SNP genotypes of olfactory receptor genes associated with olfactory ability in German Shepherd dogs

To find out the relationship between SNP genotypes of canine olfactory receptor genes and olfactory ability, 28 males and 20 females from German Shepherd dogs in police service were scored by odor detection tests and analyzed using the Beckman GenomeLab SNPstream. The representative 22 SNP loci from the exonic regions of 12 olfactory receptor genes were investigated, and three kinds of odor (human, ice drug and trinitrotoluene) were detected. The results showed that the SNP genotypes at the OR10H1‐like:c.632C>T, OR10H1‐like:c.770A>T, OR2K2‐like:c.518G>A, OR4C11‐like:c.511T>G and OR4C11‐like:c.692G>A loci had a statistically significant effect on the scenting abilities (P < 0.001). The kind of odor influenced the performances of the dogs (P < 0.001). In addition, there were interactions between genotype and the kind of odor at the following loci: OR10H1‐like:c.632C>T, OR10H1‐like:c.770A>T, OR4C11‐like:c.511T>G and OR4C11‐like:c.692G>A (P < 0.001). The dogs with genotype CC at the OR10H1‐like:c.632C>T, genotype AA at the OR10H1‐like:c.770A>T, genotype TT at the OR4C11‐like:c.511T>G and genotype GG at the OR4C11‐like:c.692G>A loci did better at detecting the ice drug. We concluded that there was linkage between certain SNP genotypes and the olfactory ability of dogs and that SNP genotypes might be useful in determining dogs' scenting potential.     

Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: , Programs: BDGP: , ESEfinder program:     

The CDKN2A/p16INK4a 5′UTR sequence and translational regulation: impact of novel variants predisposing to melanoma

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5′UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.‐27del23, c.‐93‐91delAGG) were classified as causal mutations (score ≥3), along with the c.‐21C>T, c.‐34G>T, and c.‐56G>T, which had already been studied by a subset of assays. The novel c.‐42T>A as well as the previously described c.‐67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.‐14C>T, c.‐20A>G, c.‐25C>T+c.‐180G>A, c.‐30G>A, c.‐40C>T, c.‐45G>A, c.‐59C>G, c.‐87T>A, c.‐252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5′UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16^INK4a translation.     

Analysis of non‐synonymous SNPs of the porcine SERPINA6 gene as potential causal variants for a QTL affecting plasma cortisol levels on SSC7

Recently, the SERPINA6 gene encoding corticosteroid‐binding globulin (CBG) has been proposed as a candidate gene for a quantitative trait locus (QTL) affecting cortisol level on pig chromosome 7. The QTL was repeatedly detected in different lines, including a Piétrain × (German Landrace × German Large White) cross (PiF1) and purebred German Landrace (LR). In this study, we investigated whether the known non‐synonymous polymorphisms c.44G>T, c.622C>T, c.770C>T, c.793G>A, c.832G>A and c.919G>A of SERPINA6 are sufficient to explain the QTL in these two populations. Our investigations revealed that SNPs c.44G>T, c.622C>T, c.793G>A and c.919G>A are associated with cortisol level in PiF1 (P < 0.01). Haplotype analysis showed that these associations are largely attributable to differences between a major haplotype carrying SNPs c.793G>A and c.919G>A and a haplotype carrying SNPs c.44G>T and c.622C>T. Furthermore, some SNPs, particularly c.44G>T and c.622C>T and the carrier haplotype, showed association with meat quality traits including pH and conductivity (P < 0.05). In LR, the non‐synonymous SNPs segregate at very low frequency (<5%) and/or show only weak association with cortisol level (SNPs c.832G>A and c.919G>A; P < 0.05). These findings suggest that the non‐synonymous SNPs are not sufficient to explain the QTL across different breeds. Therefore, we examined whether the expression of SERPINA6 is affected by cis‐regulatory polymorphisms in liver, the major organ for CBG production. We found allelic expression imbalance of SERPINA6, which suggests that its expression is indeed affected by genetic variation in cis‐acting elements. This represents candidate causal variation for future studies of the molecular background of the QTL.     

Sequence analysis of three canine adipokine genes revealed an association between TNF polymorphisms and obesity in Labrador dogs

Obesity is an emerging health problem in purebred dogs. Due to their crucial role in energy homeostasis control, genes encoding adipokines are considered candidate genes, and their variants may be associated with predisposition to obesity. Searching for polymorphism was carried out in three adipokine genes (TNF, RETN and IL6). The study was performed on 260 dogs, including lean (n = 109), overweight (n = 88) and obese (n = 63) dogs. The largest cohort was represented by Labrador Retrievers (n = 136). Altogether, 24 novel polymorphisms were identified: 12 in TNF (including one missense SNP), eight in RETN (including one missense SNP) and four in IL6. Distributions of five common SNPs (two in TNF, two in RETN and one in IL6) were further analyzed with regard to body condition score. Two SNPs in the non‐coding parts of TNF (c.‐40A>C and c.233+14G>A) were associated with obesity in Labrador dogs. The obtained results showed that the studied adipokine genes are highly polymorphic and two polymorphisms in the TNF gene may be considered as markers predisposing Labrador dogs to obesity.     

Polymorphism identification in goat GNRH1 and GDF9 genes and their association analysis with litter size

This study investigated the polymorphisms of GNRH1 and GDF9 genes in 641 goats of three breeds: Xinong Saanen, Guanzhong and Boer. Two allelic variants were identified in the GNRH1 gene (JN645280:g.3548A>G and JN645281:g.3699G>A) and one allelic variant was found in the GDF9 gene (JN655693:g.4093G>A). Furthermore, g.4093G>A was a missense mutation (p.Val397Ile of GDF9). Results of an association analysis indicated that SNPs g.3548A>G and g.4093G>A had significant effects on litter size (P < 0.05). The combination genotypes of SNPs g.3548A>G, g.3699G>A and g.4093G>A also affected litter size with the C₅ genotype having the highest litter size in the first, third, fourth and average parity. Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the GNRH1 and GDF9 genes could serve as genetic markers for litter size in goat breeding.     

Examination of structural changes in the transforming growth factor β receptor 1 (TGFβR1) gene in patients with chronic heart failure

We examined nine exons of transforming growth factor β receptor type 1 (TGFβR1) gene in patients with chronic heart failure with different types of heart remodelling. We identified two missense mutations (c.457G>A (p.V1531) and c.1285A>C (p.Y229S)) and two synonym substitutions (c.1125A>C (p.Y377Y) and c.516A>G (p.S172S)), as well as polymorphisms at splicing site c.1024+24G>A (rs334354). Substitutions c.1285A>C (p.Y229S) and c.516A>G (p.S172S) were not previously described.     

An association between polymorphism of the heme oxygenase-1 and -2 genes and age-related macular degeneration

Iron may be implicated in the generation of oxidative stress by the catalyzing the Haber–Weiss or Fenton reaction. On the other hand, oxidative stress has been implicated in the pathogenesis of age-related macular degeneration (AMD) and heme oxygenase-1 (HO-1), encoded by the HMOX1 gene and heme oxygenase-2 (HO-2), encoded by the HMOX2 gene are important markers of iron-related oxidative stress and its consequences. Therefore, variability of the HMOX1 and HMOX2 genes might be implicated in the pathogenesis of AMD through the modulation of the cellular reaction to oxidative stress. In the present work, we investigated the association between AMD and a G → C transversion at the 19 position in the HMOX1 gene (the 19G>C-HMOX1 polymorphism, rs2071747) and a A → G transition at the −42 + 1444 position in the HMOX2 gene (the −42 + 1444A>G-HMOX2 polymorphism, rs2270363) and its modulation by some environmental factors. 279 patients with AMD and 105 controls were recruited in this study and the polymorphisms were typed by restriction fragment length polymorphism and allele-specific polymerase chain reaction (PCR). We observed an association between the occurrence of dry AMD and the G/A genotype of the −42 + 1444A>G-HMOX2 polymorphism (odds ratio (OR) 2.72), whereas the G/G genotype reduced the risk of dry AMD (OR 0.41). The G/C genotype and the C allele of the 19 G>C-HMOX1 polymorphism and the G/G genotype and the G allele of the −42 + 1444A>G-HMOX2 polymorphism were associated with progression of AMD from dry to wet form (OR 4.83, 5.20, 2.55, 1.69, respectively). On the other hand, the G/G genotype and the G allele of the 19 G>C-HMOX1 polymorphism and the A/G genotype and the A allele of the −42 + 1444A>G-HMOX2 polymorphism protected against AMD progression (OR 0.19, 0.19, 0.34, 0.59, respectively). Therefore, the 19G>C-HMOX1 and the −42 + 1444A>G-HMOX2 polymorphisms may be associated with the occurrence and progression of AMD.     

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes,and depressive disorders

Tryptophan catabolites pathway disorders are observed in patients with depression. Moreover, single nucleotide polymorphisms of tryptophan hydroxylase genes may modulate the risk of depression occurrence. The objective of our study was to confirm the association between the presence of polymorphic variants of TPH1 and TPH2 genes, and the development of depressive disorders. Six polymorphisms were selected: c.804‐7C>A (rs10488682), c.‐1668T>A (rs623580), c.803+221C>A (rs1800532), c.‐173A>T (rs1799913)—TPH1, c.‐1449C>A (rs7963803), and c.‐844G>T (rs4570625)—TPH2. A total of 510 DNA samples (230 controls and 280 patients) were genotyped using TaqMan probes. Among the studied polymoorphisms, the G/G genotype and G allele of c.804‐7C>A—TPH1, the T/T homozygote of c.803+221C>A—TPH1, the A/A genotype and A allele of c.1668T>A—TPH1, the G/G homozygote and G allele of c.‐844G>T—TPH2, and the C/A heterozygote and A allele of c.‐1449C>A—TPH2 were associated with the occurrence of depression. However, the T/T homozygote of c.‐1668T>A—TPH1, the G/T heterozygote and T allele of c.‐844G>T—TPH2, and the C/C homozygote and C allele of c.‐1449C>A—TPH2 decreased the risk of development of depressive disorders . Each of the studied polymorphisms modulated the risk of depression for selected genotypes and alleles. These results support the hypothesis regarding the involvement of the pathway in the pathogenesis of depression.     

Genetic polymorphism c.1438A>G of the 5-HT<Subscript>2A</Subscript> receptor is associated with abdominal obesity in Chinese Northern Han population

Objective We examined the potential impact of the 5-hydroxytryptamine 2A receptor (5-HT_2AR) c.1438A>G promoter polymorphism on obesity and estimates of insulin, glucose as well as lipid metabolism. Methods The genotypes and allelic frequencies of the 5-HT_2AR c.1438A>G were examined with polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 210 patients with overweight/obesity and 216 unrelated healthy subjects. Results The genotype (AA, AG, and GG) distribution of c.1438A>G polymorphism of the 5-HT_2AR gene promoter was 35%, 46%, and 19% in patients, and 32%, 56%, 12% in controls, respectively, no significant difference was found between two groups. Association of genetic diversity of 5-HT_2AR c.1438A>G with the total body fat, fat distribution and clinical characteristics revealed that overweight/obese men carrying G allele were associated with increased body mass index (P = 0.043), waist circumference (P = 0.038), waist-to-hip ratio (P = 0.045), in comparison with patients who carrying A allele, but there were no significant difference between the c.1438A>G genotype groups in overweight/obese women. Conclusion No significant associations were detected. However, the present study suggests the possibility that an abnormal production rate of the 5-HT_2AR c.1438A>G gene product might lead to the development of abdominal obesity in men but not in women. Su Ying and Yan-Ming Sun equally contributed to this study.     

Genetic variation in catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (<Emphasis Type="Italic">COMT</Emphasis>) and obesity in the prostate,lung, colorectal,and ovarian (PLCO) cancer screening trial

Catechol-O-methyltransferase (COMT) is an important modulator in the catabolism of extraneural dopamine, which plays an important role in drug reward mechanisms. It is hypothesized that genetic variations in the COMT gene, which can result in a three to fourfold difference in COMT enzyme activity, may be associated with several reward-motivated behaviors. The aim of our study was to examine the relationship between COMT polymorphisms with smoking, obesity and alcohol. Three single nucleotide polymorphisms (SNPs) in COMT were genotyped in 2,371 participants selected randomly from the screening arm of the PLCO Cancer Screening Trial after stratifying by sex, age, and smoking status. Smoking, obesity, and alcohol consumption were assessed by questionnaire. SNP and haplotype associations were estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from conditional logistic regression models, adjusted for race/ethnicity. The COMT Ex4-76C > G (Leu136Leu) polymorphism was statistically significantly associated with individuals who had >30% increases in BMI from ages 20 to 50 years, compared to those with 0–5% increase in BMI (0–5%) over the same age period: (CC is referent; OR_CG = 1.42, OR_GG = 1.46, P _trend = 0.06). By sex, the increased risk was further pronounced among females (OR_CG = 1.50, OR_GG = 2.10, P _trend = 0.03). Consistent with our analyses of single polymorphisms, individuals whose BMI increased >30% from ages 20 to 50 years were more likely than individuals with 0–5% increases in BMI to possess COMT haplotypes [COMT Ex3-104C > T–COMT Ex4-76 C > G–COMT Ex4-12 A > G] that included the variant allele for COMT Ex4-76 C > G: C-G-G (T-C-A is referent: OR_C-G-G = 1.33, 95% CI 1.01–1.77) and C-G-A (OR_C-G-A = 1.79, 95% CI 0.72–4.49). We observed no association between any of the COMT polymorphisms with smoking behavior or alcohol intake. The COMT Ex4-76C > G (Leu136Leu) polymorphism appears to play a role in large increases in BMI. The null association with smoking and alcohol and the pronounced association with increasing BMI among women further implicates COMT’s role in estrogen metabolism as a potentially culpable pathway. Our results support a need for comprehensive evaluation of COMT variations and their functional relevance as COMT may be an important molecular target to evaluate for new treatments regarding obesity.     

Polymorphism and methylation of the <Emphasis Type="Italic">MC4R</Emphasis> gene in obese and non-obese dogs

The dog is considered to be a useful biomedical model for human diseases and disorders, including obesity. One of the numerous genes associated with human polygenic obesity is MC4R, encoding the melanocortin 4 receptor. The aim of our study was to analyze polymorphisms and methylation of the canine MC4R in relation to adiposity. Altogether 270 dogs representing four breeds predisposed to obesity: Labrador Retriever (n?=?187), Golden Retriever (n?=?38), Beagle (n?=?28) and Cocker Spaniel (n?=?17), were studied. The dogs were classified into three groups: lean, overweight and obese, according to the 5-point Body Condition Score (BCS) scale. In the cohort of Labradors a complete phenotypic data (age, sex, neutering status, body weight and BCS) were collected for 127 dogs. The entire coding sequence as well as 5′ and 3′-flanking regions of the studied gene were sequenced and six polymorphic sites were reported. Genotype frequencies differed considerably between breeds and Labrador Retrievers appeared to be the less polymorphic. Moreover, distribution of some polymorphic variants differed significantly (P?<?0.05) between small cohorts with diverse BCS in Golden Retrievers (c.777T>C, c.868C>T and c.*33C>G) and Beagles (c.-435T>C and c.637G>T). On the contrary, in Labradors no association between the studied polymorphisms and BCS or body weight was observed. Methylation analysis, using bisulfite DNA conversion followed by Sanger sequencing, was carried out for 12 dogs with BCS?=?3 and 12 dogs with BCS?=?5. Two intragenic CpG islands, containing 19 cytosines, were analyzed and the methylation profile did not differ significantly between lean and obese animals. We conclude that an association of the MC4R gene polymorphism with dog obesity or body weight is unlikely, in spite of the fact that some associations were found in small cohorts of Beagles and Golden Retrievers. Also methylation level of this gene is not related with dog adiposity.     

Sample-to-SNP kit: A reliable,easy and fast tool for the detection of HFE p.H63D and p.C282Y variations associated to hereditary hemochromatosis

Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G > A; rs1800562) and HFE p.H63D (c.187C > G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G > A, Coagulation factor V-gene F5 p.R506Q (c.1517G > A; rs121917732), Mitochondria SNP: mt7028 G > A, Mitochondria SNP: mt12308 A > G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G > T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A > G; rs1695), LXR g.-171 A > G, ZNF202 g.-118 G > T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility.     

Identification of novel single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) gene associated with fatty acid composition in Korean cattle

The lipoprotein lipase (LPL) gene can be considered a functional candidate gene that regulates fatty acid composition. In this study, genetic associations between fatty acid composition and exonic single nucleotide polymorphisms (SNPs) in the LPL gene were examined using 612 Korean cattle. We investigated the relationship between unsaturated fatty acids and five novel SNPs (c.322G>A, c.329A>T, c.527T>G, c.988C>T and c.1591G>A), and confirmed that three polymorphic SNPs (c.322G>A, c.329A>T and c.1591G>A) were associated with fatty acid composition. Korean cattle with an AA genotype of c.322G>A, c.329A>T, and GA genotype of c.1591G>A had higher levels of monounsaturated fatty acids and carcass traits (P < 0.05). Our findings confirmed that three novel SNPs we identified in the LPL gene can affect fatty acid composition and carcass traits. Therefore, selection for AA and GA genotypes should be recommended to genetically improve beef quality and flavor.     

Association between MT-CO3 haplotypes and high-altitude adaptation in Tibetan chicken

Genetic mutation in cytochrome c oxidase subunit III gene (MT-CO3) could influence the kinetics of cytochrome c oxidase (COX), which catalyzes oxygen transport capacity in oxidative phosphorylation. However, the potential relationship between MT-CO3 variants and high-altitude adaptation remains poorly understood in Tibetan chicken. Here, we sequenced MT-CO3 gene of 125 Tibetan chickens and 144 Chinese domestic chickens in areas at a low elevation (below 1000 m). Eight single nucleotide polymorphisms (SNPs) were detected; and five of them (m.10081A>G, m.10115G>A, m.10270G>A, m.10336A>G and m.10447C>T) shared by Tibetan chicken and lowland chicken with the significant difference in their respective allele frequencies. Nine haplotypes (H1–H9) were finally defined. Among them, haplotype H4 was positively associated with high-altitude adaptation whereas haplotypes H6, H7 and H8 had negative association with high-altitude adaptation. The Median-joining profile suggested that haplotype H5 had the ancestral position to the other haplotypes but had no significant relationship with high-altitude adaptation. However, there was only m.10081A>G mutation differed from haplotype H4 and H5. Results also suggested that chickens with A allele at m.10081A>G, had over 2.6 times than those with G allele in the probability of the ability to adapt hypoxia. It suggests that the synonymous mutation m.10081A>G may be a prerequisite for shaping high-altitude adaptation-specific haplotypes.     

Targeted Molecular Sequencing Revealed Allelic Heterogeneity of <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">PTPN11</Emphasis> Genes among Arab Noonan Syndrome Patients

Noonan syndrome (NS) is a very rare heterogenous genetic disorder often characterized by short stature, facial dysmorphisms, congenital heart defects and learning disabilities in affected children. In the current study, we sought to discover the disease causal mutations, inherited or de novo, for Noonan Syndrome among Arab patients. We screened the coding regions and splice sites of 10 known RAS/MAP Kinase pathway genes in 17 NS-trios and 100 random healthy volunteers by oilgonucleotide chip testing and Sanger sequencing methods. We found pathogenic heteroallelic de novo mutations in BRAF or PTPN11 gene in 7/17 (41.17%) of NS patients. None of the 200 chromosomes of healthy volunteers had those pathogenic mutations. Genotype-phenotype analysis showed positive correlation between BRAF and PTPN11 gene mutations and classical NS clinical manifestations. Characteristic facies is the major observed clinical manifestation among PTPN11-mutation positive cases (c.236A>G, c.854T>C, c.923A>G), whereas both characteristic facies and ectodermal manifestations are seen as dominant clinical features among BRAF-mutation positive cases (c.730A>C, c.770A>G, c.1406G>A). In addition to genotyping and clinical phenotyping, we performed computational structural analysis to examine the impact of amino acid substitution mutations on the conformation and folding of BRAF and PTPN11 proteins. Our results suggested that BRAF (c.730A>C, c.770A>G, c.1406G>A) and PTPN11 (c.236A>G, c.854T>C, c.923A>G) gene mutations elicits structural and functional alterations at protein level, which would eventually lead to dysregulation of RAS-MAPK signaling cascade, which plays critical a role in cell cycle and senescence. In conclusion, our study suggest that molecular screening of BRAF and PTPN11 genetic mutations in Arab NS patients may assist in deriving competitive outcomes related to clinical phenotyping and disease diagnosis.     

Nucleotide variants in prion-related protein (testis-specific) gene (PRNT) and effects on Chinese and Mongolian sheep phenotypes

ABSTRACT

Studies of the ovine prion-related protein (testis-specific) gene (PRNT), including studies of genetic diversity, have highlighted its potential relationship to scrapie infection and economically important ovine traits. PRNT was previously reported to be highly polymorphic in Portuguese sheep. To characterize genetic polymorphisms in this gene in Asian sheep, a direct sequencing method was used to detect polymorphic loci in PRNT in 285 individual sheep from four Chinese and one Mongolian breeds. Seven SNP variants in PRNT were identified, including three novel variants (g.93G>A, g.162G>T, and g.190A>G) and four previously reported variants (g.17 C>T, g.112G>C, g.129C>T, and g.144A>G). In the five breeds that we analyzed, the mutation frequencies of g.190A>G in Lanzhou Fat-tail sheep (LFTS) and g.129C>T in the other four varieties were high (F>0.5). Moreover, thirteen different haplotypes that had a comparable distribution in the tested breeds were also identified; ‘C-G-G-C-A-G-A’ occurred at the highest frequency in the five sheep breeds. Additionally, we previously explored the significance of relationships between polymorphisms in PRNP or PRND and ovine growth performance. Here, we also performed correlation analysis in all tested loci. These loci polymorphisms were significantly associated with ten different growth traits (P<0.05), except for g.93G>A. Meanwhile, in contrast to a previous study, there was no significant association between the seven SNP loci analyzed and our previously reported sheep PRND or PRNP insertion/deletion mutations. Our findings may provide new insights into polymorphic variation in ovine PRNT, which may contribute to genetic improvements in economic traits that are important for sheep breeding.     

Characterization of the porcine transferrin gene (TF ) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection

Transferrin (TF)‐mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron‐depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra‐ and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed‐specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.     

The prion‐related protein (testis‐specific) gene (PRNT) is highly polymorphic in Portuguese sheep

The objective of this study was to search for polymorphisms in the ovine prion‐related protein (testis‐specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene‐coding region was analyzed by single‐strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine‐X‐X‐serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C‐112G‐129T‐144A,17CT‐112GC‐129CT‐144AG and 17T‐112C‐129C‐144G), and the only three animals found homozygous at c.78A had the 17C‐112G‐129C‐144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).