The Journal of Heredity 2001:92(5)
© 2001 The American Genetic Association 92:433-435
Brief Communication |
Breed Differences in Allele Frequency of the Dopamine Receptor D4 Gene in Dogs
From the United Graduate School of Agricultural Science (Niimi) and Faculty of Agriculture (Inoue-Murayama, Kato, Matsuura, and Ito), Gifu University, Gifu 501–1193, Japan, National Institute of Animal Health, Tsukuba, Japan (Murayama), and Department of Veterinary Internal Medicine, Tokyo University of Agriculture and Technology, Fuchu, Japan (Momoi, Konno, and Iwasaki).
Address correspondence to M. Inoue-Murayama at the address above or e-mail: miho-i;cacc.gifu-u.ac.jp.
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Abstract |
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We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles—447a, 447b, 486, 498, and 549—and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.
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Introduction |
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The dog (Canis familiaris) has a close living association with human beings, and more than 400 breeds have been established for various purposes, for example, hunting dogs, watch dogs, family dogs, etc. Since different morphological characters and behavioral traits have been selected during establishment of the respective breeds, some of the above characteristics are considered to be peculiar to modern dog breeds. The diversity among breeds is demonstrated also in their genetic backgrounds: the allelic distributions of the genes encoding D blood type (Ejima et al. 1994), complement C6 protein (Shibata et al. 1995), and major histocompatibility antigen class II molecules (Kennedy et al. 1999) are significantly different among breeds, and genetic variations among breeds have also been detected using microsatellites (Koskinen and Bredbacka 1999; Morera et al. 1999; Zajc and Sampson 1999) and arbitrary primer polymerase chain reaction (PCR) (Ezer et al. 1996).
To clarify the variation in neurotransmitter-related genes of dogs, we previously examined the dopamine receptor D4 (DRD4) gene and demonstrated that polymorphism existed in dog DRD4 as observed in humans (Niimi et al. 1999). Human DRD4 is polymorphic in terms of the repeat numbers of the 48 bp sequence in the third cytoplasmic loop of the receptor (Van Tol et al. 1992). It has been shown that the allelic distribution is considerably different among human populations (Chang et al. 1996) and that human DRD4 helps to modulate the personality trait known as novelty seeking (Benjamin et al. 1996; Ebstein et al. 1996). The polymorphic region of dog DRD4 corresponding to the repeated region in human DRD4 is composed of three types of units with 39, 27, and 12 bp lengths. We previously identified four alleles (A–D) based on the number and order of the 12 and 39 bp units, and found that the allele frequency varied significantly between the golden retriever and shiba.
Analysis of the diversity of dog DRD4 and allelic variations among dog breeds may yield useful information for elucidating the relationships among dog breeds. Such data may also provide a clue toward understanding the genetic background of the behavioral traits of dogs. We therefore investigated the allelic variations of the DRD4 gene in the beagle and Shetland sheepdog, as well as in the golden retriever and shiba. These breeds are popular in Japan and some distinct breed differences were noted in their behavioral traits (Hart and Hart 1985). The present report describes the DNA sequences of three novel alleles and demonstrates distinct differences in the allelic frequency of the dog DRD4 gene in the above four breeds.
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Materials and Methods |
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Genomic DNA was extracted from the peripheral blood or buccal mucous mem brane obtained from 102 unrelated individuals of the four dog breeds: 38 beagles, 40 Shetland sheepdogs, 11 golden retrievers, and 13 shibas. Four of the beagle samples were collected from dogs bred at the International Pet Culture Association (IPC), Okazaki, Japan. Other samples were obtained from pet animals at the Veterinary Medical Teaching Hospital, Gifu University, and Tsuda Animal Hospital, Gifu, Japan. Data for 19 golden retrievers and 15 shibas examined in a previous study (Niimi et al. 1999) were also included in the present statistical analysis of allelic variations.
The dog DRD4 polymorphic region was amplified by PCR according to Niimi et al. (1999). The primers were synthesized based on the dog DRD4 sequence previously determined. The sequences of the forward D4F and reverse D4dogR primers are 5'-TTCTTCCTACCCTGCCCGCTCATG-3' and 5'-CCGCGGGGGCTCTGCAGGGTCG-3', respectively. To distinguish alleles with the same length, D4dogBR (5'-TGGGCTGGGGGTGCCGTCC-3') primer was employed with D4F primer for the subsequent PCR. The D4dogBR primer was designed on the basis of nucleotide substitution at the 30th position in the first unit (see Figure 1). After initial incubation at 98°C for 2 min, PCR amplification was performed for 30 cycles consisting of 98°C for 30 sec, 65°C for 1 min, and 74°C for 1 min, followed by 74°C for 10 min to permit extension of uncompleted chains, employing Pfu polymerase (Stratagene, La Jolla, CA). The PCR products were separated by electrophoresis on 2.5% agarose gel. They were then extracted from the gel and directly sequenced by the dye termination method using an ABI377 DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster, CA). PCR amplification was also performed employing fluorescent-labeled D4F primer, and the resultant solution was separated by electrophoresis on 6% polyacrylamide gel to examine many samples simultaneously and to estimate the size of the PCR product accurately.
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Results |
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Six alleles with different sizes were detected by PCR amplification employing the D4F and D4dogR primers. Alleles of 435 and 549 bp in length correspond to the previously reported A and D alleles, respectively (Niimi et al. 1999). In the present study, each allele was designated by the length of the PCR products, including the 5' upstream and 3' downstream sequences (201 bp length) together with the repetitive region. Since alleles with a 447 bp length could not be distinguished by PCR with D4F and D4dogR primers, further analysis was carried out to identify these alleles. PCR employing D4F and D4dogBR primers in the samples with 447 bp length alleles yielded a PCR product with 168 bp (447a, described as B previously) or 180 bp (447b, C). In addition to the above alleles, we identified three new alleles in the present study. That is, an allele with a 396 bp length was found in the homozygote state in a beagle and an allele with a 486 bp length was observed in the present samples of shibas. An allele with a 498 bp length was detected in a Shetland sheepdog and the shibas. The 486 and 498 alleles were observed only in heterozygote states.
We determined the nucleotide sequences of the new three alleles with 396, 486, and 498 bp lengths. Figure 1 shows the alignment of the repetitive region of the seven dog DRD4 alleles. This region was composed of three types of units with different lengths of 39, 27, and 12 bp, and the number and order of the 12 and 39 bp units produced the observed allelic variation. The 498 allele shared the same sequence as that lacking the sixth and seventh units (serial number of units) from the 549 allele. The 396 allele shared a similar sequence to that lacking the second and fifth units from the 447b allele, and nucleotide substitutions were observed at the 26th (G
C) and 30th (CG) positions in the first unit. In the 486 allele, additional units appeared to be inserted in the 8th and 9th unit positions of the sequence of the 435 allele, and the same nucleotide substitution (CA) was found in the 11th unit as observed in the 447a allele. We sequenced all observed alleles in each dog breed, and the same nucleotide sequences were obtained in the respective alleles.
The allelic variation in the four dog breeds is summarized in Table 1. The data obtained previously from golden retrievers and shibas (Niimi et al. 1999) have also been included in the table. The allelic distribution differed among the four dog breeds. In beagles, four alleles—396, 435, 447a, and 447b—were observed, and the 435 and 447a alleles were common in this breed. Golden retrievers had the 435 and 447a alleles, of which the 435 allele was common as demonstrated by us previously (Niimi et al. 1999). In Shetland sheepdogs, the 447a allele was most frequent among the three alleles of 435, 447a, and 498. Shibas shared five alleles and the 447b allele was most common in the present study. The shiba exhibited the highest value of heterozygosity in the DRD4 gene (53.6%), while the Shetland sheepdog showed the lowest value (25.0%) among these breeds. In contrast with European breeds, short alleles such as 396 and 435 could not be found in shibas, while the long 549 allele was significantly distributed in shibas. The difference in the actual number of alleles among the four breeds was highly significant based on the chi-squared test for independence (
2 = 290.728, df = 18, P < .001).
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Discussion |
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In the present study, we examined the DRD4 gene in dogs and found that DRD4 was highly polymorphic in the dog as observed in humans. In the polymorphic region of the dog DRD4 gene, deletion/insertion of units was observed at the second and fifth to ninth unit positions, suggesting that it was these events which gave rise to the allelic variation of the dog DRD4. Differences of allelic distribution among the dog breeds were noted, as demonstrated previously (Niimi et al. 1999). That is, the 435 and/or 447a alleles were common in the beagle, golden retriever, and Shetland sheepdog, and the frequencies of other alleles, if present, were low. In the shiba, which is an indigenous Japanese dog breed, the frequency of alleles including 447b, 486, 498, and 549 was higher than those of the other three breeds. In addition, the shiba exhibited the highest value for the heterozygosity in the DRD4 gene among the breeds examined. A high genetic diversity in Japanese dog breeds has also been inferred from their mitochondrial DNA sequences (Okumura et al. 1996).
DRD4 is mainly expressed in neural cells, having an effect upon cognitive and emotional behaviors (Van Tol et al. 1991). In humans, individuals with long repeats (six or more) in the DRD4 gene have been found to achieve higher scores in personality tests for novelty seeking than those with shorter repeats (Benjamin et al. 1996; Ebstein et al. 1996). However, the functional differences of the receptors encoded by each allele remain obscure. Since both short- and long-repeat forms of human DRD4 have been found to display similar binding affinities for ligands (Asghari et al. 1994), the length or structure of the repetitive region located in the cytoplasmic portion may affect the efficiency of intracellular signal transduction. The sequence of the DRD4 polymorphic region is considerably different between humans and dogs, and the close association between DRD4 and behavioral traits observed in humans is therefore not necessarily valid for dogs. However, it has been demonstrated that DRD4 knock out (DRD4-/-) mice are significantly less behaviorally responsive to novelty than are DRD4+/+ wild-type mice (Dulawa et al. 1999), suggesting that DRD4 may play an important role in behavioral responses to novelty in animals. Further analysis of the DRD4 gene in other dog breeds is needed in order to elucidate whether or not the DRD4 gene is affecting behavioral traits in dogs.
Tandem-repeated sequences with a 48 bp unit have also been demonstrated in the DRD4 genes of several nonhuman primates (Livak et al. 1995; Matsumoto et al. 1995), and the frequency of alleles with long repeated sequences has tended to increase during primate evolution (Inoue-Murayama et al. 1998). We have clarified that polymorphism of DRD4 exists in dogs as well as in primates. Since the sequence and repeated structure of the polymorphic region of the DRD4 gene differ considerably between primates and dogs, these sequences appear to have had different ancestral origins. Analysis of various species of the order Carnivora may provide useful information for understanding the origin of the repetitive region and the diversity of the DRD4 gene in dogs.
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Acknowledgments |
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We are indebted to Dr. A. Hayakawa, Gifu University, and Dr. T. Tsuda, Tsuda Animal Hospital, for their gifts of dog samples, and to the staff of the Veterinary Hospital, Gifu University, and the IPC for their cooperation in obtaining blood samples from dogs. We gratefully acknowledge the assistance of T. Noguchi, Tokyo University of Agriculture and Technology, in DNA extraction. We would like to thank B. B. Kayang, Gifu University, for reading our manuscript and making invaluable comments. This work was supported by the Companion Animal Information and Research Center.
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Footnotes |
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Corresponding Editor: Robert Wayne
Received September 19, 2000
Accepted April 30, 2001
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References |
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