Identification of selective signatures associated with gastrointestinal atresia in Holstein calves

Introduction: Obstructive gastrointestinal (GI) malformations are one of the most important congenital problems resulting in calf mortality within a few days of birth. The most common site for atresia, after the esophagus, is the jejunum. Jejunum atresia is the congenital absence or complete blockage of a part of the jejunum lumen. Early detection of intestinal obstruction is essential to prevent further complications. Intestinal atresia is an underdiagnosed congenital defect in cattle. It results in complete occlusion of the intestinal lumen and, unless surgically corrected, results in death or euthanasia of the affected calf. There is limited information on the incidence of this condition or risk factors, including predisposing alleles, associated with the defect. Atresia is a well-known congenital defect of the gastrointestinal system in calves and investigations into the etiology of this condition are warranted. Domestication and selection have significantly changed the behavioral and phenotypic traits in modern domestic animals. The selection of animals by humans left detectable signatures on the genome of modern dairy cattle. The identification of these signals can help us to improve the genetic characteristics of economically important traits in goats. Over the last decade, interest in the detection of genes or genomic regions that are targeted by selection has been growing. Identifying signatures of selection can provide valuable insights about the genes or genomic regions that are or have been under selection pressure, which in turn leads to a better understanding of genotype-phenotype relationships. This study aimed to identify the selection signatures using the unbiased theta method associated with gastrointestinal atresia in Holstein dairy calves.
Materials and methods: For calves with intestinal atresia, muscle tissue (>1 g) was collected from the Latissimus dorsi muscle postmortem, and submerged in RNA later solution. DNA samples from 91 atresia cases and 377 control animals were genotyped using the Illumina 777K BovineHD beadchip (Illumina Inc). The work described here is a case–control association study. Single nucleotide polymorphism (SNP) missing 5% of data, with MAF of <1% and Hardy–Weinberg equilibrium P-values <10⁻⁶ were removed. The genotyping efficiency for samples was also verified, and samples with more than 5% missing data were removed. Grouping was done to infer selection signatures based on F_ST statistic. The bioinformatics investigations were carried out using the Ensembl database for bovine genes (assembly ARS-UCD1.2), to identify potential candidate genes which already have been reported in/or surrounding genomic regions containing the peak of absolute extreme F_ST values. The regions corresponding to the upper and lower 0.01% of positive and negative obtained F_ST scores were considered regions under selection. Genes within a 500-kb span of the start and end of the QTL were identified using Ensembl 108 on the ARS-UCD1.2 bovine genome assembly implemented in biomart. Then, using the PANTHER database, the general biological function of the genes was checked. At this stage, it is assumed that genes that belong to a functional class can be considered as a group of genes that have some specific and common characteristics, and the quantitative trait loci (QTLs) in the selected region were extracted using the Animalgenome database, and the genes were compared with other researches. GeneCards (http://www.genecards.org) and UniProtKB (http://www.uniprot.org) databases were also used to interpret the function of the obtained genes.
Results and discussion: with a 99.90 percentile threshold of the obtained theta (θ) values, eight genomic regions on chromosomes 7, 12, 13, 21, 22, 23 (two regions), and 29 in the Holstein calves were identified. Further investigation using bioinformatics tools showed these genomic regions overlapped with the genes (CSF2, SIAH3, TMEM14A, and SKIV2L) associated with embryonic development, small intestine length, apoptosis, and several tumors. The population used in our study is small, owing to the challenge of collecting a substantial amount of blood on calves on commercial herds having received the diagnosis of gastrointestinal atresia and ready to be culled. Diagnosis and culling of gastrointestinal atresia animals are ineffective preventive measures.  Further work is required to identify which farm-specific or management risk factors contribute to the incidence of intestinal atresia.
Conclusions: The results of this study may provide an important source to facilitate the identification of genomic regions and then, the genes affecting gastrointestinal atresia in claves. However, further studies are warranted to refine the findings using a larger sample size, whole-genome sequencing, and/or high-density genotyping.Materials and Methods: For calves with intestinal atresia, muscle tissue (>1 g) was collected from the Latissimus dorsi muscle postmortem, submerged in RNA later solution. DNA samples from 91 atresia cases and 377 control animals were genotyped using the Illumina 777K BovineHD beadchip (Illumina Inc). The work described here is a case–control association study. SNP missing 5% of data, with MAF of <1% and Hardy–Weinberg equilibrium p-values <10−6 were removed. The genotyping efficiency for samples was also verified, and samples with more than 5% missing data were removed. Grouping was done to infer selection signatures based on FST statistic. The bioinformatics investigations were carried out using the Ensembl database (Cunningham et al., 2022) for bovine genes (assembly ARS-UCD1.2), to identify potential candidate genes which already have been reported in/or surrounding genomic regions containing the peak of absolute extreme FST values. The regions corresponding to the upper and lower 0.01% of positive and negative obtained FST scores were considered as regions under selection.
Genes within a 500-kb span of the start and end of the QTL were identified using Ensembl 108 on the ARS-UCD1.2 bovine genome assembly implemented in biomart. Then, using the PANTHER database, the general biological function of the genes was checked. At this stage, it is assumed that genes that belong to a functional class can be considered as a group of genes that have some specific and common characteristics, and the QTLs in the selected region were extracted using the Animalgenome database, and the genes were compared with other researches. GeneCards (http://www.genecards.org) and UniProtKB (http://www.uniprot.org) databases were also used to interpret the function of the obtained genes.
Results and Discussion with 99.90 percentile threshold of the obtained Theta (θ) values, eight genomic regions on chromosomes 7, 12, 13, 21, 22, 23 (2 regions), and 29 in Holstein calves breed were identified. Further investigation using bioinformatics tools showed these genomic regions overlapped with the genes (CSF2, SIAH3, TMEM14A, SKIV2L) associated with embryonic development, small intestine length, apoptosis, and several tumours. The population used in our study is small, owing to the challenge of collecting a substantial amount of blood on calves on commercial herds having received the diagnosis of gastrointestinal atresia and ready to be culled. Diagnosis and culling of gastrointestinal atresia animals are ineffective preventive measures. Further work is required to identify which farm-specific or management risk factors contribute to the incidence of intestinal atresia.
Conclusions: In conclusion, the results of this study may provide an important source to facilitate the identification of genomic regions and then, the genes affecting gastrointestinal atresia in claves. However, further studies are warranted to refine the findings using a larger sample size, whole-genome sequencing, and/or high density genotyping