demonstrated to cause gastroschisis in rabbits.13 This rabbit kit had a single fused kidney (horseshoe kidney) and a separate well-formed supernumerary kidney that was grossly and histologically normal, with the exception of mild hydronephrosis. This rare anomaly is often an incidental finding; however in two-thirds of cases, symptoms such as urinary tract infections and renal or ureteral calculi develop. During normal embryologic development of the kidneys, the mesonephric duct bilaterally gives rise to the ureteric bud, which grows cranially into the metanephric blastema. This metanephric mass ultimately becomes the adult kidney. A supernumerary kidney is developed when there is a secondary outpouching of the nephrogenic cord into 2 metanephric blastemas, resulting in a partial or completely duplicated ureteral bud. The lower branch of this division often fuses with the left kidney, giving it a horseshoe appearance.31,36 Renal fusion has been induced experimentally in rabbits that were exposed to teratogens, such as thalidomide and L-asparaginase.13 Male pseudohermaphroditism is defined as the presence of male gonads with female genitalia. This type of reproductive development may occur with any genetic defect that impairs androgen synthesis or activity and with failure of Müllerian duct regression.24,32 The SRY gene on the Y chromosome is responsible for the initiation of testis development.4 Sertoli cells secrete Müllerian inhibiting factor, which causes regression of the Müllerianassociated structures. When Müllerian inhibiting factor is deficient, the affected subject develops reproductive structures of both Müllerian and Wolffian origin.32 The production of testosterone and 5α-dihydrotestosterone are responsible for the development of Wolffian duct structures and external male genitalia. Mutation of the androgen receptor causes complete or incomplete testicular feminization, in which the affected subject develops external female genitalia but internal male reproductive structures and gonads.23,24,29 Disorders of sexual differentiation in rabbits have not been previously described, but the case we present is consistent with persistent Müllerian duct. The defect in the caudodorsal body wall near the supernumerary limb was of undetermined etiology. Although spina bifida was suspected in light of the gross appearance, a simple musculocutaneous defect or postmortem trauma from the dam could not be excluded. Surprisingly, no cardiovascular malformations were present. Neither this breeding pair nor the entire breeding colony have been associated with any other reports of kits with congenital malformations. Given the clinical and breeding history of the rabbits in this colony, the case we present was most likely an isolated and rare spontaneous event that undoubtedly had some genetic influence. Whether the anomalies of the caudal end of the kit were due to monozygotic twinning or another pathologic entity such as caudal duplication anomaly is unclear. Karyotyping for chromosomal abnormalities and gene sequencing for mutations that are known to be associated with the witnessed anomalies in this rabbit would have been ideal. However, we did not have
any unfixed tissues or specimens available for testing nor any gene candidate(s) for sequencing. Genetic tools for testing in rabbits are very limited and only available on a research basis, thus presenting many difficulties for definitive diagnoses. In addition, rabbit gene polymorphisms are poorly documented.
Acknowledgments
We thank Dr Ronald Wilson, Chair of the Department of Comparative Medicine and director of the laboratory animal medicine residency training program, and the Comparative Medicine histology lab staff, Weifang Lin and Ellen Mullady. Jean Copper, Steve Peckins, and Dr Keith Cheng administer the Penn State Zebrafish Atlas, funded by NIH grant R24 OD011152 (formerly RR017441).
References
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