PCR Genotyping of a Novel FGF4 Retrogene in Pit …...Pit Bull Terriers and related breeds. N = 217...
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Materials and Methods Sample Identification Screened Bannasch Lab DNA database and the William R. Pritchard Veterinary Medical Teaching Hospital records for IVDD surgical cases from mixed breeds, Pit Bull Terriers and related breeds. N = 217 Non IVDD dogs N = 83 dogs with IVDD 626bp Introduction Intervertebral Disc Disease (IVDD) is an incredibly devastating and painful disease that affects a wide variety of dog breeds. Chondrodystrophic dog breeds such as Dachshunds and French Bulldogs exhibit the highest frequency of IVDD [1]. Chondrodystrophy is characterized by shortened long bones and prematurely calcified intervertebral discs [2]. IVDD is the result of herniation of this intervertebral disc into the spinal canal often causing pain and paralysis. A recently discovered fibroblast growth factor 4 (FGF4) retrogene on chromosome (Chr) 12 is responsible for canine chondrodystrophy and a leading contributor to a majority of IVDD cases [3]. However, not all dogs affected with IVDD have the Chr 12 retrogene [Brown et al. and K. Batcher and D. Bannasch unpublished results]. Recently the laboratory identified a second FGF4 retrogene on Chr 13 in two IVDD affected Pit Bull Terriers that did not have the Chr 12 retrogene. The purpose of this study was to determine if this FGF4 retrogene may be a contributing cause of IVDD in dogs. Phenotype A classic example of IVDD is shown in Figure 1 and Figure 2 below. The CT images show different angles of the same calcified disc that has ruptured and is compressing the spinal canal dorsally. The yellow arrow points to the herniated degenerative disc material. Herniated intervertebral discs can cause clinical signs ranging from sudden onset paralysis to chronic back pain. Treatment includes surgery to reduce the spinal compression and crate rest. Frequency of FGF4 Retrogene on Chr 13 Acknowledgments References 1. Bellumori, T.P., et al., Prevalence of inherited disorders among mixed-breed and purebred dogs: 27,254 cases (1995–2010). Journal of the American Veterinary Medical Association, 2013. 242(11): p. 1549-1555. 2. Hansen, H.-J., A Pathologic-Anatomical Interpretation of Disc Degeneration in Dogs. Acta Orthopaedica Scandinavica, 1951. 20(4): p. 280-293. 3. Brown, E.A., et al., FGF4 retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc disease in dogs. Proceedings of the National Academy of Sciences, 2017. 114: p. 11476-11481. 4. Kaessmann, H., N. Vinckenbosch, and M. Long, RNA-based gene duplication: mechanistic and evolutionary insights. Nature reviews. Genetics, 2009. 10(1): p. 19-31. 5. Parker, H.G., et al., An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in domestic dogs. Science, 2009. 325(5943): p. 995-8. 6. For corgi picture www.adoptapet.com https://www.adoptapet.com/s/adopt-a-corgi 7. For Staffordshire Terrier picture http://iworkdogs.com/3943234-american-staffordshire-terrier-puppy-3- months-in-front-of-a-white-background/ Conclusion • The rare Chr 13 FGF4 retrogene appears to cause IVDD in dogs. Future Directions • Look at the dogs that have the previously identified retrogene on Chr 12 and see if there are any dogs that have both Chr 12 and Chr 13 retrogenes. • Identify more FGF4 retrogenes that could cause IVDD in other unexplained cases. Genotyping Used a three primer PCR to amplify retrogene and non retrogene DNA (see next box). Evaluated PCR product sizes using agarose gel electrophoresis SC 3 Primer PCR The FGF4 retrogene on Chr 13 came from the endogenous FGF4 gene on chromosome 18. FGF4 is composed of three exons and two introns. When FGF4 is transcribed the introns are removed and a poly-A tail is added. This entire transcript is approximately 3.2kb long. Retrogene formation is a rare event that can occur when RNA is reverse transcribed into DNA and integrated back elsewhere in the genome [4]. Several FGF4 retrogenes have been identified in the dog genome [3,5]. The focus of this study was a FGF4 retrogene on chromosome 13 identified in two Pit Bull Terriers affected by IVDD. In order to identify dogs containing the FGF4 retrogene on chromosome 13 a three primer PCR reaction was used as shown above. One primer pair was designed flanking the retrogene insertion site and an additional forward primer was designed within the retrogene. When no retrogene insertion is present, the product between the forward genomic (F1) and reverse genomic primer (R1) is 451bp in length. When the retrogene insert is present, the product between the forward primer within the retrogene (F2) and R1 produces a product 626bp in length. FGF4 Retrogene ex 2 ex 3 ex 1 AAAAAA Chr13 FGF4 Retrogene F1 F2 R1 Thank you to the dog owners that submitted samples. Financial support was provided by the Students Training in Advanced Research (STAR) Program through the NIH T35 OD010956-19 grant. Only two copies of the retrogene on Chr 13 were found, and both were in Pit Bull Terriers. As Pit Bull Terrier is not a well defined breed we expanded the search to include Staffordshire Terriers and other mixed breeds that were both affected and unaffected by IVDD. The novel Chr 13 retrogene was rare and appeared in only 0.995% of the Pit Bull Terriers tested. The only dogs that had the Chr 13 retrogene had IVDD and did not have any other identified retrogenes. Disc SC Figure 1: sagittal CT image of herniation at T11-T12; SC= spinal canal Figure 2: cranial-caudal CT image of herniation at T11- T12; SC= spinal canal PCR Genotyping of a Novel FGF4 Retrogene in Pit Bull Terriers and Mixed Breed Dogs Affected by Disc Disease KA Maciejczyk 1 , KL Batcher 1 , DL Bannasch 1 1 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis Chr13 Without Retrogene 451bp F1 R1 Pit Bull Terrier Staffordshire Terrier Mixes Chr 13 Retrogene Pos Neg Pos Neg Pos Neg IVDD 2 20 0 0 0 61 Not IVDD 0 179 0 14 0 24
Transcript of PCR Genotyping of a Novel FGF4 Retrogene in Pit …...Pit Bull Terriers and related breeds. N = 217...
Materials and MethodsSample Identification
Screened Bannasch Lab DNA database and the William R. Pritchard Veterinary Medical Teaching Hospital records for
IVDD surgical cases from mixed breeds, Pit Bull Terriers and related breeds.
N = 217 Non IVDD
dogs
N = 83 dogs with
IVDD
626bp
IntroductionIntervertebral Disc Disease (IVDD) is an incredibly devastating andpainful disease that affects a wide variety of dog breeds.Chondrodystrophic dog breeds such as Dachshunds and FrenchBulldogs exhibit the highest frequency of IVDD [1].Chondrodystrophy is characterized by shortened long bones andprematurely calcified intervertebral discs [2]. IVDD is the result ofherniation of this intervertebral disc into the spinal canal oftencausing pain and paralysis. A recently discovered fibroblast growthfactor 4 (FGF4) retrogene on chromosome (Chr) 12 is responsiblefor canine chondrodystrophy and a leading contributor to amajority of IVDD cases [3]. However, not all dogs affected withIVDD have the Chr 12 retrogene [Brown etal. and K. Batcher and D. Bannaschunpublished results]. Recently thelaboratory identified a second FGF4retrogene on Chr 13 in two IVDD affectedPit Bull Terriers that did not have the Chr 12retrogene. The purpose of this study was todetermine if this FGF4 retrogene may be a
contributing cause of IVDD in dogs.
PhenotypeA classic example of IVDD is shown in Figure 1 and Figure 2 below.The CT images show different angles of the same calcified discthat has ruptured and is compressing the spinal canal dorsally. Theyellow arrow points to the herniated degenerative disc material.Herniated intervertebral discs can cause clinical signs ranging fromsudden onset paralysis to chronic back pain. Treatment includessurgery to reduce the spinal compression and crate rest.
Frequency of FGF4 Retrogene on Chr 13
Acknowledgments
References1. Bellumori, T.P., et al., Prevalence of inherited disorders among mixed-breed and purebred dogs: 27,254
cases (1995–2010). Journal of the American Veterinary Medical Association, 2013. 242(11): p. 1549-1555.2. Hansen, H.-J., A Pathologic-Anatomical Interpretation of Disc Degeneration in Dogs. Acta Orthopaedica
Scandinavica, 1951. 20(4): p. 280-293.3. Brown, E.A., et al., FGF4 retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc
disease in dogs. Proceedings of the National Academy of Sciences, 2017. 114: p. 11476-11481.4. Kaessmann, H., N. Vinckenbosch, and M. Long, RNA-based gene duplication: mechanistic and evolutionary
insights. Nature reviews. Genetics, 2009. 10(1): p. 19-31.5. Parker, H.G., et al., An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in
domestic dogs. Science, 2009. 325(5943): p. 995-8.6. For corgi picture www.adoptapet.com https://www.adoptapet.com/s/adopt-a-corgi7. For Staffordshire Terrier picture http://iworkdogs.com/3943234-american-staffordshire-terrier-puppy-3-
months-in-front-of-a-white-background/
Conclusion• The rare Chr 13 FGF4 retrogene
appears to cause IVDD in dogs.
Future Directions• Look at the dogs that have the
previously identified retrogeneon Chr 12 and see if there areany dogs that have both Chr 12and Chr 13 retrogenes.
• Identify more FGF4 retrogenesthat could cause IVDD in otherunexplained cases.
Genotyping
Used a three primer PCR to amplify retrogene and non
retrogene DNA (see next box).
Evaluated PCR product sizes using agarose gel electrophoresis
SC
3 Primer PCR
The FGF4 retrogene on Chr 13 came from the endogenous FGF4 gene on chromosome 18. FGF4 is composed ofthree exons and two introns. When FGF4 is transcribed the introns are removed and a poly-A tail is added. Thisentire transcript is approximately 3.2kb long. Retrogene formation is a rare event that can occur when RNA isreverse transcribed into DNA and integrated back elsewhere in the genome [4]. Several FGF4 retrogenes havebeen identified in the dog genome [3,5]. The focus of this study was a FGF4 retrogene on chromosome 13identified in two Pit Bull Terriers affected by IVDD.
In order to identify dogs containing the FGF4 retrogene on chromosome 13 a three primer PCR reaction wasused as shown above. One primer pair was designed flanking the retrogene insertion site and an additionalforward primer was designed within the retrogene. When no retrogene insertion is present, the productbetween the forward genomic (F1) and reverse genomic primer (R1) is 451bp in length. When the retrogeneinsert is present, the product between the forward primer within the retrogene (F2) and R1 produces a product626bp in length.
FGF4 Retrogene
ex 2 ex 3ex 1 AAAAAAChr13
FGF4 Retrogene
F1 F2
R1
Thank you to the dog owners that submitted samples. Financial support was provided by the Students Training inAdvanced Research (STAR) Program through the NIH T35 OD010956-19 grant.
Only two copies of the retrogene on Chr 13 were found, and both were in Pit Bull Terriers. As Pit Bull Terrier isnot a well defined breed we expanded the search to include Staffordshire Terriers and other mixed breedsthat were both affected and unaffected by IVDD. The novel Chr 13 retrogene was rare and appeared in only0.995% of the Pit Bull Terriers tested. The only dogs that had the Chr 13 retrogene had IVDD and did not haveany other identified retrogenes.
Disc
SC
Figure 1: sagittal CT image of herniation at T11-T12; SC= spinal
canal
Figure 2: cranial-caudal CT image of herniation at T11-
T12; SC= spinal canal
PCR Genotyping of a Novel FGF4 Retrogene in Pit Bull Terriers and Mixed Breed Dogs Affected by Disc Disease
KA Maciejczyk1, KL Batcher1, DL Bannasch1
1Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis
Chr13
Without Retrogene
451bpF1
R1
Pit Bull Terrier Staffordshire Terrier Mixes
Chr 13 Retrogene Pos Neg Pos Neg Pos Neg
IVDD 2 20 0 0 0 61
Not IVDD 0 179 0 14 0 24
![Original Article Preliminary study of presumptive...Intervertebral disc disease (IVDD) is one of the most common vertebral and spinal disorders in dogs [1] and is traditionally categorized](https://static.fdocuments.us/doc/165x107/5fa13d5d472acd3a2b427e9c/original-article-preliminary-study-of-presumptive-intervertebral-disc-disease.jpg)
