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Genetic Screening of the Canine Transcription Factor AP-2 Beta (TFAP2B) Gene in Dogs with Patent Ductus Arteriosus (PDA)
So-Jeong Nam and Changbaig Hyun1
Section of Small Animal Internal Medicine, School of Veterinary Medicine, Kangwon National University, Chuncheon 201-100, Korea
(Accepted : April 06, 2009)
Abstract : Patent ductus arteriosus (PDA) is an abnormal shunt between the descending aorta and pulmonary artery through the incompletely closed ductus arteriosus and is the most common congenital heart defect in dogs. Recent human genetic studies found that a the gene mutation in transcription factor AP-2 beta (TFAP2B) was responsible for syndromic cases of PDA. Mutations in the TFAP2B gene are associated with certain congenital cardiac defects in humans that include PDA. In this study, we isolated the entire coding exons of canine TFAP2B gene for genetic screening in dogs with PDA. Analysis of the deduced amino acid sequence suggested that the canine TFAP2B are phylogenetically closer to the human TFAP2B (100% identity in amino acid sequence) than mouse and rat. In cTFAP2B gene screening, one single c.936 + 203G > A base change was found in affected Maltese dogs with PDA. However, further screening found the same base change in one unaffected control dog, suggesting this base change might be polymorphism. No other base changes were found in other dog breeds enrolled in this study. Because the base change was located in the intronic region and found in an unaffected control dog, TFAP2B might not be responsible for familial PDA in Malteses and sporadic cases of other dog breeds, although the gene promoter region should be investigated before reaching to this conclusion. A future study that may take this study further would be to collect more samples and to screen TFAP2B in various breeds of dogs with PDA and other various congenital heart defects.
Key words : dog, TFAP2B, CHD, patent ductus arteriosus.
Introduction
Patent ductus arteriosus (PDA) is the most common con- genital heart defect in dogs where it is 3 times more preva- lent in females and in small dog breeds (3). Familial PDA has been found in several dog breeds including Bishon Frise, Maltese, Pomeranian, Toy Poodle and Yorkshire Terrier (4).
PDA is an incomplete closure of the ductus arteriosus (the temporary fetal blood vessel that connects the descending aorta and the pulmonary artery), causing abnormal arterio- venous blood communication. Normally, immediately after birth, inflation of the lungs allows the shunt between the main pulmonary artery (MPA) and the descending aorta (dAo) to close. The rapid increase in arterial oxygen tension in the left ventricle (when the puppy starts to breathe) results in rapid decrease of prostaglandin E2 and subsequently trig- gers ductal constriction of the ductus arteriosus (DA;
11,14,15). PDA is caused by the failure of the ductus arterio- sus to spontaneous close after birth. The disturbances involved in the prostaglandin inhibited ductal constriction of the DA were found to be the cause of experimentally induced PDA in rats (10). In a recent human genetic study, the
genetic disturbance in the transcriptional pathway involved in cardiomorphogenesis was found to cause PDA (17). Given the wealth of information on TFAP2B implicated as the gene responsible for human familial PDA, the TFAP2B gene was considered a good candidate for our investigation into canine PDA.
Transcription factor AP-2 beta (TFAP2B) is a member of the AP-2 family of transcription factors that has been found to be associated with PDA in humans (13). TFAP2B was also found to be expressed in the neural crest and that it stimu- lated cell proliferation (16), and suppressed terminal differen- tiation of specific cell types during embryonic development (8,9). The gene expression of TFAP2B has also been sug- gested to modulate the development of the facial skeleton, heart and kidney (9,13). The TFAP2B protein interacts with other TFAP2B proteins including other members of the AP-2 family of proteins to produce homo- or hetero-dimers in order to bind to specific DNA sequences. The different com- binations either stimulate cell proliferation or suppress termi- nal differentiation of specific cell types during embryonic development. The expression of the AP-2 family of proteins varies; therefore, the binding affinity for different promoters is heavily dependent on the combinations of AP-2 dimers assembled. Therefore, TFAP2B can act as a transcriptional activator or repressor, depending on the transcriptional path-
1Corresponding author.
E-mail: hyun5188@kangwon.ac.kr
124 So-Jeong Nam and Changbaig Hyun
ways that TFAP2B is utilized.
In human studies, mutations in this gene resulted in an autosomal dominant Char syndrome, which produced a num- ber of congenital cardiac defects, including PDA (13,17). An investigation of a Palestinian family with Char syndrome having PDA, clinodactyly, typical facial features and super- numerary nipple, a missense mutation (c.673C> T) in the TFAP2B gene was identified that resulted in a c.225R > C amino acid substitution (17).
Materials and Methods
Animals
Twelve affected dogs (8 Malteses, 1 Toy Poodles, 1 Minia- ture Schnauzers, and 2 German Shepherds), 3 related control dogs, and 20 unrelated breed control dogs (five healthy normal dogs for each breed) were screened in this study (Table 1). The twelve affected dogs were clinical cases pre- sented to the KWVTH (Kangwon National University Veter- inary Teaching Hospital) for either medical treatment or trans-jugular coil embolization. All affected dogs were phe- notypically confirmed by cardiac auscultation, thoracic radi- ography, angiography and echocardiography (two dimensional, M-mode and color Doppler), before being enrolled in this study.
Isolation of the canine TFAP2B gene
The heart tissue was taken from a pound dog (6 month old female Kelpie-cross). Total RNA was extracted from the heart tissue and treated with DNase I, according to the man- ufacturer’s instructions (SV RNA extraction kit, Promega, USA). The cDNA was synthesized by reverse transcription from the total RNA sample (SuperScriptIII ® RT-Kit, Invit- rogen, USA). A primer set was designed from the conserved regions of the 5’ and 3’ end sequences of the human TFAP2B mRNA sequences (Table 2). PCR amplifications were car- ried out using 1µL cDNA, 500 µM dNTP, 0.5 µM of each primer, 1 X PCR buffer (ProofStart PCR buffer, Qiagen, Ger- many), 1.5 mM magnesium chloride and 0.5U of DNA poly- merase (ProofStart DNA Polymerase, Qiagen, Germany).
The PCR-amplificationprotocol involved an initial denatur- ation at 94oC for 5 min, followed by 35 cycles: 94oC, 30 s;
60oC,30 s; 72oC, 1 min in an automated thermal cycler (PCR Tetrad, MJ research, UK).
Table 1. Signalments and clinical diagnosis of dogs used in this study
Dog ID Breed Sex Age Clinical diagnosis
AI:2 Maltese F 5 yr PDA
AII:1 Maltese F 1 yr Normal
AII:3 Maltese F 1 yr PDA
BI:2 Maltese F 3.5 yr PDA
BII:1 Maltese F 1 yr Normal
BII:2 Maltese F 1 yr PDA
BII:3 Maltese M 1 yr PDA
BII:4 Maltese M 1 yr Normal
C1 Maltese M 1.5 yr PDA
C2 Maltese F 2 yr PDA
C3 Maltese F 0.5 yr PDA
C4 Toy Poodle M 3 yr PDA
C5 Miniature Schnauzer F 2.5 yr PDA
C6 German Shepherd F 1 yr PDA
C7 German Shepherd F 0.5 yr PDA, PHT PDA: Patent ductus arteriosus, PHT: Pulmonary hypertension
*.AI:2, AII:1 and AII:3 are family A; BI:2, BII:1, BII:2, BII:3 and BII:4are family B
Table 2. Primer sequences and PCR protocols used in this study
Primer Name Primer sequence PCR annealing Tm(oC)
TFAP2B ex1-F 5’-TCCAGCCTATTGTTTGAG-3’
TFAP2B ex1-R 5’-CGGCCGGGAATCTACGAC-3’ 55
TFAP2B ex2-F 5’-CCGAGCTTCTCCATTAGTCG-3’
TFAP2B ex2-R 5’-CTCCTATCTCCTGGGGCATT-3’ 61
TFAP2B ex3-F 5’-CTCATAGTTTCCTTTTTCCTCTGC-3’
TFAP2B ex3-R 5’-CCTCCCCCAATTTCCTTCTTC-3’ 59
TFAP2B ex4-F 5’-ACGGGGTAAGTTTCCAGAGG-3’
TFAP2B ex4-R 5’-GAGGGAGCCCTCTTCCTTC-3’ 58
TFAP2B ex5-F 5’-GCAGAAATTAAGCAAACCT-3’
TFAP2B ex5-R 5’-GCCTTAGAGTCTGGAACCGAGCAC-3’ 54
TFAP2B ex6-F 5’-ACATGCCTGCCCACCTTTCCTT-3’
TFAP2B ex6-R 5’-TGCCGCTCCCCAACACCT-3’ 64
TFAP2B ex7-F 5’-CTTTCTAATGCCAATGACAACGAC-3’
TFAP2B ex7-R 5’-CAAAAGCACTGACACCAACAATG-3’ 61
TFAP2B mRNA-F 5’-ATGCACTCACCTCCTAGAGAC-3’
TFAP2B mRNA-R 5’-TCTTCCAAAGCCAATCCAAC-3’ 52
Mutation screening of canine TFAP2B in a family of PDA
Seven sets of mutation screening primers were designed from the intron boundary of each exon of the canine TFAP2B gene in the canine TFAP2B genomic clone (GenBank No.
NW_876254, Table 2). All exons of TFAP2B gene were amplified by polymerase chain reaction (PCR) from 50 ng of genomic DNA extracted from blood, purified with PCR (Cleanup Plates, Millipore, USA) and sequenced using Big Dye Terminator v3.1 kit (Applied Biosystems, USA) and ABI PRISM® 3700 DNA Analyzer USA. PCR amplifica- tions for exon 1-7 of TFAP2B gene screening were carried out using 1µL DNA, 500 µM dNTP, 0.5 µM of each primer, 1 X PCR buffer, 1.5 mM magnesium chloride and 0.5U of Taq polymerase (NeoThem™ DNA polymerase, Germany).
The PCR-amplificationprotocol involved initial denaturation
at 94oC for 3 min, followed by 35 cycles: 94oC, 15 s; spe- cific annealing temperature,30 s; 72oC, 1 min in an auto- mated thermal cycler (PCR Tetrad, MJ research, UK).
Results
In this study, we initially examined two Maltese dog fami- lies. In Maltese family A, the mother (AI:2 in Fig 1) and one female offspring (AII:3 in Fig 1) had the typical left-right shunted PDA which was clearly demonstrated by color and pulse Doppler echocardiography (Fig 2), while the other female offspring (AII:1) did not have PDA and one male off- spring (AII:2) died immediately after birth (unidentified cause). In Maltese family B, the mother (BI:2 in Fig 1) and two offsprings (BII:2 and BII:3 in Fig 1) had PDA, while the rest of offspring did not have PDA. Seven more dogs with Fig 1. Color Doppler echocardiogram of an affected dog (AII: 3 in Fig 2). There was no communication between the descending aorta (dAO) and main pulmonary artery (MPA) in normal dogs. Patent ductus arteriosus (PDA) is abnormal communication between AO and MPA caused by the incomplete closure of the ductus arteriosus. In the color Doppler echocardiogram, mosaic colored blood flow implied turbulent blood flow between dAO and MPA (left panel), which were further confirmed as abnormal blood flow between dAO and MPA in the pulsed Doppler echocardiography (right panel).
Fig 2. Affected Maltese dog family used in this study. Square: male, Circle: female. Filled: affected with patent ductus arteriosus, Clear: unaffected with patent ductus arteriosus. Slashed: dead, Question mark: not determined.
126 So-Jeong Nam and Changbaig Hyun
PDA (either sporadic or un-pedigreed; confirmed by the echocardiography and angiography) were also included in the genetic screening (Table 1).
For genetic screening of canine TFAP2B, we successfully isolated the entire coding region of mRNA sequence (1347 bp; Genbank No. EU541501) of canine TFAP2B (Fig 3).
Using deduced amino acid sequence, we analysed the sequence homology to other TFAP2B sequences and found that it was highly conserved across species (Fig 4; Table 3).
The canine TFAP2B was closer to human TFAP2B (Genbank No. NM003221) in both mRNA sequences as well as amino acid sequence than TFAP2Bs from any other species.
In cTFAP2B gene mutation screening, one single homozygous c.936+203G>A base change was found in two affected Maltese dogs (AII:3 in Fig 1, one sporadic case) with PDA (Fig 5). Furthermore heterozygous c.936+203G>A base change was also found in one affected German shepherd dog (Fig 5). However, further screening found either homozygous or heterozygous c.936+203G>A base change in
unrelated control dogs. Frequencies of A/A, A/G and G/G in this study population were 68.6%, 20% and 11.4%, respectively. No other base changes were found in this study population
Discussion
Closure of the DA after birth is dependent on the oxygen tension (e.g. well oxygenated blood from the left side of the heart) and the levels of hormones (e.g. decreased level of prostaglandin E2). Lack of inhibitors of prostaglandin bio- synthesis and persistent pressure overload from the right car- diac chambers after birth often result in patent ductus arteriosus (15). In humans, PDA typically has not been regarded as a genetic disorder, because it most often occurs sporadically. However over 5% of PDA cases in humans were found to be familial (6,12), suggesting there might be a genetic etiology associated with the disease. In humans, PDA may be multifactorial; however, recent mouse studies revealed that single genes could influence this trait (10). The disruption of the prostaglandin E2 receptor produced mice with PDA. Furthermore, mutations in the transcription factor TFAP2B were found to be associated with the syndromic forms of PDA (Char syndrome) in humans (7). Breed predi- lection for canine PDA has been well documented in the lit- erature (2,3). The high prevalence rate of PDA in Maltese dogs in Korea, strongly suggested a single gene etiology in this dog breed. As a result, Maltese dogs (5 clinical cases including one familial case) were heavily outnumbered by other dog breeds in this screening. A possible explanation for Table 3. Sequence comparison of TFAP2B in canine, human,
mouse and rat. The percent homology of the amino acid sequences are in bold. The percent sequence identities of the mRNA sequences are in parentheses.
Percent of identify in amino acid and mRNA sequence H. sapiens M. musculus R. norvegicus C. familiaris 100 (96.1) 98 (90.6) 97.1 (90.8)
H. sapiens - 98 (90.6) 97.1 (89.2)
M. musculus - - 95.2 (93.4)
Fig 3. Predicted structure (A) and conserved domains (B) of canine TFAP2B. TF_AP-2; AP-2 proteins- transactivation domain.
the high prevalence rate in this dog breed is the contribution of recessive loci with reduced penetrance. A population genetics study in Iran for congenital heart defects performed by Mani et al. (7) compared the prevalence of PDA in popu- lations with differing rates of consanguinity, and demon-
strated a single gene association for the population with high rates of PDA. Subsequent genome-wide analysis of linkage in this Iranian population revealed the linkage of PDA to a 3- cM interval of chromosome 12q24 (Lod score of 6.27), sug- gesting a single gene etiology responsible for at least one- third of the PDA cases in this population. The situation was similar to our Maltese population where over 50% of the clinical cases of PDA in Korea were Maltese dogs (1). Fur- thermore, this dog breed is highly inbred (a few stud dogs from Japan were intensively used for breeding in the past).
Our premise of a genetic screen of this dog breed was based on findings described previously (1).
The transcription factor AP-2 Beta (TFAP2B) family appears to regulate the expression of genes required for development of tissues of ectodermal origin (16). Satoda et al. (13) found that TFAP2B played a role in ductal, facial, and limb development in humans. Several mutations in TFAP2B were found to be responsible for human syndromic PDA (Char syndrome) (7,13,17). The mutations were located in the basic domain, the conserved PY motif in the transacti- vation domain and splicing site of exon 3 (7,17). Affected individuals with the PY motif mutation had a high preva- lence of patent PDA (17).
Our preliminary screening of TFAP2B in dogs with PDA found one single c.936+203G>A base change in two unre- Fig 4. The alignment of canine TFAP2B protein sequences from different sources. GenBank accession number for the sequences used:
H. sapiens (NP_003212), M. musculus (NP_033360), R. norvegicus (XP_217356), and G. gallus (NP_990495). The amino acid sequence in the three major domains of TFAP2B is highly conserved across these species. Red: 100% homology, Blue: 50% homology, and Green: 75% homology.
Fig 5. DNA sequence analysis showing c.936+203G>A base change in dogs with PDA.
128 So-Jeong Nam and Changbaig Hyun
lated affected Maltese dogs and one unaffected control dog.
Since the base change was located at the intronic region and the base change was also found in an unaffected control dog, the direct effect of this base change may not be responsible for the PDA. Furthermore the same base change was also found in dogs without PDA, suggesting this base change may be a single polymorphism. Because we only screened the exonic region of the TFAP2B, future study should be directed towards identifying the mutations in the regulatory and pro- moter regions of TFAP2B or performing a direct comparison of the expression level of TFAP2B in the unaffected and affected groups. This may help to understand the role of TFAP2B in the pathogenesis of PDA.
Acknowledgements
This study was supported by a research fund from Korean Research Foundation (KRF-2008-331-E00369).
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동맥관 개존증(PDA)에 이환된 개에서의 전사 인자 AP-2 beta(TFAP2B) 유전자 스크리닝
남소정·현창백1
강원대학교 수의(학부)대학 소동물 내과교실
요 약 : 동맥관 개존증(PDA)은 동맥관의 불완전한 폐쇄로 인해 하행 대동맥과 폐동맥 사이에 비정상적인 단락이 형 성된 것이다. 최근 인간의 유전자 연구에서 전사인자 AP-2 beta (TFAP2B)의 유전자 변이가 PDA 증후적 사례를 초 래함을 발견하였다. TFAP2B 유전자의 변이는 사람의 PDA와 같은 특정 선천성 심장 기형과 관련되어 있다. 본 연구 에서는 PDA에 이환된 개의 유전자 스크리닝을 하기 위해 개 TFAP2B유전자의 전체 exon 부위를 분리하였다. 개 TFAP2B유전자는 사람의 TFAP2B와 아미노산 서열이 매우 유사하였다. PDA에 이환된 말티즈견의 TFAP2B유전자 스 크리닝에서 단일 c.936+203G>A 염기 변화가 발견되었다. 그러나 대조군의 유전자 스크리닝에서도 동일한 염기 변화 가 발견되었다. 이 염기의 변화는 인트론 지역에 위치해 있었으며 이환되지 않은 대조군 개에서 발견된 것으로 보아 TFAP2B는 말티즈 견의 유전성 PDA와 다른 종의 PDA 환자를 초래하지 않을 것으로 보인다. 향후 더 많은 샘플을 모으고 PDA에 이환된 다양한 종과 다른 선천성 심장 기형을 가진 환축에서 TFAP2B를 스크리닝하는 연구가 필요하다.
주요어 : 개, TFAP2B, 선천성 심장병, 동맥관 개존증.
