Investigation of single‐nucleotide polymorphisms in the NR3C1a glucocorticoid receptor gene in Cocker Spaniels with primary immune thrombocytopenia

BackgroundIn dogs, 6 single‐nucleotide polymorphisms (SNPs) have been described in the glucocorticoid receptor gene NR3C1a, 2 of which were nonsynonymous SNPs in exons 2 and 8. The clinical importance of these SNPs is unknown.ObjectivesTo investigate whether SNPs in NR3C1a are associated with clinical outcome in Cocker Spaniels with primary immune thrombocytopenia (pITP).AnimalsTwenty‐four Cocker Spaniels with pITP presented to a referral center. Dogs were classified as slow (n = 11) or fast responders (n = 12) based on time required after initiating glucocorticoid treatment to achieve a platelet count >70 000/μL.MethodsDeoxyribonucleic acid was extracted from stored blood samples before amplification by PCR and sequencing of exons 2 and 8 of NR3C1a. Associations between genotype and clinical response variables were investigated.ResultsNeither previously identified nonsynonymous SNPs were identified. The synonymous SNP NR3C1a:c.798C>T in exon 2 was found at an increased prevalence compared to a previous report. No difference was found in prevalence of any genotype at NR3C1a:c.798C>T between fast and slow responders (P = .70).Conclusions and Clinical ImportanceNone of the previously reported nonsynonymous SNPs in exons 2 and 8 of the NR3C1a gene were detected in our cohort of Cocker Spaniels with pITP. The synonymous SNP NR3C1a:c.798C>T in exon 2 was reported at a higher frequency than previously, but was not associated with outcome measures that estimated responsiveness to glucocorticoids.     

Polymorphism of glutathione S-transferase P1 of dogs with mammary tumours

Mammary tumours constitute more than half of neoplasms in female dogs from different countries. Genome sequences are associated with cancer susceptibility but there is little information available about genetic polymorphisms of glutathione S-transferase P1 (GSTP1) in canine cancers. The aim of this study was to find single nucleotide polymorphisms (SNPs) in GSTP1 of dogs (Canis lupus familiaris) with mammary tumours compared to healthy dogs and to determine the association between GSTP1 polymorphisms and the occurrence of these tumours. The study population included 36 client-owned female dogs with mammary tumours and 12 healthy female dogs, with no previous diagnosis of cancer. DNA was extracted from blood and amplified by PCR assay. PCR-products were sequenced by Sanger method and analysed manually. The 33 polymorphisms were found in GSTP1: 1 coding SNP (exon 4), 24 non-coding SNPs (9 in exon 1), 7 deletions and 1 insertion. The 17 polymorphisms have been found in introns 1, 4, 5 and 6. The dogs with mammary tumours have significant difference from healthy in SNPs I4 c.1018 + 123 T > C (OR 13.412, 95%CI 1.574–114.267, P = .001), I5 c.1487 + 27 T > C (OR 10.737, 95%CI 1.260–91.477, P = .004), I5 c.1487 + 842 G > C (OR 4.714, 95% CI 1.086–20.472, P = .046) and I6 c.2481 + 50 A > G (OR 12.000, 95% CI 1.409–102.207, P = .002). SNP E5 c.1487 T > C and I5 c.1487 + 829 delG also differed significantly (P = .03) but not to the confidence interval. The study, for the first time, showed a positive association of SNPs in GSTP1 with mammary tumours of dogs, that can possibly be used to predict the occurrence of this pathology.     

The polymorphisms of bovine melanocortin-3 receptor pseudogene

Mammalian melanocortin-3 receptor (MC3R) plays an important role in the central control of energy homeostasis, and several functional polymorphisms of mc3r have been detected. Interestingly, the bovine mc3r was a pseudogene, and its polymorphisms and function remain to be investigated. Single-strand conformation polymorphism (SSCP) showed 5, 2 and 3 genotypes in fragment F1, F2 and F3 of mc3r in seven cattle breeds, respectively. All genotypes revealed novel sequences. Three SNPs 657G > T, 756C > T, 822T > C were detected in fragment F1, five SNPs 1091T > C, 1133T > C, 1144C > T, 1259T > C and 1319G > A were detected in fragment F2, and two SNPs 1687G > A, 1860C > T were detected in F3. The SNPs in fragment F1 and F2 were located at exon 2. The five SNPs in fragment F2 demonstrated a tight linkage disequilibrium status. Variation detected here might have an impact on the function of bovine mc3r pseudogene.     

Association of PCSK1 gene polymorphisms with abdominal fat content in broilers

Protein proteolytic enzymes (Proprotein Convertase, PC) is a Ca²⁺‐dependent serine protease family, whose main function is to cleave precursors of biologically inactive proteins or peptide chains into active functional molecules. Proprotein convertase subtilisin/kexin type 1 (PCSK1) gene is mainly expressed in nerve and endocrine tissues. In this study, PCSK1 was selected as an important candidate gene for abdominal fat content in broilers. We cloned the exon region of chicken PCSK1 gene and found six single‐nucleotide polymorphisms (SNPs). Association analysis was carried out and we found that the polymorphisms of these six SNPs were significantly associated with abdominal fat content in G19 and G20 populations. Five of these SNPs were significantly associated with abdominal fat content in G19 and G20 combined population. The polymorphism of these five SNPs was significantly correlated with the abdominal fat content of AA broilers. Together, our study demonstrated that c.927T>C, c.1880C>T, c.*900G>A, and c.*1164C>T were significantly associated with abdominal fat content in populations used in this study, which means that these SNPs in PCSK1 gene could be used as candidate markers to select lean broiler lines.     

Polymorphisms in the canine glutamate transporter-1 gene: identification and variation among five dog breeds

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.     

MC1R c.310G>‐ and c.871G > A determine the coat color of Kumamoto sub‐breed of Japanese Brown cattle

Coat color is one of the important factors characterizing breeds for domestic animals. Melanocortin 1 receptor (MC1R) is a representative responsible gene for this phenotype. Two single‐nucleotide polymorphisms (SNPs) in bovine MC1R gene, c.296T > C and c.310G>‐, have been well characterized, but these SNPs are not enough to explain cattle coat color. As far as we know, MC1R genotypes of Kumamoto sub‐breed of Japanese Brown cattle have not been analyzed. In the current study, genotyping for c.296T > C and c.310G>‐ was performed to elucidate the role of MC1R in determining the coat color of this sub‐breed. As a result, most animals were e/e genotype, suggesting the coat color of this sub‐breed is derived from the e allele of MC1R gene. However, we found six animals with E/e genotype, which coat color would be black theoretically. Subsequently, sequence comparison was performed with these animals to identify other polymorphisms affecting coat color, elucidating that these animals possessed the A allele of c.871G > A commonly. c.871G > A was a non‐synonymous mutation in the seventh transmembrane domain, suggesting alteration of the function and/or the structure of MC1R protein. Our data indicated that the A allele of c.871G > A might be a loss‐of‐function mutation.     

The g.841G>C SNP of FASN gene is associated with fatty acid composition in beef cattle

The objective of the current study is to evaluate the association between fatty acid composition and fatty acid synthase gene polymorphisms as responsible mutations. For this purpose, we selected seven previously reported single nucleotide polymorphisms (SNPs) in FASN gene, including one within promoter region (g.841G>C) and six non‐synonymous SNPs (g.8805C>T, g.13126C>T, g.15532A>C, g.16024A>G, g.16039C>T, g.17924A>G), and genotyped them in Japanese Black cattle. Genotyping results revealed that g.8805 C>T and g.17924 A>G were monomorphic loci. Genome‐wide association analysis including the other five SNPs revealed that only g.841G>C showed significant associations with the percentages of C14:0, C14:1, C16:1 and C18:1 at 5% genome‐wide significance level. In order to further evaluate the effect, we genotyped g.841G>C using additional three populations, including two Japanese Black populations and a Holstein cattle population. g.16024A>G was also genotyped and included in the analysis because it has been reported to be associated with fatty acid composition in Japanese Black cattle. In the result of analysis of variance, g.841G>C showed stronger effects on fatty acid percentage than those of g.16024A>G in all populations. These results suggested that g.841G>C would be a responsible mutation for fatty acid composition and contribute to production of high‐grade beef as a selection marker in beef cattle.     

Associations between candidate SNPs in the calpain 1, calpastatin and leptin genes and meat tenderness among Swedish beef populations

Abstract

The contribution of three candidate genes to the variation in meat tenderness was tested in muscle samples from 243 pure-bred, young, beef bulls of Angus, Charolais, Hereford, Limousin and Simmental breeds, raised in Swedish commercial herds. The animals were genotyped for single nucleotide polymorphisms (SNPs) in the calpain 1 (CAPN1), calpastatin (CAST) and leptin genes. The frequent calpain 1 CAPN1:c.947G>C G allele showed an unfavourable association with tenderness. The calpastatin CAST:c.155C>T T allele, which was the most common allele, showed a favourable association with Warner–Bratzler shear force (WBSF) and compression tests. An association was observed between the leptin UASMS2C>T SNP and compression tests.     

Canine orosomucoid (alpha‐1 acid glycoprotein) variants and their influence on drug plasma protein binding

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed‐breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n = 12). Free and total drug plasma concentrations were quantified by liquid chromatography–mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n = 1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n = 21). Free drug fractions did not differ significantly (p > .05; Mann‐Whitney U) between plasma collected from dogs with c.70AA (n = 4) and those with c.70GG (n = 8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.     

Genetic variations of BRCA1 and BRCA2 genes in dogs with mammary tumours

Mammary tumours are the most common tumour type in female dogs. The formation of the mammary tumours is multifactorial but the high incidence of tumour disease in certain canine breeds suggests a strong genetic component. BRCA1 and BRCA2 are the most important genes significantly associated with mammary tumours. The aim of this study was to determine the association between the variations of these two genes and canine mammary tumours. 5′-untranslated region, intron 8 and exon 9 of BRCA1 and exons 12, 24, 27 of BRCA2 were sequenced in order to detect the genetic variations. In addition to six previously identified polymorphisms, six novel single nucleotide polymorphisms (SNPs) were detected. Five of the coding SNPs were synonymous and three of them were non-synonymous. The comparison of the sequences from 25 mammary tumour bearing and 10 tumour free dogs suggested that the two SNPs in intron 8 and exon 9 of BRCA1 and two SNPs in exon 24 and exon 27 of BRCA2, which are firstly identified in this study, might be associated with mammary tumour development in dogs. Especially one SNP in exon 9 of BRCA1 and one SNP in exon 24 of BRCA2 were found to be significantly associated with canine mammary tumours.     

Somatic SNPs of the BRCA2 gene at the fragments encoding RAD51 binding sites of canine mammary tumors

Mammary tumors are the most common tumor type both in women and in female dogs. In women, heritable breast cancers have been linked mutations in the breast cancer susceptibility gene BRCA2 and it contains eight BRC repeats in exon 11 that bind to RAD51. In this study, we investigated the sequence variations of BRC1‐BRC8 and C‐terminus of canine BRCA2 gene. From a total of 64 canine patients with mammary tumors, 31 mammary tumors with benign and malign carcinomas and the 3 normal mammary glands were used for the study. In this study, 19 SNPs of exon 11 of BRCA2 in canine mammary tumors were detected for the first time. The c.2383A>C (T1425P) SNP was found to be the most probable disease‐associated nsSNP. Our findings suggest that T1425P variation in BRC3 to be the most probable disease‐associated nsSNP and may affect RAD51 binding strength.     

Identification of single nucleotide polymorphisms within exon 1 of the canine mu-opioid receptor gene

Objective  To determine the presence and frequency of single nucleotide polymorphisms (SNPs) within exon 1 of the canine mu-opioid receptor (MOR) gene.
Study design  Prospective genetic analysis.
Animals  Seventy-five dogs of various breeds.
Methods  DNA was isolated from dog blood. Polymerase chain reaction (PCR) was performed to amplify exon 1 of the canine MOR gene using primers derived from a published sequence. PCR products of anticipated size were identified by gel electrophoresis, isolated and sequenced.
Results  Two SNPs were found within the examined region. One is 15 base pairs (bp) upstream (C-15A) of the protein-coding portion of the gene. The second is at position 207 (C207T); a synonymous mutation predicting unaltered protein sequence. The overall prevalence of the C-15A SNP was 43% (64/150 alleles). The overall prevalence of the C207T SNP was 26% (39/150 alleles).
Conclusions and clinical relevance  Absence of haplotypes containing both an adenosine at position −15 and a thymine at position 207 suggests that these polymorphisms occurred independently from each other. How these SNPs influence variations in responses seen after opioid administration to dogs remain to be determined, however, our data indicates the C-15A SNP may play a role in opioid dysphoria.     

Three SNPs within exons of INHA and ACVR2B genes are significantly associated with litter size in Dazu black goats

The aim of this study was to analyse the association between single-nucleotide polymorphisms within INHA and ACVR2B and litter size in Dazu black goats. In total, twenty-two SNPs were genotyped in 190 individuals by SNaPshot and resequencing. The results showed that three SNPs (SNP_1, SNP_12 and SNP_13 in this study) were detected to have significant additive genetic effect on the recorded goat litter size (p < .05). The SNP_1 (NC_030809.1), a non-synonymous substitution of G for T at chr2-g. 28314990 in the exon 2 of INHA gene (NM_001285606.1), resulted in homozygote 2 (HOM2) contributed 0.25 and heterozygote (HET) contributed 0.12 larger litter than homozygote 1 (HOM1). Meanwhile, SNP_12 (Chr22-g. 11721225 A > T) and SNP_13 (Chr22-g. 11721227 A > C) (NC_030829.1) simultaneously mutated at the first and third position of a triplet AAA (lysine, K) in the exon 4 of ACVR2B gene (XM_018066623.1) had estimated genetic effects of HOM1 (0.00) and HOM2 (0.03) larger than HET (−0.12). In conclusion, one SNPs (chr2-g. 28314990 T > G) within the exon 2 of INHA and two SNPs (Chr22-g. 11721225 A > T and Chr22-g. 11721227 A > C) i n the exon 4 of ACVR2B gene were highly recommended as candidate markers of litter size in Dazu black goats. A large-scale association study to assess the impact of these variants on litter size is still necessary.     

玉树马和德保矮马SLC36A1基因多态性研究

 控制马香槟毛色的CH（Champagne gene）基因家族包含4个候选基因（SLC36A1、SLC36A2、SLC36A3、SPARC）,研究发现SLC36A1基因外显子2的突变是造成马香槟毛色的关键位点。为揭示中国马SLC36A1基因遗传多态性,本研究以玉树马和德保矮马共74个样本为研究对象,以马DNA池为模板扩增SLC36A1基因的10个外显子及部分内含子序列并进行测序分析。共发现马SLC36A1基因5个SNPs,分别位于内含子3（g.26699953 A>G, g.26699851 G>C, g.26699850 G>C）,外显子4（g.26699562 G>A）及外显子6（g.26697018 C>T）。利用PCR RFLP方法对74个家马样本进行基因分型,发现外显子6的SNP有基因型CC、CT;外显子4的SNP有基因型GG、GA;内含子3的g.26699850 G>C突变有基因型GG、GC;内含子3的另外2个SNPs（g.26699953 A>G, g.26699851 G>C）通过测序,发现有AA、AG、GG与GG、GC、CC基因型。所有5个马SNPs均为野生型占主要优势。由此界定了马SLC36A1基因有9种单倍型（H1 H9）,其中H5是最主要的单倍型。德保矮马遗传多样度为0.4190,比玉树马（0.2228）高,表明德保矮马香槟毛色遗传多态性比玉树马更丰富。玉树马与德保矮马的平均单倍型多样度为0.3160,表明其香槟毛色遗传多态性相对较低。     

Canine tyrosine hydroxylase (TH) gene and dopamine beta -hydroxylase (DBH) gene: their sequences, genetic polymorphisms, and diversities among five different dog breeds

Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.     

Whole‐genome resequencing to identify candidate genes for the QTL for oleic acid percentage in Japanese Black cattle

In our previous study, we detected a QTL for the oleic acid percentage (C18:1) on BTA9 in Japanese Black cattle through a genome‐wide association study (GWAS). In this study, we performed whole‐genome resequencing on eight animals with higher and lower C18:1 to identify candidate polymorphisms for the QTL. A total of 39,658 polymorphisms were detected in the candidate region, which were narrowed to 1993 polymorphisms within 23 genes based on allele differences between the high and low C18:1 groups. We subsequently selected three candidate genes, that is, CYB5R4, MED23, and VNN1, among the 23 genes based on their function in fatty acid metabolism. In each candidate gene, three SNPs, that is, CYB5R4 c.*349G > T, MED23 c.3700G > A, and VNN1 c.197C > T, were selected as candidate SNPs to verify their effect on C18:1 in a Japanese Black cattle population (n = 889). The statistical analysis showed that these SNPs were significantly associated with C18:1 (p < 0.05), suggesting that they were candidates for the QTL. In conclusion, we successfully narrowed the candidates for the QTL by detecting possible polymorphisms located within the candidate region. It is expected that the responsible polymorphism can be identified by demonstrating their effect on the gene's function.     

Sequencing of canine 5-hydroxytriptamine receptor (5-HTR) 1B, 2A, 2C genes and identification of polymorphisms in the 5-HTR1B gene

Polymorphisms of human genes encoding 5-hydroxytriptamine (serotonin) receptors (5-HTRs) are thought to be associated with psychiatric disorders and behavioral traits. In the present study, we searched for corresponding polymorphisms in the dog and compared allelic frequencies for the canine 5-HTR1B, 5-HTR2A, and 5-HTR2C genes among five canine breeds. The canine genes consisted of the following: 5-HTR1B, 1170 bp; 5-HTR2A, 1413 bp; and 5-HTR2C, 1377 bp. All of these genes were highly homologous with the human genes. We found six single nucleotide polymorphisms (SNPs) in the 5-HTR1B gene (G57A, A157C, G246A, C660G, T955C, and G1146C). Genotyping of the respective SNPs revealed that there were inter-breed variations in the genotypes and allelic frequencies for four out of the six identified SNPs, suggesting that further analyses of the polymorphisms of the 5-HTR1B gene would be useful in order to gain an understanding of the genetic background underlying the diversified behavioral traits among canine species.     

Lack of GNB3 exon 9 polymorphism in primary hypertensive and normotensive dogs

The 5500T allele variant of the C5500T single nucleotide polymorphism in the human G protein beta3 subunit (GNB3) has been reported to be associated with primary hypertension. In this study, the GNB3 gene of primary hypertensive and normotensive dogs was examined for an analogous nucleotide polymorphism associated with hypertension. The genomic GNB3 dna, with 10 exons and nine introns coding for 340 amino acids, is described. PCR product sequencing of the GNB3 exon 9 from 25 dogs (including five hypertensive animals) failed to detect any nucleotide polymorphism. In contrast to human beings, there was no polymorphism at either the analogous nucleotide or in the respective exon. Only the human hypertension-associated thymine was detected, regardless of whether the dogs were hypertensive or normotensive. Furthermore, examinations of 565 dogs of 85 distinct breeds for the presence of the human 5500C nucleotide at the analogous nucleotide side failed to detect a cytosine that is present with high allele frequency in normotensive man. Owing to the lack of allele variance, it is concluded that canine primary hypertension is not associated with a polymorphism at either the respective human hypertension-associated nucleotide site or in the entire exon.