Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

Deep sequencing of a candidate region harboring the SOX9 gene for the canine XX disorder of sex development.

A disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation in the region harboring the SOX9 gene. We applied a genome-wide association study and targeted next-generation sequencing techniques to compare DSD and normal female dogs. The genome-wide association study did not indicate a significant chromosome region. Targeted next-generation sequencing of a 1.5-Mb region on canine chromosome 9 harboring the SOX9 gene revealed two putatively DSD-associated copy number variations 355 kb upstream and 691 kb downstream of SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial next-generation sequencing analysis showed an association with two SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of SOX9, that overlaps a known copy number variation region. It shows a high sequence similarity with the 5' flanking region of the MAGI2 gene located on canine chromosome 18 that encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified copy number variation region located upstream of the SOX9 gene contains potential regulatory sequences (long non-coding RNA and hfMAGI2) and led to the assumption that a multiplication of this element may alter expression of the SOX9 gene, triggering the DSD phenotype.

Germline gene polymorphisms predisposing domestic mammals to carcinogenesis.

Cancer is a complex disease caused in part by predisposing germline gene polymorphisms. Knowledge of carcinogenesis in companion mammals (dog and cat) and some livestock species (pig and horse) is quite advanced. The prevalence of certain cancers varies by breed in these species, suggesting the presence of predisposing genetic variants in susceptible breeds. This review summarizes the present understanding of germline gene polymorphisms, including BRCA1, BRCA2, MC1R, KIT, NRAS and RAD51, associated with predisposition to melanoma, mammary cancer, osteosarcoma and histiocytic sarcoma in dogs, cats, pigs and horses. The predisposing variants in these species are discussed in the context of human germline gene polymorphisms associated with the same types of cancer.

A porcine model of osteosarcoma.

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53(R167H) and KRAS(G12D), and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7-8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.

Polymorphism in 3' untranslated region of the pig PPARA gene influences its transcript level and is associated with adipose tissue accumulation.

The PPARA (peroxisome proliferator-activated receptor-α) gene encodes a nuclear receptor that plays an important role in fatty acid catabolism by transcriptional regulation of genes involved in fatty acid oxidation and can be considered as a candidate gene for fatness traits in the pig. The aim of the study was to search for a functional polymorphism in 3' untranslated region (UTR), their association with production traits, and postnatal PPARA transcript level in 2 skeletal muscles (longissimus and semimembranosus) of 5 commercial pig breeds (Polish Landrace [PL], Polish Large White [PLW], Duroc, Pietrain, and Pulawska). Altogether, 9 novel polymorphisms (8 SNP and 1 indel) were found in the 3' UTR. The in silico analysis revealed 6 putative microRNA target sequences in the analyzed region. The c.*636A>G substitution was widely distributed across breeds and located near the putative target sequence for miR-224. The relative PPARA transcript level was higher (P < 0.05) in LM of AA than in those of GG homozygous animals for SNP c.*636A>G. The luciferase assay revealed that miR-224 probably acts as a negative regulator of the PPARA expression in pig adipocytes (P = 2.9 × 10(-7)), but we did not observe the effect of the A or G alleles on the interaction between miR-224 and its putative target sequence. We hypothesize that the 2 predominant haplotypes, differing at 4 sites (including c.*636A>G), present different architecture of its 3' UTR and it could affect the level of the transcript. The c.*636A>G SNP, analyzed in PL and PLW, was significantly associated with backfat thickness at 3 points (P < 0.05) and intramuscular fat content (P < 0.01) in PL. Suggestive associations were found between 4 SNP (c.*321A>C, c.*324G>C, c.*626T>C, and c.*636A>G) and fatty acid contents in LM and subcutaneous and visceral fat tissue of PL, PLW, Duroc and Pietrain pigs. The PPARA mRNA level was higher in semimembranosus muscle than in LM (P = 8.38 × 10(-12)) in a general comparison and the same trend was found in most breeds (except for PL) and at all tested days of age (60, 90, 120, 150, 180, and 210 d). The effect of breed was highly significant in a general comparison (P = 0.48 × 10(-8)), but there was no common expression pattern in both muscles among different age groups. We conclude that the c.*636A>G SNP in the PPARA gene can be considered in PL breed as a useful genetic marker for adipose tissue accumulation.

Polymorphisms in the promoter region of the adiponectin (ADIPOQ) gene are presumably associated with transcription level and carcass traits in pigs.

The main goal of this study was to screen for polymorphisms in the porcine adiponectin (ADIPOQ) gene promoter, analyse their influence on transcription and identify any association with production traits in pigs. A 1018-bp region of the ADIPOQ gene promoter was analysed in 113 pigs, and seven novel polymorphisms found. Luciferase assays were performed in HEK293 (human embryonic kidney) cells and primary porcine adipose mesenchymal stem cells (pADMSCs) to investigate their affect on promoter activity. A 16-bp indel (c.-106_-91delGCCAGGGGTGTGAGCC) was found to influence promoter strength in vitro. In the HEK293 cell line, the Del/Del genotype showed greater luciferase activity than did the Ins/Ins genotype (P < 0.01). In pADMSCs, the insertion genotype of the ADIPOQ promoter showed greater luciferase activity than did the deletion genotype (P < 0.01). An association study performed for two novel polymorphisms, c.-67G>A and the 16-bp indel, showed significant correlation with loin measurements in Polish Landrace (P < 0.05) and synthetic line 990 (P < 0.01) pigs.

Effects of new polymorphisms in the bovine myocyte enhancer factor 2D (MEF2D) gene on the expression rates of the longissimus dorsi muscle.

Myocyte enhancer factor 2D (MEF2D), a product of the MEF2D gene, belongs to the myocyte enhancer factor 2 (MEF2) protein family which is involved in vertebrate skeletal muscle development and differentiation during myogenesis. The aim of the present study was to search for polymorphisms in the bovine MEF2D gene and to analyze their effect on MEF2D mRNA and on protein expression levels in the longissimus dorsi muscle of Polish Holstein-Friesian cattle. Overall, three novel variations, namely, insertion/deletion g.-818_-814AGCCG and g.-211C

Nucleotide sequence and variations of the bovine myocyte enhancer factor 2C (MEF2C) gene promoter in Bos taurus cattle.

Myocyte Enhancer Factor 2 (MEF2) proteins are a small family of transcription factors that play pivotal role in morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells. In vertebrates, there are four MEF2 genes, referred to as MEF2A, -B, -C, and -D, that are located on different chromosomes. After birth MEF2A, MEF2B, MEF2D transcriptions are expressed ubiquitously, whereas MEF2C transcripts are restricted to skeletal muscle, brain, and spleen. In this study, on the basis of the sequences of the bovine chromosome 7 genomic contig, available in the GenBank database, sets of PCR primers were designed and to amplify the bovine MEF2C gene promoter region, exon 1 (5'UTR) and part sequence of the intron 1. Seven overlapping fragments of the bovine MEF2C gene were amplified and then sequenced. Altogether, these fragments were composed in the 3,120-bp sequence which was deposited in the GenBank database under accession no. GU211007. The sequence fragment included the putative site of the promoter region and transcription start of the exon 1. The sequence analysis of these fragments in individual animals representing different Bos taurus breeds revealed four variations in promoter region: g.-1606C>T, g.-1336_-1335DelG, g.-818C>T, g.-613_-612DelA and four SNPs within intron 1: g.2711A>G, g. 2913A>G, g.2962G>T and g.3014A>G. No polymorphism was found within sequence of the exon 1 (5'UTR). These polymorphisms were identified for first time using these sequences and were confirmed by RFLP or MSSCP methods.

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids.

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24-25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox--Chinese raccoon dog) to 99.5% (red fox--arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3'-flanking sequence, showed a significant association (P < 0.01) with body weight.

Characterization of the porcine AMPK alpha 2 catalytic subunitgene (PRKAA2): genomic structure, polymorphism detection and association study.

AMP-activated protein kinase (AMPK), known as a key regulator of cellular energy homeostasis, plays an important role in regulation of glucose and lipid metabolism, and protein synthesis in mammals. The characterization of porcine PRKAA2 encoding the alpha 2 catalytic subunit of AMPK is reported in this study. PRKAA2 was assigned to porcine chromosome 6q by analysis of radiation hybrids (IMpRH panel), and its genomic structure was determined by BAC sequencing. PRKAA2 spans more than 62 kb and consists of nine exons and eight introns. A total of 25 polymorphisms were identified by re-sequencing approximately 7 kb, including all the exons, exon-intron boundaries and 5' and 3' gene flanking regions using twelve founder animals of a Mangalitsa x Piétrain intercross. Neither of two single nucleotide polymorphisms (SNPs) found in the coding region caused an amino acid substitution. Two SNPs (NM_214266.1: c.236+142A>G and NM_214266.1: c.630C>T) in PRKAA2 were genotyped in the Mangalitsa x Piétrain F(2) cross (n = 589) and two commercial populations [Piétrain (n = 1173) and German Landrace (n = 536)] and evaluated for association with traits of interest (muscle development and fat deposition). Single SNP and haplotype analyses revealed weak associations between the PRKAA2 genotypes and loin muscle area in the investigated populations.

Postnatal transcription profile and polymorphism of the ADIPOR1 gene in five pig breeds.

As a result of its role in energy homeostasis regulation, the ADIPOR1 gene is a candidate for fat deposition, an important production trait, in the pig. The aim of the study was to conduct a comparative analysis of the ADIPOR1 postnatal transcript level, in order to establish its promoter and 5'UTR sequences and to search the gene for polymorphisms. The transcription level was examined in longissimus dorsi and semimembranosus muscles collected from 180 pigs at 60-210 days of age, representing five pig breeds: Duroc, Polish Large White, Polish Landrace, Pietrain and Pulawska. We calculated highly significant breed by age by muscle interaction (P < 0.0001) and breed by muscle interactions (P < 0.01). The 5'UTR and promoter region of the porcine ADIPOR1 gene were amplified for the first time and their sequences were deposited in the GenBank database. In total, 21 novel and two previously described polymorphisms were found in the ADIPOR1 promoter, coding, intronic, 5' and 3' untranslated regions. The only SNP detected in the coding region was a synonymous substitution. Two polymorphisms in 3'UTR (c.*129A>C and c.*536A>G) showed no significant effect on the transcript level. Our results showed a high polymorphism of the ADIPOR1 and a complexity in its transcription level in the studied muscles. This complexity indicates that conclusions based on such studies should be carefully gradated.

A new SNP in the 3'UTR region of the bovine calpain small subunit (CAPNS1) gene.

Calpains are a ubiquitous cytoplasmic cysteine protease, the activity of which is absolutely dependent on calcium. This proteolytic system degrades myofibrillar protein under post-mortem conditions and appears to be the primary enzyme in the postmortem tenderization process. In the present study a new single nucleotide polymorphism was found in the bovine CAPNS1 gene exon 11 coding for the 3'UTR. Transition C --> T at position 6536 was detected and identified using PCR-SSCP and DNA sequencing techniques, and then analysed with PCR-RFLP using MboII nuclease. The genotype frequencies and alleles distribution were studied in 190 bulls including, Charolaise, Hereford, Limousine, Simmental, Polish Red and Fresian breeds.

Variation in neighbouring genes of the dopaminergic and serotonergic systems affects feather pecking behaviour of laying hens.

Feather pecking is a behavioural disorder of laying hens and has serious animal welfare and economic implications. One of the several aetiological hypotheses proposes that the disorder results from redirected exploratory behaviour. Variation in the gene encoding the dopamine D4 receptor (DRD4) has been shown to be associated with exploratory behaviour in several species, including in a passerine bird species. We therefore considered DRD4 as a candidate gene for feather pecking. We have annotated DRD4 in the chicken genome and have re-sequenced it in 140 animals belonging to: experimental layer lines divergently selected for high and low propensity to feather pecking; the unselected founder population; and two commercial lines with low and high propensity to feather pecking. We have identified two sub-haplotypes of DRD4 that are highly significantly associated with feather pecking behaviour in the experimental (P = 7.30 x 10(-7)) as well as in the commercial lines (P = 2.78 x 10(-6)). Linkage disequilibrium (LD) extends into a neighbouring gene encoding deformed epidermal autoregulatory factor 1 (DEAF1). The product of DEAF1 regulates the transcription of the gene encoding the serotonin (5-hydroxytryptamine) 1A receptor. Thus, DEAF1 represents another candidate gene for feather pecking. Re-sequencing of five animals homozygous for the 'low-pecking' sub-haplotype and of six animals homozygous for the 'high-pecking' sub-haplotype delineated an LD block of 14 833 bases spanning the two genes. None of the variants in the LD block is obviously functional. However, the haplotype information will be useful to select against the propensity to feather pecking in chicken and to elucidate the functional implications of the variants.

Identification of the new polymorphisms in the promoter region of the CAST gene in cattle.

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases -µ-calpain and m-calpain. This proteolytic system plays a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditions. In the present study using comparative sequencing seven novel polymorphisms located within P3 promoter region for exon 1u of the bovine CAST gene: -357 (C/G), -556 (G/T), -557 (A/G), -580 (G/C), -750 (T/C), and two InDel at position -890 (A/-) and (GTT/-) at position -353/-351 were found. This region directs the expression of type III calpastatin mRNA, encoding the prototypical calpastatin. The genotype frequencies and haplotypes distribution were studied in 191 bulls belonging to six cattle breeds. All genotypes were distributed according to the HWE test and two major combined haplotypes were identified. The frequency of the haplotype1 varied from 0.45 in Aberdeen Angus to 0.82 in Simmental.

Effect of new SNP within bovine prolactin gene enhancer region on expression in the pituitary gland.

A new single nucleotide polymorphism was revealed using PCR-SSCP and sequencing methods within the bovine prolactin distal promoter region described as a functional enhancer. The A-->G transition at position -1043 abolishes the recognition site for Hsp92II restriction endonuclease, allowing for PCR-RFLP genotyping. The application of real-time PCR revealed that the prolactin gene expression level in the pituitary was higher in cattle with the AA genotype than in those with the GG genotype. EMSA analysis, however, showed increased nuclear protein binding to the sequence variant with G, suggesting a possible inhibition event, in which the transcription factors Pit1, Oct1, and YY1 could be involved.

The polymorphism in the beta4-defensin gene and its association with production and somatic cell count in Holstein-Friesian cows.

The aim of this study was to find a polymorphism of the bovine beta4-defensin gene and search for its association with milk yield and composition and with the somatic cell count in milk. The data were from the years 1999 to 2004 on 212 Holstein-Friesian (HF) dairy cows, descended from 70 sires. Based on the sequence of the bovine beta4-defensin gene (GenBank no. AF008307) the primers were designed for the amplification of the 924-bp or 393-bp long fragments. The 924-bp long fragment was sequenced and the sequence was compared with that available in the GenBank. Ten putative nucleotide sequence polymorphisms were found in the intron of the bovine beta4-defensin gene. One of them, a C-->T transition at position 2239, that creates a new NlaIII (Hin1II) restriction site, was genotyped with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a cohort of 212 HF cows. The CC genotype was the most common (72%). The heterozygous CT genotype was found in 26% of the genotyped cows and four cows (2%) were TT homozygotes. In order to determine the relationship between the polymorphism of the beta4-defensin gene and milk production traits a multi-trait repeatability test-day animal model was used. The Derivative-free Multivariate analysis program was used for computation. The differences between estimates for genotypes were checked using Student's t-test. The model included the animal genotype, year-season of calving and parity as fixed effects and the animal additive genetic effect and permanent environmental effect of individual cows as well as dates of the tests as random effects. Significant associations were found between the RFLP-NlaIII and milk fat, protein and lactose contents. Also, a significant effect was shown of the defensin genotype on the somatic cell count in the milk.