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.     

Single nucleotide polymorphisms in bovine <Emphasis Type="Italic">PRL</Emphasis> gene and their associations with milk production traits in Chinese Holsteins

Prolactin (PRL) plays a crucial role in the initiation and maintenance of lactation in mammals. In this study, seven PCR fragments representing most important functional domains of PRL gene were screened for single nucleotide polymorphisms (SNPs) in Chinese Holsteins by single-strand conformation polymorphisms and amplicons sequencing, and their genetic effects on milk production traits were evaluated. A total of four SNPs, including two in the promoter (−1043A>G and −402A>G), one in intron 1 (+2723C>T) and one in exon 4 (+8398G>A) were identified in PRL gene. Statistical results showed significant associations between the promoter genotypes and the milk performance traits in Chinese Holsteins. Cows with genotype P1-GG showed higher milk yields (P < 0.01), while cows with genotype P1-AA showed higher fat contents (P < 0.01). Haplotype analysis of two SNPs in promoter region revealed that the Hap(AG) was significantly associated with increased milk yields and Hap(AA) was associated with increased fat contents (P < 0.01). This is the second study reporting SNPs in the 5′-regulatory region of PRL gene, which interfere with milk production traits.     

Fatty acid composition of beef is associated with exonic nucleotide variants of the gene encoding FASN

Genetic associations of fatty acid composition with exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase (FASN) were examined using 513 Korean cattle. All five individual SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A were associated with a variety of fatty acid compositions and further with marbling score (P < 0.05). Their genotypes of CC, TT, AA, TT, and GG were associated with increased monounsaturated fatty acids and with decreased saturated fatty acids (P < 0.05). The genotypes at all the SNPs also increased marbling score (P < 0.05). Further genetic associations with fatty acid composition suggested that homozygous genotype with the haplotype of ATG at g.15532, g.16907, and g.17924 in a linkage disequilibrium block increased monounsaturated fatty acids and marbling score (P < 0.05). We concluded that the five exonic SNPs of g.12870, g.13126, g.15532, g.16907, and g.17924 in the FASN gene could change fatty acid contents. Their genotypes of CC, TT, AA, TT, and GG and haplotype of ATG at g.15532, g.16907, and g.17924 were recommended for genetic improvement of beef quality.     

Contribution of SELP and PSGL-1 genotypes and haplotypes to the presence of coronary heart disease in Tunisians

P-selectin (SELP) and its counter-receptor, P-selectin glycoprotein ligand-1 (PSGL-1), play key role in the transient attachment of leukocytes to endothelial cells predisposing to coronary heart disease (CHD). In the current report, 293 angiographically proven CHD patients and 327 age, gender, and race-matched controls were included. Our aim was to evaluate the contribution to CHD of the following SNPs: C-2123G, G-1969A and T715P in SELP, Met62Ile and the VNTR variants in PSGL-1 gene in a North African population from Tunisia. While there were no significant differences in the distribution of SELP or PSGL-1 alleles or genotypes between patients and controls, a trend for a significant association of the C-2123G genotypes distribution with incident CHD was observed (P = 0.06). Assuming an additive model of transmission, the risk was 74% higher among subjects carrying the GG genotypes in comparison to those carrying the CC genotype (OR = 1.74 [1.01–2.98], P = 0.04) and 80% higher in the recessive model (OR = 1.80 [1.08–3.01], P = 0.02). Haplotype analysis did not identify any specific SELP or PSGL-1 haplotypes to be associated with CHD. The present study demonstrated no evidence of association between individual SELP or PSGL-1 SNPs or haplotypes with incident CHD. However, this study replicates absence of association of the mostly studied SNP, T715P, previously reported in individuals with African origin.     

Identification of four SNPs and association analysis with meat quality traits in the porcine <Emphasis Type="Italic">Pitx2c</Emphasis> gene

The association of the porcine Pitx2c gene with meat quality traits was investigated in the present study. A total of eight single nucleotide polymorphisms (SNPs) were found. Allele frequencies of four SNPs were further detected in four commercial breeds and eight Chinese indigenous breeds. Single SNP and meat quality associations were analyzed in a Yorkshire×Meishan F₂ population. The SNPs c.474C>T (P<0.01) and c.636C>T (P<0.05) showed a significant association with meat color (MCV1). The SNPs c.*37G>A and c.*47G>A were significantly associated with drip loss rate (DLR), water holding capacity (WHC) and meat color value (MCV1) consistently (P<0.05). Linkage disequilibrium (LD) analysis revealed that the adjacent SNPs were in LD. Two major haplotypes were identified, and association analysis between haplotype combinations and meat quality indicated that the presence of two copies of haplotype 1 -CCGG- may improve meat quality.     

Association of the variants in the BUD13‐ZNF259 genes and the risk of hyperlipidaemia

The single nucleotide polymorphisms (SNPs) in the BUD13 homolog (BUD13) and zinc finger protein 259 (ZNF259) genes have been associated with one or more serum lipid traits in the European populations. However, little is known about such association in the Chinese populations. Our objectives were to determine the association of the BUD13/ZNF259 SNPs and their haplotypes with hypercholesterolaemia (HCH)/hypertriglyceridaemia (HTG) and to identify the possible gene–gene interactions among these SNPs. Genotyping of 6 SNPs was performed in 634 hyperlipidaemic and 547 normolipidaemic participants. The ZNF259 rs2075290, ZNF259 rs964184 and BUD13 rs10790162 SNPs were significantly associated with serum lipid levels in both HCH and non‐HCH populations (P < 0.008–0.001). On single locus analysis, only BUD13 rs10790162 was associated with HCH (OR: 2.23, 95% CI: 1.05, 4.75, P = 0.015). The G‐G‐A‐A‐C‐C haplotype, carrying rs964184‐G‐allele, was associated with increased risk of HCH (OR: 1.35, 95% CI: 1.10, 1.66, P = 0.005) and HTG (OR: 1.75, 95% CI: 1.39, 2.21, P = 0.000). The A‐C‐G‐G‐C‐C and A‐C‐A‐G‐T‐C haplotypes, carrying rs964184‐C‐allele, were associated with reduced risk of HCH (OR: 0.77, 95% CI: 0.61, 0.99, P = 0.039 and OR: 0.66, 95% CI: 0.47, 0.94, P = 0.021 respectively). On multifactor dimensionality reduction analyses, the two‐ to three‐locus models showed a significant association with HCH and HTG (P < 0.01–0.001). The BUD13/ZNF259 SNPs, which were significant in the European populations, are also replicable in the Southern Chinese population. Moreover, inter‐locus interactions may exist among these SNPs. However, further functional studies are required to clarify how these SNPs and genes actually affect the serum lipid levels.     

IL-10 promoter single nucleotide polymorphisms are significantly associated with resistance to leprosy

The minor haplotype −3575A/-2849G/-2763C in IL-10 promoter has been defined as a marker of disease resistance to leprosy and its severity in Brazilian population. Our investigation of six single-nucleotide polymorphisms (SNPs) in IL-10 promoter in 282 Indian leprosy patients and 266 healthy controls by direct PCR sequencing, however, showed that the extended haplotype: −3575T/-2849G/-2763C/-1082A/-819C/-592C was associated with resistance to leprosy per se and to the development of severe form of leprosy, using either a binomial (controls vs cases, P=0.01, OR=0.58, CI=0.37–0.89) or ordinal (controls vs paucibacillary vs multibacillary, P=0.004) model. Whereas, IL-10 haplotype −3575T/-2849G/-2763C/-1082A/-819T/-592A was associated with the risk of development of severe form of leprosy (P=0.0002) in contrast to the minor risk haplotype −3575T/-2849A/-2763C in the Brazilian population. The role of IL-10 promoter SNPs in Brazilian and Indian population strongly suggests the involvement of IL-10 locus in the outcome of leprosy.     

Identification of three novel SNPs and association with carcass traits in porcine <Emphasis Type="Italic">TNNI1</Emphasis> and <Emphasis Type="Italic">TNNI2</Emphasis>

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR–RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F₂ pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax–waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.     

Polymorphism of chicken myocyte-specific enhancer-binding factor 2A gene and its association with chicken carcass traits

Myocyte-specific enhancer-binding factor 2A (MEF2A) gene is a member of the myocyte-specific enhancer-binding factor 2 (MEF2) protein family which involved in vertebrate skeletal muscle development and differentiation. The aim of the current study is to investigate the potential associations between MEF2A gene SNPs (single nucleotide polymorphisms) and the carcass traits in 471 chicken samples from four populations. Three new SNPs (T46023C, A72626G, and T89232G) were detected in the chicken MEF2A gene. The T46023C genotypes were associated with live body weight (BW), carcass weight (CW), eviscerated weight, semi-eviscerated weight (SEW), and leg muscle weight (LMW) (P < 0.05); the A72626G genotypes were associated with BW, CW, LMW (P < 0.01) and breast muscle weight (BMW), leg muscle percentage (LMP) (P < 0.05); whereas the T89232G genotypes were associated with carcass percentage (CP) and semi-eviscerated percentage (SEP) (P < 0.05). The haplotypes constructed on the three SNPs were associated with BW, CW, LMW (P < 0.01), SEW, BMW, CP (P < 0.05). Significantly and suggestive dominant effects of diplotype H1H2 were observed for BW, CW, SEW, BMW and CP, whereas diplotype H5H5 had a negative effect on BW, CW, SEW, BMW and LMW. Our results suggest that the MEF2A gene may be a potential marker affecting the muscle trait of chickens.     

Association of <Emphasis Type="Italic">Cytotoxic T Lymphocyte-associated antigen-4</Emphasis> gene haplotype with the susceptibility to gastric cancer

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was widely accepted as a pivotal molecule in downregulating T-cell mediated immune responses. In this study we investigated the polymorphisms which would impact the CTLA-4 gene expression and function to assess the association with the risk of gastric cancer. 205 gastric cancer patients and 262 healthy controls were included in the case-control study. PCR and restriction fragment length polymorphism (RFLP) methods were performed to identify the +49A/G and promoter −1661A/G polymorphisms. The promoter −1772T/C polymorphism was detected by PCR amplification refractory mutation system (ARMS) technique. A significant difference was observed between case and control groups. The frequency of +49A/G polymorphism AG and −1661A/G polymorphism GG genotype were significantly higher in patients than in controls (OR = 2.15, OR = 1.88, respectively). No significant difference was found in the allelic frequency of −1772T/C polymorphism between cases and controls (P = 0.478). By the haplotype analysis, logistic regression showed the frequency of haplotype A (GAT) and D (AGT) in the case group revealed significant difference compared with in control group(OR = 2.00, P < 0.001; OR = 1.62, P = 0.043, respectively). Our findings implied the genetic variations within CTLA-4 gene would be a critical risk factor to the susceptibility of gastric cancer.     

<Emphasis Type="Italic">Interleukin13</Emphasis> haplotypes and susceptibility of Iranian women to breast cancer

Interleukin-13 (IL-13) is a TH2 cytokine with direct and indirect immunoregulatory functions on cancer cells. The cytokine has been reported to have some polymorphic variations at the gene level associated with some immune related diseases including asthma and allergy. In the present study, association of three IL13 gene polymorphisms at positions −1512 A/C and −1055 C/T in the promoter and +2044 G/A in exon-4 was investigated in Iranian women with breast cancer and healthy controls. Genotyping of IL13 gene polymorphisms were performed by PCR–RFLP methods. Serum level of IL-13 was assessed by ELISA. Haplotypes were constructed from genotypic data using Arlequin 3.1 software package. Haplotype analysis revealed higher frequency of a three-locus haplotype, ACA (−1512A/−1055C/+2044A), in normal women than breast cancer patients (P < 0.025). Haplotype CCA, from the other hand, was observed with more frequency among patients than controls (P < 0.03). No statistically significant differences were found in the frequency of genotypes and alleles between patients and control group. No association was observed between investigated genotypes and other prognostic factors including tumor type, lymph node involvement and tumor size. IL-13 serum level was undetectable in both patients and control subjects. Despite observing no association between breast cancer and the single SNPs, results of this investigation suggest that the presence of CCA haplotype of IL13 gene may be associated with susceptibility of Iranian women to breast cancer.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

Novel SNPs of the mtDNA ND5 Gene and Their Associations with Several Growth Traits in the Nanyang Cattle Breed

Polymorphism of the mtDNA ND5 gene was detected by PCR-SSCP and DNA sequencing methods in 714 individuals from six Chinese cattle breeds. The results showed that at the P2 locus, two kinds of haplotypes, named A and B, with three SNPs (T12900C, A12923T, C12924T) were detected. The frequencies of haplotype A in six breeds were 0.041–0.167. Polymorphism of the ND5 gene was shown to be associated with growth in the Nanyang breed. Individuals with haplotype B had greater hucklebone width than those with haplotype A when 6 months old (P < 0.01), as well as better body height, body length, body weight, and average daily gain at 6 months (P < 0.05). Therefore, haplotype B of the ND5 gene was likely to have a positive effect on growth traits at 6 months in the Nanyang breed.     

The IGF1 pathway genes and their association with age of puberty in cattle

Insulin‐like growth factor I (somatomedin C) (IGF1) influences gonadotrophin‐releasing hormone (GnRH) neurons during puberty, and GnRH release guides pubertal development. Therefore, genes of the IGF1 pathway are biological candidates for the identification of single‐nucleotide polymorphisms (SNPs) affecting age of puberty. In a genome‐wide association study, genotyped heifers were Tropical Composite (TCOMP, n = 866) or Brahman (BRAH, n = 843), with observation of age at first corpus luteum defining puberty. We examined SNPs in or near genes of the IGF1 pathway and report seven genes associated with age at puberty in cattle: IGF1R, IGFBP2, IGFBP4, PERK (HUGO symbol EIF2AK3), PIK3R1, GSK3B and IRS1. SNPs in the IGF1 receptor (IGF1R) showed the most promising associations: two SNPs were associated with puberty in TCOMP (P < 0.05) and one in BRAH (P = 0.00009). This last SNP explained 2% of the genetic variation (R² = 2.04%) for age of puberty in BRAH. Hence, IGF1R was examined further. Additional SNPs were genotyped, and haplotypes were analysed. To test more SNPs in this gene, four new SNPs from dbSNP were selected and genotyped. Single SNP and haploytpe analysis revealed associations with age of puberty in both breeds. There were two haplotypes of 12 IGF1R SNPs associated with puberty in BRAH (P < 0.05) and one in TCOMP (P < 0.05). One haplotype of two SNPs was associated (P < 0.01) with puberty in BRAH, but not in TCOMP. In conclusion, the IGF1 pathway appeared more relevant for age of puberty in Brahman cattle, and IGF1R showed higher significance when compared with other genes from the pathway.     

Haplotype combinations of <Emphasis Type="Italic">AdPLA</Emphasis> gene polymorphisms associate with growth traits in Chinese cattle

Ablation of AdPLA in mice could prevent obesity, which implicated AdPLA was a new candidate gene for growth traits. In this study, we screened the polymorphisms of AdPLA gene and revealed three novel SNPs (NC_007330.4:g.43638506 C>T, 43658457 T>C and 43661404 T>C) by DNA sequencing and forced PCR-RFLP methods in 1340 individuals from six breeds. A preliminary single SNP-traits association analysis demonstrated that CC genotype was significantly higher than TT genotype at two growth traits in P6-EcoRII locus (P < 0.05). Moreover, the P8-FbaI locus had a significant association with some growth traits, individuals with TT genotype were higher than CC genotype (P < 0.05). A further analysis confirmed that these two SNPs were in linkage disequilibrium, and haplotype H2, both advantage allelic in P6-EcoRII and P8-FbaI loci, was better than the others for bovine growth traits, which was consistent with single SNP-traits association results. Therefore, this study not only proved that these SNPs were in association with bovine growth traits, but also contributed to evaluate them as genetic markers in bovine genetics and breeding and had potential application in breeding programs.