Fear, a crucial survival mechanism, becomes pathological when excessive or disproportionate. Anxiety disorders and phobias are significant public health concerns in humans, exhibiting heritability but complex genetic architectures, hindering the identification of causal genes. Dogs, with their diverse behavioral variations and close human interaction, offer a valuable animal model for studying anxiety. Their shared living environment facilitates detailed behavioral assessments, and their distinct breeding history results in significant genetic diversity. Previous research has linked fear in dogs to shorter lifespans and potential risks to human safety. Fear in dogs can be categorized as social (e.g., fear of strangers, other dogs) and non-social (e.g., fear of novel situations), with overlaps observed between these categories. While some candidate loci and genes have been suggested for canine fear, aggression, and other behavioral traits, the challenges of measuring behavior and its heritable component along with the complex genetic architecture have slowed down progress. Environmental factors, particularly early socialization, also play a crucial role in shaping fearful behaviors in dogs. This study focuses on the genetic basis of stranger fear in Great Danes, utilizing a validated behavioral survey, GWAS, and whole exome and genome sequencing to identify a novel locus on chromosome 11 potentially harboring candidate genes.

The literature review section of the paper examines prior studies investigating the genetic and environmental underpinnings of fear and anxiety in both humans and dogs. Studies on human anxiety disorders highlight the heritable nature of these conditions but emphasize their genetic complexity, underscoring the need for animal models. The authors review existing research on canine behavioral genetics, noting previous findings of candidate genes and loci associated with fear, aggression, social behavior, and obsessive-compulsive disorder. The literature also emphasizes the significant influence of environmental factors, particularly early socialization experiences, on the development of fearful behavior in dogs. The existing literature supports the use of dogs as a valuable model for studying fearfulness due to their diverse behavioral phenotypes, readily available behavioral data from their owners, and the potential for identifying genetic and environmental interactions. This study builds on this foundation by utilizing a large cohort of Great Danes and implementing comprehensive genetic analyses.

The study employed a multi-stage approach to identify genetic factors associated with stranger fear in Great Danes. First, 124 Great Dane dogs were phenotyped using a validated owner-completed behavioral survey. This survey yielded a quantitative “stranger fear score” (SFS) ranging from 0 to 48, reflecting the frequency and intensity of fearful reactions towards strangers. Dogs with an SFS of 0 were classified as controls, while dogs with SFS values above 0 were classified as cases. A socialisation score (SS), reflecting the extent and frequency of socialisation events during puppyhood, was also calculated and used as a covariate. Genotyping was performed on all 124 dogs using Illumina Canine HD SNP arrays. GWAS was conducted using both PLINK (quantitative trait analysis and linear regression) and GenABEL (mixed model association analysis), with and without the SS as a covariate. Quality control procedures were implemented to filter out low-quality SNPs and individuals. To account for population stratification and relatedness, genomic control approaches were applied. The significance threshold was adjusted using Bonferroni correction and by estimating the effective number of independent tests using simpleM. Following GWAS, whole exome sequencing (WES) was performed on 8 cases and 8 controls, selected based on contrasting genotypes at the most significant SNP identified in the GWAS. Four cases and four controls were also subject to whole genome sequencing (WGS). The sequencing data were analyzed using Genotype Query Tools and an in-house analysis pipeline. Variants were filtered based on a dominant model, comparing cases and controls. Variant effects were predicted using Ensembl’s Variant Effect Predictor, with allele frequencies assessed using a database of dog exomes and genomes. Sanger sequencing was performed on 40 dogs (15 cases, 25 controls) to validate three candidate variants identified through WES and WGS. The CanFam 3.1 genome build was used as a reference throughout the study.

The GWAS analyses consistently identified a significant association on canine chromosome 11, regardless of whether socialisation score was included as a covariate. The strongest associations were observed using PLINK with a linear regression model in both cohorts (with and without the covariate). The association remained significant even after correcting for multiple testing using Bonferroni correction and the effective number of independent tests. The genotype plots showed distinct differences in allelic distributions between cases and controls in the associated region on chromosome 11. Whole exome and genome sequencing further revealed extensive regions of contrasting homozygosity in this region between cases and controls. Several candidate genes were identified within the associated region, including *MAPK9/JNK2*, a known regulator of anxiety in the hippocampus. Sanger sequencing successfully validated three candidate variants identified through WES and WGS in a subset of the dogs.

The findings provide compelling evidence for a novel genomic region on chromosome 11 associated with stranger fear in Great Danes. The consistent identification of this locus across different analytical approaches and the contrasting homozygosity patterns between cases and controls strongly support its involvement in fearfulness. The identification of *MAPK9/JNK2* as a candidate gene is biologically plausible given its known role in anxiety regulation in the hippocampus. The study’s inclusion of socialization as a covariate strengthens the findings by controlling for a known environmental factor impacting fearfulness. This work contributes to the molecular understanding of canine fear and provides a valuable animal model for studying human anxiety disorders. Future research should focus on functional validation of the candidate genes and variants and investigation of the interactions between genetic and environmental factors.

This study identified a novel locus on canine chromosome 11 associated with stranger fear in Great Danes, highlighting candidate genes including *MAPK9/JNK2*. This provides a valuable resource for understanding the genetic basis of canine fear and offers a potential animal model for human anxiety. Future work should focus on functional characterization of identified variants and exploring gene-environment interactions.

The study focused on a single breed, Great Danes, which may limit the generalizability of the findings to other breeds. The use of owner-reported behavioral data, while practical and readily available, might introduce some subjective bias. The sample size, while substantial for a canine genetics study, could be further increased to enhance statistical power and potentially identify additional associated loci.