Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs.

INTRODUCTION: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. METHODS: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. RESULTS: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. CONCLUSION: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.

A joint analysis using exome and transcriptome data identifiescandidate polymorphisms and genes involved with umbilical hernia in pigs.

BACKGROUND: Umbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design. RESULTS: In the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others. CONCLUSIONS: We have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated.

Differentially expressed genes in the femur cartilage transcriptome clarify the understanding of femoral head separation in chickens.

Locomotor problems are among one of the main concerns in the current poultry industry, causing major economic losses and affecting animal welfare. The most common bone anomalies in the femur are dyschondroplasia, femoral head separation (FHS), and bacterial chondronecrosis with osteomyelitis (BCO), also known as femoral head necrosis (FHN). The present study aimed to identify differentially expressed (DE) genes in the articular cartilage (AC) of normal and FHS-affected broilers by RNA-Seq analysis. In the transcriptome analysis, 12,169 genes were expressed in the femur AC. Of those, 107 genes were DE (FDR < 0.05) between normal and affected chickens, of which 9 were downregulated and 98 were upregulated in the affected broilers. In the gene-set enrichment analysis using the DE genes, 79 biological processes (BP) were identified and were grouped into 12 superclusters. The main BP found were involved in the response to biotic stimulus, gas transport, cellular activation, carbohydrate-derived catabolism, multi-organism regulation, immune system, muscle contraction, multi-organism process, cytolysis, leukocytes and cell adhesion. In this study, the first transcriptome analysis of the broilers femur articular cartilage was performed, and a set of candidate genes (AvBD1, AvBD2, ANK1, EPX, ADA, RHAG) that could trigger changes in the broiler´s femoral growth plate was identified. Moreover, these results could be helpful to better understand FHN in chickens and possibly in humans.

Differential Expression of Myogenic and Calcium Signaling-Related Genes in Broilers Affected With White Striping.

White Striping (WS) has been one of the main issues in poultry production in the last years since it affects meat quality. Studies have been conducted to understand WS and other myopathies in chickens, and some biological pathways have been associated to the prevalence of these conditions, such as extracellular calcium level, oxidative stress, localized hypoxia, possible fiber-type switching, and cellular repairing. Therefore, to understand the genetic mechanisms involved in WS, 15 functional candidate genes were chosen to be analyzed by quantitative PCR (qPCR) in breast muscle of normal and WS-affected chickens. To this, the pectoral major muscle (PMM) of 16 normal and 16 WS-affected broilers were collected at 42 days of age and submitted to qRT-PCR analysis. Out of the 15 genes studied, six were differentially expressed between groups. The CA2, CSRP3, and PLIN1 were upregulated, while CALM2, DNASE1L3, and MYLK2 genes were downregulated in the WS-affected when compared to the normal broilers. These findings highlight that the disruption on muscle and calcium signaling pathways can possibly be triggering WS in chickens. Improving our understanding on the genetic basis involved with this myopathy might contribute for reducing WS in poultry production.

Genes and SNPs Involved with Scrotal and Umbilical Hernia in Pigs.

Hernia is one of the most common defects in pigs. The most prevalent are the scrotal (SH), inguinal (IH) and umbilical (UH) hernias. We compared the inguinal ring transcriptome of normal and SH-affected pigs with the umbilical ring transcriptome of normal and UH-affected pigs to discover genes and pathways involved with the development of both types of hernia. A total of 13,307 transcripts was expressed in the inguinal and 13,302 in the umbilical ring tissues with 94.91% of them present in both tissues. From those, 35 genes were differentially expressed in both groups, participating in 108 biological processes. A total of 67 polymorphisms was identified in the inguinal ring and 76 in the umbilical ring tissue, of which 11 and 14 were novel, respectively. A single nucleotide polymorphism (SNP) with deleterious function was identified in the integrin α M (ITGAM) gene. The microtubule associated protein 1 light chain 3 γ (MAP1LC3C), vitrin (VIT), aggrecan (ACAN), alkaline ceramidase 2 (ACER2), potassium calcium-activated channel subfamily M α 1 (KCNMA1) and synaptopodin 2 (SYNPO2) genes are highlighted as candidates to trigger both types of hernia. We generated the first comparative study of the pig umbilical and inguinal ring transcriptomes, contributing to the understanding of the genetic mechanism involved with these two types of hernia in pigs and probably in other mammals.

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage.

The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.

Transcriptome analysis identifies genes involved with the development of umbilical hernias in pigs.

Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation.

Stable reference genes for expression studies in breast muscle of normal and white striping-affected chickens.

The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.

Proximal femoral head transcriptome reveals novel candidate genes related to epiphysiolysis in broiler chickens.

BACKGROUND: The proximal femoral head separation (FHS) or epiphysiolysis is a prevalent disorder affecting the chicken femur epiphysis, being considered a risk factor to infection which can cause bacterial chondronecrosis with osteomyelitis in broilers. To identify the genetic mechanisms involved in epiphysiolysis, differentially expressed (DE) genes in the femur of normal and FHS-affected broilers were identified using RNA-Seq technology. Femoral growth plate (GP) samples from 35-day-old commercial male broilers were collected from 4 healthy and 4 FHS-affected broilers. Sequencing was performed using an Illumina paired-end protocol. Differentially expressed genes were obtained using the edgeR package based on the False Discovery Rate (FDR < 0.05). RESULTS: Approximately 16 million reads/sample were generated with 2 × 100 bp paired-end reads. After data quality control, approximately 12 million reads/sample were mapped to the reference chicken genome (Galgal5). A total of 12,645 genes were expressed in the femur GP. Out of those, 314 were DE between groups, being 154 upregulated and 160 downregulated in FHS-affected broilers. In the functional analyses, several biological processes (BP) were overrepresented. Among them, those related to cell adhesion, extracellular matrix (ECM), bone development, blood circulation and lipid metabolism, which are more related to chicken growth, are possibly involved with the onset of FHS. On the other hand, BP associated to apoptosis or cell death and immune response, which were also found in our study, could be related to the consequence of the FHS. CONCLUSIONS: Genes with potential role in the epiphysiolysis were identified through the femur head transcriptome analysis, providing a better understanding of the mechanisms that regulate bone development in fast-growing chickens. In this study, we highlighted the importance of cell adhesion and extracellular matrix related genes in triggering FHS. Furthermore, we have shown new insights on the involvement of lipidemia and immune response/inflammation with FHS in broilers. Understanding the changes in the GP transcriptome might support breeding strategies to address poultry robustness and to obtain more resilient broilers.

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs.

The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.