Identification and Functional Annotation of Genes Related to Bone Stability in Laying Hens Using Random Forests

Abstract

Skeletal disorders, including fractures and osteoporosis, in laying hens cause major welfare and economic problems. Although genetics have been shown to play a key role in bone integrity, little is yet known about the underlying genetic architecture of the traits. This study aimed to identify genes associated with bone breaking strength and bone mineral density of the tibiotarsus and the humerus in laying hens. Potentially informative single nucleotide polymorphisms (SNP) were identified using Random Forests classification. We then searched for genes known to be related to bone stability in close proximity to the SNPs and identified 16 potential candidates. Some of them had human orthologues. Based on our findings, we can support the assumption that multiple genes determine bone strength, with each of them having a rather small effect, as illustrated by our SNP effect estimates. Furthermore, the enrichment analysis showed that some of these candidates are involved in metabolic pathways critical for bone integrity. In conclusion, the identified candidates represent genes that may play a role in the bone integrity of chickens. Although further studies are needed to determine causality, the genes reported here are promising in terms of alleviating bone disorders in laying hens.

Keywords: Random Forests; bone breaking strength; bone mineral density; gene set enrichment analysis; osteoporosis; single nucleotide polymorphism; skeletal integrity.

Figure 1

Schematic illustration of the study design. The data set included 524 laying hens phenotyped for bone stability traits. The corresponding genotypes included 490,745 SNP markers. Association analysis was performed applying Random Forests (RF) classification. Genes harbouring significant SNPs were extracted and screened for links to bone stability. Gene set analyses were performed considering all genes obtained from the RF classification. Retrospectively, SNP effects were estimated for a subset of candidate genes identified in gene sets obtained from the RF classifier.

Figure 2

Venn diagram showing the overlap of genes for the bone breaking strengths (BBS) and bone mineral densities (BMD) of the tibiotarsus and humerus.

Figure 3

Significantly enriched Gene Ontology terms of the category biological processes (GO:BP; top 15 with the highest -log₁₀ p-values) and KEGG pathways for the bone breaking strength (A) and bone mineral density (B) of the tibiotarsus. The dot size represents the absolute number of genes enriched in the term. The proportion of enriched genes in all queried genes is represented on the x-axis. The colour represents the −log₁₀ transformed adjusted p-values.

Figure 4

Significantly enriched Gene Ontology terms of the category biological processes (GO:BP; top 15 with the highest -log₁₀ p-values) and KEGG pathways for the bone breaking strength (A) and bone mineral density (B) of the humerus. The dot size represents the absolute number of genes enriched in the term. The proportion of enriched genes in all queried genes is represented on the x-axis. The colour represents the −log₁₀ transformed adjusted p-values.