The association of C789A polymorphism in the dopamine beta-hydroxylase gene ( DBH ) and aggressive behaviour in dogs

The genetic basis of aggressive behaviour has been examined extensively, including analysis of genes encoding neurotransmitters, signalling molecules and regulatory enzymes, as well as their synthesis and degradation. Dopamine beta-hydroxylase, an enzyme catalysing the conversion of dopamine into norepinephrine in synaptic endings, significantly affects the modulation of emotional states and behaviour. The aim of this study was to determine the association of C789A polymorphism in the canine dopamine beta-hydroxylase gene ( DBH ) and aggressive behaviour in dogs. A total of 110 dogs of different breeds were analysed. All animals were classified according to their individual behavioural characteristics, defined by a veterinary interview and observation. Polymorphism was analysed using ACRS-PCR (amplification created restriction site-polymerase chain reaction) method. Significant differences in DBH genotypes and allele frequency between aggressive and non-aggressive dogs were observed (χ 2 = 16,232, P = 0.0003). In aggressive dogs, the CC genotype (0.788) and C allele (0.815) were most frequent while in non-aggressive dogs, their frequencies were significantly lower (0.361 and 0.404, respectively). The obtained results indicate that DBH is a promising candidate gene for canine behavioural study.

Aggression is one of the most common behavioural problem in dogs. Results of studies on human populations indicate that genetic factors may contribute nearly 50% of the determination of behavioural traits, suggesting the multigenic (quantitative) nature of personality phenotypes (Bouchard et al. 1990). Neurotransmitters in the central nervous system are a very important factor in the modulation of emotional states and behavioural patterns in both humans and animals (Steimer 2002). Dopamine beta-hydroxylase (DβH) is an enzyme catalysing the conversion of dopamine into norepinephrine -one of the main neurotransmitters of the peripheral and central nervous system. Dopamine beta-hydroxylase is located almost entirely inside the synaptic vesicles of postganglionic sympathetic neurons (norepinephrinic) and the adrenal medulla (Gonzalez-Lopez and Vrana 2020).
Polymorphisms in genes encoding regulatory enzymes of neurotransmitters can be associated with various behaviours, including aggression. DBH, the structural locus encoding the DβH enzyme is the major quantitative trait locus influencing plasma DβH activity (Cubells and Zabetian 2004;Kopecková et al. 2006). An analysis showed that the C-1021T polymorphism, located 1,021 bp upstream of the transcriptional start site in the 5'-flanking region of the human DBH gene, is associated with 35-52% of the total variance in DBH plasma activity (Zabetian et al. 2001). Further studies took into consideration C-1021T single nucleotide polymorphism (SNP) in relation to affective disorders and personality styles. Results indicated that the TT genotype was correlated with personality features linked to impulsiveness and aggressive hostility. However, no relations were observed for affective disorders and DBH variants (Hess et al. 2009).
In canines, the DBH gene is located on chromosome 9 (Cfa9) in the position 50,004,806-50, 024,130. It consists of 12 exons, whereas the transcript length is 2346 bp (Cunningham et al. 2019). Takeuchi et al. (2005) detected two SNPs in the canine DBH gene -C789A and A1819G. Of them, only C789A showed significant variation (P < 0.01) between five analysed breeds. A1819G polymorphism, however, was present only in one breed -Shiba. The C789A SNP (rs851198876) is located in exon 4 of the DBH gene and causes asparagine to lysine (Asn263Lys) substitution (Cunningham et al. 2019). A study performed later by Våge and Lingaas (2008) confirmed presence of the C789A variant as well as 5 additional nonsynonymous SNPs in the canine DBH gene. The authors also predicted a possible damaging effect of the amino acid substitutions caused by the C789A polymorphism as leading to positively charged/polar uncharged change. Because the C789A polymorphism in dogs was investigated only in relation to differences between breeds, we decided to evaluate its relation to different behaviours. The aim of this study was to determine the association of C789A polymorphism in the canine dopamine beta-hydroxylase gene (DBH) and aggression in dogs.

Materials and Methods
The study involved 110 individuals of different breeds of dogs (Canis familiaris) classified into 9 groups according to the Fédération Cynologique Internationale (FCI). The dogs (n =110) were assessed based on their individual behavioural characteristics defined by veterinary interview and observation, and then divided into those exhibiting aggressive behaviour (n = 27) and those not showing aggression (n = 83) ( Table 1).
Peripheral blood was collected by a veterinarian into tubes containing K 3 EDTA. DNA isolation from the blood was performed using the MasterPure™ DNA Purification Kit for Blood II (Epicentre Biotechnologies, Illumina, Madison, Wisconsin, USA), according to the methodology given by the manufacturer. For polymorphism screening, the ACRS-PCR method was applied. In the close distance (12 bp) of the analysed DBH gene polymorphism is the second recognition site for the BsrI enzyme, so we designed a forward primer to abolish this site, allowing for correct genotyping. The primers were as follows: forward 5'-GGTCCACCACATAGAGATCTTACA-3', reverse 5'-GAGGTGAGGGGACACAGGT-3' (underlined nucleotide introduces mismatch).

Results
In the analysed dogs, three genotypes of the DBH gene were identified: CC (176, 34 bp), CA (210, 176, 34 bp), and AA (210 bp). The CC genotype was most frequent in aggressive dogs (0.778), while in non-aggressive it reached significantly lower frequency (0.361). In case of the heterozygous genotype (CA), reverse tendency was observed. Frequency of the AA genotype was similar in both groups (Table 2). Analysing the numbers of aggressive dogs within groups, the highest were found in Terriers (41.2%) as well as Pinscher and Schnauzer -Molossoid and Swiss Mountain and Cattle dogs (28.6%).
Significant differences in DBH genotypes and allele frequency between aggressive and non-aggressive dogs were observed (χ 2 = 16,232, P = 0.0003). In aggressive dogs, the CC genotype (0.788) and C allele (0.815) were the most frequent whereas in non-aggressive dogs, their frequency was significantly lower (0.361 and 0.404, respectively).

Discussion
The difference between aggressive and non-aggressive dogs could be affected by genes that control the synthesis of the neurotransmitter and its reuptake, their receptors, enzymes that inactivate the neurotransmitters or genes which control expression of any of the abovementioned genes (Houpt 2007;Proskura et al. 2013). Våge et al. (2010) studied the expression profiles in the brains of aggressive and non-aggressive dogs and found the increased expression of two genes (UBE2V2 and ZNF227) strongly associated with the aggression phenotype. In another research, Wang et al. (2018) identified differentially expressed genes in brain tissues between tame and aggressive fox populations, with the top two significant candidate genes PCDHGA1 and DKKL1. In the genome wide association (GWA) mapping of canine fear and aggression, Zapata et al. (2016) found that the IGF-1 and HMGA2 loci were associated with separation anxiety, touch sensitivity, owner-directed aggression and dog rivalry. The authors reported also two additional loci on the canine chromosome 18 and X (GNAT3 and CD36, respectively) associated with several behavioural traits, including fear and aggression directed toward unfamiliar dogs and humans.
In this study, we analysed a gene encoding enzyme that catalyses the synthesis of norepinephrine from dopamine. The C789A polymorphism detected in the DBH gene was investigated earlier in 5 different breeds of dogs. Among them, the AA genotype and A allele were most frequent in Golden and Labrador Retrievers (Takeuchi et al. 2005). Golden Retrievers are characterized by low aggression toward other dogs, low snapping at children, low dominance over the owner and low territorial deference (Hart and Miller 1985). They are recognized as trainable and playful dogs but not aggressive (Prakash 2009). We did not find any differences regarding the AA genotype in both groups. Interestingly, when analysing the CC genotype, Takeuchi et al. (2005) found that it was The DBH gene was also investigated in relation to behavioural traits in other domestic animals. Ren et al. (2017) studied four candidate genes for temperament behaviour in the Mongolian horse. They identified polymorphism of the DBH gene in a similar position (G758A) to that found in our study (C789A). Different equine DBH variants were associated significantly with horse-friendly behaviour which is directly translated into other traits, e.g. comprehension ability, crossing the river, and swimming. Other studies were also conducted in pigs, where behavioural characteristics were analysed in relation to neurotransmitter-related genes in the Chinese indigenous Mi breed and Landrace × Large White crossbreeds. After backtesting and assessing aggressive behaviour, it was confirmed that Mi pigs are less active and less aggressive than European pigs. Genetic analysis confirmed that the most frequent haplotypes were significantly different between Mi and Landrace × Large White pigs for the DBH gene and few others (Chu et al. 2017). The last domestic animals include cows, where TH (tyrosine hydroxylase) and DBH genes polymorphisms were analysed in four different breeds. Significant DBH haplotype differences between Brahman cattle and the three B. t. taurus breeds, Charolais, Holstein, and Lidia, were found. The authors stated that detected polymorphism is interesting from a behavioural point of view because of the observed differences in temperament between these breeds (Lourenco-Jaramillo et al. 2012).
In conclusion, we found that DBH genotypes and allele frequencies differ significantly between aggressive and non-aggressive dogs. Thus, further studies should be conducted to confirm the usefulness of the DBH gene as a marker for behavioural traits in this species.