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Mamm Genome (2007) 18:871–879 Novel repeat polymorphisms of the dopaminergicneurotransmitter genes among dogs and wolves Krisztina Hejjas Æ Judit Vas Æ Eniko Kubinyi Æ Maria Sasvari-Szekely ÆAdam Miklosi Æ Zsolt Ronai Received: 21 May 2007 / Accepted: 21 September 2007 / Published online: 30 November 2007Ó Springer Science+Business Media, LLC 2007 Genetic polymorphisms of the neurotransmis- difference of allele and genotype frequencies was demon- sion systems are intensively studied in the human because of a possible influence on personality traits and the risk of association analysis was also carried out between the psychiatric disorders. The investigation of genetic varia- activity–impulsivity phenotype and the described VNTRs.
tions of the dog genome has recently been a promising Preliminary findings are presented that polymorphisms of approach, as a considerable similarity can be observed the DRD4, DBH, and DAT genes can be associated with between dogs and humans, in both genetic and social attention deficit among Belgian Tervuerens.
aspects, suggesting that the dog could become an appro-priate animal model of human behavioral genetic studies.
The aim of our study was the identification and analysis ofvariable (VNTRs) in the genes of the dopaminergic neurotrans-mitter system of dogs. The in silico search was followed by The investigation of genetic variations has recently been the development of PCR-based techniques for the analysis the focus of interest because they play a significant role in of the putative VNTRs. Highly variable repetitive sequence the development of inherited features (i.e., psychological regions were found in the tyrosine hydroxylase (TH), traits) and they are supposed to code for risk or protective dopamine transporter (DAT), and dopamine b-hydroxylase factors of complex diseases. One of the most often used (DBH) genes. Allele frequency and genotype distribution approaches for the identification of genetic components of of these novel polymorphisms together with the exon 3 and multifactorial traits is the analysis of candidate genes in exon 1 VNTR of the dopamine D4 receptor gene were association studies; this methodology has been used so far determined in a large sample involving four dog breeds in studies of dogs. Single nucleotide polymorphisms of the (German Shepherd, Belgian Tervueren, Groenandael, and cytokine genes were investigated in canine malignant his- Malinois) and European Grey Wolves. A significant tiocytosis (Soller et al. ) and in rupture of the cranialcruciate ligament (Wilke et al. Microsatellitemarkers were employed for the analysis of dog breedphylogeny, and significant divergence was shown in each K. Hejjas
M. Sasvari-Szekely
Z. Ronai (&)Department of Medical Chemistry, Molecular Biology and of the seven tested American Kennel Club groups (Irion Pathobiochemistry, Semmelweis University, Budapest, Hungary The genetic variations of the human dopaminergic neu- rotransmitter system are major targets in human psychiatric J. Vas
E. Kubinyi
A. MiklosiDepartment of Ethology, Eotvos Lorand University, Budapest, genetic studies, involving the investigation of polymor- phisms in the dopamine D4 receptor, dopamine transporter,and dopamine-b-hydroxylase genes. The dopamine D4 receptor (DRD4) gene has recently been the focus of Department of Biochemistry, Eotvos Lorand University,Budapest, Hungary interest, as this protein is highly abundant in the limbic K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs system, which is responsible for emotions and cognitive polymorphic VNTRs, which were analyzed as possible functions, and it is the target of atypical antipsychotic drugs genetic components of disruptive behavior disorders (Lee (e.g., clozapine) used in the therapy of several psychiatric et al. alcoholism (Kohnke et al. ), and attention illnesses (Andreasen ). Moreover, both the 50 region deficit hyperactivity disorder (Purper-Ouakil et al. and the coding sequence of the DRD4 gene are highly Moreover, polymorphisms in the tyrosine hydroxylase polymorphic in humans (Szantai The most thor- (TH) and dopamine-b-hydroxylase (DBH) genes have also oughly investigated polymorphism of the gene is located in been studied in genetic association analyses. These genes exon 3, where the repeat number of a 48-base pair (bp)-long code for enzymes that play a role in the metabolism of segment varies from 2 to 10. A large number of studies deal with this variable number of tandem repeats (VNTR) as a investigated the putative association between DBH poly- possible risk factor for several psychiatric disorders (for morphisms and attention deficit hyperactivity (Tang et al.
recent reviews see Eisenberg et al. Jonsson et al.
; Wigg et al. ), and polymorphisms of the TH ; Lopez Leon et al. ; Lusher et al. ). Inter- gene were also suggested to be genetic risk factors for estingly, an analog of this polymorphism was described in various disorders such as schizophrenia (Kurumaji et al.
dogs (Canis familiaris) as well (Niimi et al. More- ), dependent smoking (Olsson et al. ), and mood over, it is notable that no similar genetic variation can be disorders (Serretti et al. found in rodent models such as mice or rats (O'Malley et al.
Although researchers have been accumulating a signif- ). In contrast to the human VNTR, the repetitive seg- icant body of knowledge about the DRD4 polymorphisms ment of the dog DRD4 exon 3 polymorphism consists of among dogs, nothing is known about the canine genetic modules of 12 and 39 bp, respectively. The distribution of variations related to the other dopaminergic genes. Here we the different alleles was analyzed in several dog breeds report the identification and analysis of several novel repeat (Niimi et al. Our group recently investigated the polymorphisms in the DAT, DBH, and TH genes and a effect of this polymorphism on the behavior of German detailed study of the known DRD4 polymorphisms among Shepherds (Hejjas et al. ), as the DRD4 exon 3 VNTR four dog breeds as well as in European Grey Wolves.
was demonstrated to be associated with the novelty-seeking Because dysfunction of the dopaminergic system is known personality trait in human (Ebstein and in a nonhu- to be in the background of attention deficit hyperactivity man primate's behavior (Bailey et al. ).
disorder (ADHD) (e.g., Faraone et al. ), behavioral Several further repeat variants have also been described data of Belgian Tervuerens were also collected and in the coding region of the human DRD4 gene. A 12-bp investigated in a genetic association study.
duplication and a 13-bp deletion were demonstrated andanalyzed simultaneously (Chang and Kidd ) in exon 1,and it was shown that the latter results in a nonfunctional Materials and methods truncated protein (Nothen et al. ). Polymorphisms areabundant in the 50 noncoding region of the DRD4 gene as well. A 120-bp duplication was investigated as a possiblerisk factor for attention deficit hyperactivity disorder Four dog breeds (Belgian Tervueren, N = 102; Belgian (Kereszturi et al. ; McCracken et al. ), and the Groenandael, N = 105; Belgian Malinois N = 50; and impact of this polymorphism on gene expression was also German Shepherd, N = 240) and 22 European Grey analyzed (D'Souza et al. Kereszturi et al. Wolves (Canis lupus) were involved in our study. For the Besides the DRD4 exon 3 VNTR of dogs, an insertion/ most common characteristics of the investigated breeds, deletion polymorphism was described in exon 1 of the see Supplementary Table DNA sampling was performed canine gene. This variation is supposed to be the analog of by applying a noninvasive approach; buccal smear was the human 12-bp duplication based on its location in the collected with cotton swabs from the inner surface of the gene (Ito et al. Although there is a considerably high cheek, and DNA purification was carried out using the variance between allele frequencies of these polymor- Gentra DNA Purification Kit (Qiagen, Valencia, CA).
phisms in different canine breeds (Ito et al. ), little isknown about their effect on either receptor function orbehavior.
In silico analysis Another major element of the dopaminergic neuro- transmission is the dopamine transporter (DAT) playing a The sequences of the investigated genes (except that of role in the reuptake of dopamine. This protein is the target DRD4) were downloaded from the GenBank of amphetamine-like prescription stimulants (e.g., methyl- /) and Ensembl /) databases. GenBank accession numbers were as follows: K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs Table 1 The primers used for the amplification of the investigated repetitive regions GCA CCT CTC CCG CCC TCT TTA ACG CCA TCT ACC CTG TGA CTC CTG TGT CCC CGC TGT CTT GAC AGA GCA GGG CAG GGA GG CCC CTC ACC TCC AAG CAG AGG GTG ATG TGG GCA GGA T GTG AAG CCG ACC AGC CAT TG CTG ATT TCT CCA GCC GTG TTG GTC TGT CTG CTG TCT GGC TCC C TGG AGA GGC TTC CTG ACA CCC CGC GCG TCG GGC CAA GCT G GCG GGG GGC AGG GGG CG TGG GCT GGG GGT GCC GTC C CGC CAT GGG GAA CCG CAG CGG CTC ACC TCG GAG TAG A DAT = dopamine transporter; DBH = dopamine-b-hydroxylase; TH = tyrosine hydroxylase; DRD4 = dopamine receptor D4a TA shows the applied annealing temperature tyrosine hydroxylase: NM_001002966, AB097058, and with an allele-specific reverse primer D4dogBR. The PCR ENSCAFG00000010099; dopamine-b-hydroxylase: NM_0 analysis of the DRD4 exon 1 polymorphism was carried 01005263, AB097057, and ENSCAFG00000019783; and out as described by (Ito et al. ).
dopamine transporter: ENSCAFG00000010574. Exon-intron boundaries were determined based on the data fromEnsembl. The sequence of the DRD4 gene was obtained from the publication of Niimi et al. (The investi-gated genes were in silico searched for repetitive sequences PCR products were analyzed by conventional submarine applying the Web-based Tandem Repeats Finder search agarose gel electrophoresis (Biocenter, Szeged, Hungary), using 1.5% agarose + 2% Metaphore composite gel andvisualized by ethidium bromide staining.
PCR amplification Investigation of the activity–impulsivity Polymerase chain reaction (PCR) primers were designed by endophenotype of dogs, association study Oligo 5.0 software (Plymouth, MN). The Qiagen Hot-StarTaq polymerase kit was used for PCR amplification in The human ADHD Rating Scale (ADHD RS) Parent Ver- each analysis except the investigation of exon 1 of the sion questionnaire (DuPaul ) was adapted and DRD4 gene. The reaction mixture contained 1 lM of each validated for dog owners (Vas et al. 2006) and was used primer (Table ), approximately 5 ng of DNA template, here to characterize activity-impulsivity and attention of 200 lM dATP, dCTP, dTTP, and 100 lM of dGTP and dogs. Owners filled out the dog ADHD RS Owner Version dITP, 0.025 U HotStarTaq DNA polymerase, 19 buffer, questionnaire, containing 13 items (7 items form the and 19 Q-solution supplied together with the enzyme. The activity-impulsivity scale and 6 items form the inattention total volume of the PCR mixture was 10 ll. The PCR cycle scale). Scale scores were calculated for each dog as the consisted of an initial denaturation at 95°C for 15 min, 35 sum of scores given by the owner (range = 0–3). Fifty-nine cycles of 1-min denaturation at 95°C, an 1-min annealing Belgian Tervueren (24 males, 35 females, age mean ± at various temperatures (see TA in Table ), an 1-min SD = 5.5 ± 3.6 years) were characterized.
extension at 72°C, and a 10-min final extension at 72°C. A SPSS for Windows (SPSS, Inc., Chicago, IL) was used second, independent PCR was performed to identify the for all statistical analyses. Associations of the ADHD test ‘‘3a'' and ‘‘3b'' alleles of DRD4 exon 3 according to Niimi scores with the investigated polymorphisms were assessed et al. (using the forward primer D1C in combination by one-way ANOVA and independent-samples t test.

K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs determine the length (i.e., repeat number) of the alleles(Fig. A). A second PCR was necessary to distinguish In silico search for dog analogs of human VNTRs in the between variants 3a and 3b because they differ only in the dopaminergic genes order of the 12- and 39-bp-long modules (Niimi et al.
). Figure B represents a typical electropherogram of As a first step, the selected dopaminergic genes were in a homozygote for allele 3a, but no allele 3b was found in silico searched for repetitive sequences in the dog genome.
the studied populations. Comparing the allele frequencies Both 50 (1000-bp long), intronic and exonic segments were of 2 and 3a, the latter was most frequent in Tervuerens included in our study because exon variations may result in and Malinois, whereas allele 2 was dominant in German alteration of the protein structure, whereas polymorphisms Shepherds. The two variants had nearly the same fre- in introns or in the 50 region may influence the activity of quencies in Groenandaels. No other described variations gene expression. Regions were chosen for further analysis were found in these breeds, while allele 5 was the major if (1) the length of the repeated module was larger than 6 variant among wolves (Table ). Moreover, a novel allele bp (i.e., not microsatellites) and (2) the sequence similarity (we named/signed allele 8) was demonstrated to be of the repeated units was at least 85%. Based on the above present at a large frequency in this group. This form is considerations, the following regions were involved in the longer than any of those described earlier, presumably by remaining parts of our study: 36-bp duplication in intron 4 the insertion of an extra 39-bp unit, demonstrated in of the TH gene; a 17-bp duplication in intron 4 and a 24-bp Figure The v2 test showed a significant difference in duplication in exon 11 of the DBH gene; a 59-bp dupli- the genotype distributions among the studied breeds cation in intron 5 and a 38-bp duplication in intron 9 of the (v2 = 523.28; df = 32; p 0.0001).
DAT gene, besides the known polymorphisms of DRD4 The 24-bp insertion/deletion polymorphism in exon 1 of exon 1 and exon 3.
the dopamine D4 receptor (DRD4) gene described by Itoet al. ) was shown to be polymorphic in all dogs;however, wolves possessed only the long variant. Fig- Pilot studies for selection of polymorphic repeat regions ure shows the electrophoretic analysis of three samples among dog breeds and European Grey Wolves with L/L, S/L, and S/S genotypes, respectively (‘‘L'' stands A subset of samples (N = 48) of each of the four investi-gated breeds (Tervueren, Groenandael, Malinois, GermanShepherd) and all wolves (N = 22) was used for a pilotanalysis to decide which of the in silico identified andselected repetitive regions are polymorphic in our dogpopulations. No polymorphic variations were found in therepeat regions identified in silico in exon 11 of the DBHgene and in intron 5 of the DAT gene. On the other hand,variable repeat numbers were shown in intron 4 of the THand DBH genes, in intron 9 of the DAT gene, and in exon 1of the DRD4 gene in our breeds. These regions, togetherwith the exon 3 polymorphism of the DRD4 gene describedearlier by (Niimi et al. were subjected to down-stream analysis on the whole population (N = 495) ofvarious dog breeds. The accurate chromosomal localizationof the VNTRs is shown in Table .
Analysis of the known VNTRs in the dopamine D4 Fig. 1 Analysis of the VNTRs in the dopamine D4 receptor gene. A DRD4 exon 3 amplicons in the ‘‘first PCR'': Identification of therepeat number (2 or 3). B Differentiation between alleles 3a and 3b The VNTR in exon 3 of the DRD4 gene was shown to be (allele 2 and 8 are also shown) in the ‘‘second PCR.'' C Identification polymorphic in each of the investigated breeds as well as of allele 8 in homo- and heterozygous form D Genotyping of 24-bpinsertion/deletion polymorphism in exon 1 (L = insertion, S = dele- among European Grey Wolves. Figure A, B depicts the tion). Genotypes are shown under the electropherograms, and the electropherogram of the obtained PCR products. In a first estimated lengths of the PCR fragments are depicted on the right. A 100-bp DNA ladder was used as size marker K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs Table 2 Distribution of DRD4 exon 1 and exon 3 genotypes among dog breeds and wolves Belgian Tervueren Belgian Groenandael This table shows the number of individuals in each category as well as frequency values (percentage), which are in parentheses for the insertion allele and ‘‘S'' for the deletion allele). The lowest frequency among German Shepherds (3.12%).
L allele was the dominant form in German Shepherds; on Genotype (v2 = 77.212; df = 8; p 0.0001) distribution of the other hand, approximately the same frequencies have this polymorphism also showed a significant difference been found among Groenandael and Malinois, while the among the five breeds. The measured genotype frequencies long form was the minor variant in Tervuerens. The fit the Hardy-Weinberg equilibrium.
genotype frequencies were thus highly variable among the The results of the PCR-based investigation of the 38-bp breeds (Table ), and the difference was statistically sig- repetitive sequence in intron 9 of the DAT gene can be seen nificant (v2 = 280.58; df = 8; p 0.0001).
in Figure Interestingly, German Shepherds possessedonly the longer variant (allele 2), and this form was themost common allele in the other breeds (82.61–100%), Analysis of the novel VNTRs except the Malinois (Table In this latter group, thefrequency of the two alleles was almost the same. The The 36-bp-long sequence in the intron 4 region of the TH difference of the genotype frequencies among the breeds gene was demonstrated to be present either as a single copy was also statistically significant (v2 = 248.06; df = 8; or in a duplicated form. Figure A depicts the result of the p 0.0001), and the measured genotype frequencies fit the PCR analysis of this region. The presence of allele 1 was rare in German Shepherds, Malinois, and Grey Wolves, butthis allele was fairly frequent in Tervuerens and Groe-nandaels (Table ). Thus, genotype frequencies differed in Pilot study of associations between the VNTRs and the a fairly wide range in the various populations (v2 = 218.61; activity-impulsivity and attention deficit rating scale in df = 8; p 0.0001). However, the measured genotype Belgian Tervuerens frequencies did not show statistically significant deviationfrom the Hardy-Weinberg equilibrium.
Belgian Tervuerens were used for the association analyses The 17-bp variation in the fourth intron of the DBH gene of the investigated polymorphisms because (1) almost all was demonstrated to be polymorphic in each breed; the genetic variants could be identified in this group and (2) a copy number was interestingly either 1 or 3, while no relatively large number of individuals were available for duplication could be observed in our samples. The elec- trophoretic analysis of samples with 1/1, 1/3, and 3/3 One-way ANOVA was used to compare the three genotypes is shown in Figure B. The variant containing genotype groups in the case of TH, DRD4 exon 1, and three repeats was the minor allele in all breeds. This variant DRD4 exon 3 polymorphisms. In the case of DBH and DAT occurred most frequently in Malinois (23.96%) and had the polymorphisms, allele 3 and allele 1 were rather rare,

K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs Fig. 2 Genotyping of the novel VNTRs. A Tyrosine hydroxylase 2 repeats). Genotypes are shown under the electropherograms, and the intron 4 VNTR (1 or 2 repeats). B Dopamine-b-hydroxylase intron 4 estimated lengths of the PCR fragments are depicted on the right. A VNTR (1 or 3 repeats). C Dopamine transporter intron 9 VNTR (1 or 100-bp DNA ladder was used as the size marker Table 3 Genotype frequencies of the polymorphisms in the TH, DBH, and DAT genes among the investigated dog breeds and wolves Belgian Tervueren Belgian Groenandael The number of individuals belonging to each category is displayed, and percentage values are given in parentheses respectively (Tables and ); consequently, individuals On the other hand, genotype-dependent differences were possessing only the minor allele in homozygous form and found in the case of the attention-deficit scale of the dog heterozygote animals were merged into a single group for ADHD Rating Scale. Individuals possessing at least one reliable statistical analysis. Therefore, two groups have DBH allele 1 (N = 13), at least one DAT allele 1 (N = 9), or been defined in case of both polymorphisms: (1) samples dogs carrying the short variant in homozygous form in possessing the minor allele in homozygous or heterozygous DRD4 exon 1 (N = 10) were rated to have a higher degree form (DBH 3+ and DAT 1+ groups, respectively) and (2) of attention deficit (DRD4 exon 1: F(2,56) = 4.1120, dogs lacking this variant (DBH 3– and DAT 1– groups, p = 0.0216; DBH: t(13.8) = –2.2238, p = 0.0434; and DAT: respectively). These genotype groups were compared by t(57) = 2.8763, p = 0.0057, respectively). There was, how- using independent-samples t test.
ever, no difference in TH (F(2,56) = 0.3037, p = 0.7393) Our results demonstrated that there was no difference and DRD4 exon 3 (F(2,56) = 1.7938, p = 0.1758) variants.
between genotype groups in the activity-impulsivity scaleof the dog ADHD Rating Scale (the three TH groups:F(2,56) = 0.2246, p = 0.7996; the three DRD4 exon 1 groups: F(2,56) = 1.3066, p = 0.2789; the three DRD4exon3 groups: F(2,56) = 0.3576, p = 0.7010; the two DBH Genetic variants presumably responsible for inherited traits groups: t(57) = 1.4049, p = 0.1655; and the two DAT and disorders spread throughout the whole genome. The groups: t(57) = 1.6171, p = 0.1114).
size of these polymorphisms varies in an extremely wide K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs range from affecting a single nucleotide expanding to database. More studies are needed, however, to establish large, microscopically visible chromosome alterations with the functional significance of the identified nonexonic a length of several millions of base pairs (Redon et al.
polymorphisms of the dopaminergic genes of the dog ). The former variant is referred to as single nucleotide polymorphism (SNP), and a large number of these varia- Our results also illustrate that the genetic architecture of tions have already been discovered and investigated various dog breeds is quite different. A high divergence (Shianna and Willard as their high throughput and was observed even within the Belgian Shepherd breed, automated analysis is readily accessible. These variations between Belgian Malinois and the two other Belgian can be used as markers in linkage (Amos et al. ) and Shepherd subgroups (Table , tyrosine hydroxylase intron genome-wide analyses (John et al. ; Middleton et al.
4, dopamine transporter intron 9). Another interesting ). Moreover, they were investigated as functional finding is that the genetic difference among dog breeds was variants in both coding (Ollerenshaw et al. and shown to be larger than the variability between dogs and noncoding (Kereszturi et al. ; Okuyama et al. wolves. This observation is in agreement with the findings gene regions. Repeat polymorphisms (i.e., microsatellites, of Vila et al. ), who investigated mitochondrial DNA.
deletions, duplications, and VNTR), on the other hand, are They suggested that the genetic difference between dogs of great practical and functional significance for several and wolves may be not so significant because of the fact reasons. First, the application of multiallelic microsatellites that the two species continued to exchange genetic material can improve the efficiency of whole-genome scans by after their divergence, which happened more than 100,000 increasing the power of the analysis compared with the use of biallelic SNPs (Vignal et al. Moreover, a large set Our study also suggested that the investigated length of repeat polymorphisms have been investigated in asso- polymorphisms could have a functional role in determining ciation analyses, and their functional effect was also behavior in Belgian Tervuerens. Although the sample size demonstrated (Nakamura et al. ). VNTRs in coding in this association study is not too high, these are the first regions can result in either frameshift and consequently a results showing the effect of a few gene polymorphisms in truncated protein (e.g., 13-bp deletion in the human DRD4 a physiologic system on observable behavior in a relatively gene [Nothen et al. or in a repeated amino homogeneous sample, i.e., in a single breed. Furthermore, acid sequence (e.g., human and canine DRD4 exon 3 in an earlier study we found some effect of DRD4 exon 3 polymorphism on attention deficit in police German Here we described several novel VNTRs of the dopa- Shepherds but not in pet German Shepherds (Hejjas et al.
minergic genes in the canine genome besides the extended ). It is important to note that genetically homogeneous study of the known polymorphisms in the DRD4 gene sample populations are preferred to be analyzed in geno- (Table ). Our results show a considerable similarity type–phenotype association studies to avoid false-positive between human and canine VNTRs only in exonic poly- correlations (Hamer and Sirota ).
morphisms, while the intronic polymorphic variations have These findings can be supported in the future by large- quite a different nature between the dog and the human scale studies. Such genetic effects on behavior could have, genome (Table ). It is obvious that the nonexonic regions in principle, an important consequence in dog breeding.
of the genome have a lower selection pressure and there- Attention skills have significant relevance in trainability fore a higher variability among species. On the other hand, and communicative behavior, both of which contribute to intronic sequences might have an important regulatory role the everyday challenges of dog-human interaction. Present in gene expression, as was shown for a VNTR in intron 2 of results suggest that the process of selecting a dog for a the human serotonin transporter gene (Fiskerstrand et al.
definite purpose such as therapy, sport, police work, or pets ). Moreover, a considerable homology was demon- can be based on the animal's genetic composition, so a strated between the repeat sequences in the noncoding large amount of money and energy can be saved if the region of the human dopamine and serotonin transporters individual with appropriate genotype is chosen. Further- in spite of their differential localizations (Michelhaugh more, dogs can offer an opportunity to model human et al. Functional significance of the canine repeat dopaminergic diseases as a natural animal model (Overall regions analyzed in our study is also conceivable, as the binding site of the human Sp1 transcription factor can be In summary, we developed and applied a PCR-based demonstrated in the VNTR of tyrosine hydroxylase intron technique for the investigation of in silico identified puta- 4, whereas the DBH intron 4 VNTR contains an AML-1a tive repeat polymorphisms and verified the presence of transcription factor binding site according to TFSEARCH VNTRs in some candidate genes of the dopaminergic (Searching Transcription Factor Binding Sites ver 1.3; neurotransmitter system in four dog breeds (Belgian Ter- vueren, Belgian Groenandael, Belgian Malinois, and K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs Table 4 Repeat sequences and their polymorphisms among the genes of dopaminergic neurotransmitter system chr 18: 28722804–28723009 chr 34: 142 411 027–142 411 102 chr 9: 53 348 625–53 348 675 chr 18: 49 355 111–49 355 075 ? = sequence is unknown. Numbers in the allele columns indicate the repeat number except in the case of dog DRD4 exon 3 polymorphismwhere alleles are designated according to Niimi et al. (); np = nonpolymorphic repeats. The length of the repeated modules is shown in thelength column. Chromosomal localization of the polymorphic VNTRs is also shown according to the Ensembl databasea The whole sequence of the DRD4 gene is not available in Ensembl; sequence information was obtained from NCBI, accurate chromosomallocalization cannot be obtained however German Shepherd) and in wolves. We presented some Ebstein RP (2006) The molecular genetic architecture of human preliminary results that DRD4 exon 1, DBH, and DAT personality: beyond self-report questionnaires. Mol Psychiatry11(5):427–445 polymorphisms can be associated with attentive behavior Eisenberg J, Zohar A, Mei-Tal G, Steinberg A, Tartakovsky E, et al.
in Belgian Tervuerens. These novel variants are subjects (2000) A haplotype relative risk study of the dopamine D4 for further analysis in other populations as well as for receptor (DRD4) exon III repeat polymorphism and attention behavioral association studies, which can clarify their role deficit hyperactivity disorder (ADHD). Am J Med Genet96(3):258–261 in the development of various phenotypic traits in dogs and Faraone SV, Perlis RH, Doyle AE, Smoller JW, Gorlanick JJ, et al.
(2005) Molecular genetics of attention-deficit/hyperactivitydisorder. Biol Psychiatry 57:1313–1323 This research has been supported by the Euro- Fiskerstrand CE, Lovejoy EA, Quinn JP (1999) An intronic pean Union (NEST 012787) and the OTKA T029705.
polymorphic domain often associated with susceptibility toaffective disorders has allele dependent differential enhanceractivity in embryonic stem cells. FEBS Lett 458(2):171–174 Hamer D, Sirota L (2000) Beware the chopsticks gene. Mol Psychiatry 5:11–13 Hejjas K, Vas J, Topal J, Ronai Z, Szekely A, et al. (2007) Amos CI, Chen WV, Lee A, Li W, Kern M, et al. (2006) High-density Association of the dopamine D4 receptor gene polymorphism SNP analysis of 642 Caucasian families with rheumatoid arthritis and the ‘‘activity-impulsivity'' endophenotype in dogs. Anim identifies two new linkage regions on 11p12 and 2q33. Genes Immun 7(4):277–286 Irion DN, Schaffer AL, Famula TR, Eggleston ML, Hughes SS, et al.
Andreasen NC, Black DW (1995) Introductory Textbook of Psychi- (2003) Analysis of genetic variation in 28 dog breed populations atry, 2nd ed. (Washington, DC: American Psychiatry Press) with 100 microsatellite markers. J Hered 94(1):81–87 Bailey JN, Breidenthal SE, Jorgensen MJ, McCracken JT, Fairbanks Ito H, Nara H, Inoue-Murayama M, Shimada MK, Koshimura A, LA (2007) The association of DRD4 and novelty seeking is et al. (2004) Allele frequency distribution of the canine found in a nonhuman primate model. Psychiatr Genet 17(1):23– dopamine receptor D4 gene exon III and I in 23 breeds. J Vet Med Sci 66(7):815–820 Benson G (1999) Tandem repeats finder: a program to analyze DNA John S, Shephard N, Liu G, Zeggini E, Cao M, et al. (2004) Whole- sequences. Nucleic Acids Res 27(2):573–580 genome scan, in a complex disease, using 11,245 single- Chang FM, Kidd KK (1997) Rapid molecular haplotyping of the first nucleotide polymorphisms: comparison with microsatellites.
exon of the human dopamine D4 receptor gene by heteroduplex Am J Hum Genet 75(1):54–64 analysis. Am J Med Genet 74(1):91–94 Jonsson EG, Sedvall GC, Nothen MM, Cichon S (2003) Dopamine D'Souza UM, Russ C, Tahir E, Mill J, McGuffin P, et al. (2004) D4 receptor gene (DRD4) variants and schizophrenia: meta- Functional effects of a tandem duplication polymorphism in the analyses. Schizophr Res 61(1):111–119 50 flanking region of the DRD4 gene. Biol Psychiatry 56(9):691– Kereszturi E, Kiraly O, Barta C, Molnar N, Sasvari-Szekely M, et al.
(2006a) No direct effect of the -521 C/T polymorphism in the DuPaul GJ (1998) ADHD Rating Scale-IV: Checklist, Norms and human dopamine D4 receptor gene promoter on transcriptional Clinical Interpretations (New York: Guilford Press) activity. BMC Mol Biol 7:18 K. Hejjas et al.: Novel VNTRs of the dopaminergic genes in dogs Kereszturi E, Kiraly O, Csapo Z, Tarnok Z, Gadoros J, et al. (2006b) Ollerenshaw M, Page T, Hammonds J, Demaine A (2004) Polymor- Association between the 120-bp duplication of the dopamine D4 phisms in the hypoxia inducible factor-1alpha gene (HIF1A) are receptor gene and attention deficit hyperactivity disorder: associated with the renal cell carcinoma phenotype. Cancer Genetic and molecular analyses. Am J Med Genet B Neuropsy- Genet Cytogenet 153(2):122–126 chiatr Genet 144:231–236 Olsson C, Anney R, Forrest S, Patton G, Coffey C, et al. (2004) Kohnke MD, Batra A, Kolb W, Kohnke AM, Lutz U, et al. (2005) Association between dependent smoking and a polymorphism in Association of the dopamine transporter gene with alcoholism.
the tyrosine hydroxylase gene in a prospective population-based Alcohol Alcoholism 40(5):339–342 study of adolescent health. Behav Genet 34(1):85–91 Kurumaji A, Kuroda T, Yamada K, Yoshikawa T, Toru M (2001) An O'Malley KL, Harmon S, Tang L, Todd RD (1992) The rat dopamine association of the polymorphic repeat of tetranucleotide (TCAT) D4 receptor: sequence, gene structure and demonstration of in the first intron of the human tyrosine hydroxylase gene with expression in the cardiovascular system. New Biologist 4:137– Overall KL (2000) Natural animal models of human psychiatric Lee SS, Lahey BB, Waldman I, Van Hulle CA, Rathouz P, et al.
conditions: Assessment of mechanism and validity. Prog Neu- (2006) Association of dopamine transporter genotype with ropsychopharmacol Biol Psychiatry 24:727–776 disruptive behavior disorders in an eight-year longitudinal study Purper-Ouakil D, Wohl M, Mouren MC, Verpillat P, Ades J, et al.
of children and adolescents. Am J Med Genet B Neuropsychiatr Genet 141(4):398–402 between the dopamine transporter gene and attention deficit Lopez Leon S, Croes EA, Sayed-Tabatabaei FA, Claes S, Van hyperactivity disorder. Psychiatr Genet 15(1):53–59 Broeckhoven C, et al. (2005) The dopamine D4 receptor gene Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, et al. (2006) 48-base-pair-repeat polymorphism and mood disorders: a meta- Global variation in copy number in the human genome. Nature analysis. Biol Psychiatry 57(9):999–1003 Lusher JM, Chandler C, Ball D (2001) Dopamine D4 receptor gene Serretti A, Cusin C, Cristina S, Lorenzi C, Lilli R, et al. (2003) (DRD4) is associated with Novelty Seeking (NS) and substance Multicentre Italian family-based association study on tyrosine abuse: the saga continues. Mol Psychiatry 6(5):497–499 hydroxylase, catechol-O-methyl transferase and Wolfram syn- McCracken JT, Smalley SL, McGough JJ, Crawford L, Del'Homme drome 1 polymorphisms in mood disorders. Psychiatr Genet M, et al. (2000) Evidence for linkage of a tandem duplication polymorphism upstream of the dopamine D4 receptor gene Shianna KV, Willard HF (2006) Human genomics: in search of (DRD4) with attention deficit hyperactivity disorder (ADHD).
normality. Nature 444(7118):428–429 Mol Psychiatry 5(5):531–536 Soller JT, Murua Escobar H, Janssen M, Fork M, Bullerdiek J, et al.
Michelhaugh SK, Fiskerstrand C, Lovejoy E, Bannon MJ, Quinn JP (2006) Cytokine genes single nucleotide polymorphism (SNP) (2001) The dopamine transporter gene (SLC6A3) variable screening analyses in canine malignant histiocytosis. Anticancer number of tandem repeats domain enhances transcription in Res 26(5A):3417–3420 dopamine neurons. J Neurochem 79(5):1033–1038 Szantai E, Szmola R, Sasvari-Szekely M, Guttman A, Ronai Z (2004) Middleton FA, Pato MT, Gentile KL, Morley CP, Zhao X, et al.
The polymorphic nature of the human dopamine D4 receptor (2004) Genome wide linkage analysis of bipolar disorder by use gene: a comparative analysis of known variants and a novel 27 of a high-density single-nucleotide-polymorphism (SNP) geno- bp deletion in the promoter region. BMC Genet 6(1):39 typing assay: a comparison with microsatellite marker assays Tang Y, Buxbaum SG, Waldman I, Anderson GM, Zabetian CP, et al.
and finding of significant linkage to chromosome 6q22. Am J (2006) A single nucleotide polymorphism at DBH, possibly Hum Genet 74(5):886–897 associated with attention-deficit/hyperactivity disorder, associ- Nakamura Y, Koyama K, Matsushima M (1998) VNTR (variable ates with lower plasma dopamine beta-hydroxylase activity and number of tandem repeat) sequences as transcriptional, transla- is in linkage disequilibrium with two putative functional single tional, or functional regulators. J Hum Genet 43(3):149–152 nucleotide polymorphisms. Biol Psychiatry 60(10):1034–1038 Niimi Y, Inoue-Murayama M, Murayama Y, Ito S, Iwasaki T (1999) Vas J, Topa´l J, Pe´ch E ´ , Miklo´si A´ (2007) Measuring attention deficit Allelic variation of the D4 dopamine receptor polymorphic and activity in dogs: A new application and validation of a human region in two dog breeds, Golden retriever and Shiba. J Vet Med ADHD questionnaire. Appl Anim Behav Sci 103:105–117 Sci 61(12):1281–1286 Vignal A, Milan D, SanCristobal M, Eggen A (2002) A review on Niimi Y, Inoue-Murayama M, Kato K, Matsuura N, Murayama Y, SNP and other types of molecular markers and their use in et al. (2001) Breed differences in allele frequency of the animal genetics. Genet Select Evol 34(3):275–305 dopamine receptor D4 gene in dogs. J Hered 92(5):433–436 Vila C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, et al.
Nothen MM, Cichon S, Hemmer S, Hebebrand J, Remschmidt H, (1997) Multiple and ancient origins of the domestic dog. Science et al. (1994) Human dopamine D4 receptor gene: frequent occurrence of a null allele and observation of homozygosity.
Wigg K, Zai G, Schachar R, Tannock R, Roberts W, et al. (2002) Hum Mol Genet 3(12):2207–2212 Attention deficit hyperactivity disorder and the gene for dopa- Okuyama Y, Ishiguro H, Toru M, Arinami T (1999) A genetic mine Beta-hydroxylase. Am J Psychiatry 159(6):1046–1048 polymorphism in the promoter region of DRD4 associated with Wilke VL, Conzemius MC, Rothschild MF (2005) SNP detection and expression and schizophrenia. Biochem Biophys Res Commun association analyses of candidate genes for rupture of the cranial cruciate ligament in the dog. Anim Genet 36(6):519–521

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