Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumoniae and bacterial LPS challenge

Toll-like receptors (TLRs) play a crucial role in innate immunity, serving as pattern-recognition receptors and the first barrier in host defense against microbial infections. Genetic variations of TLR2 and TLR4 are closely associated with a variety of infectious diseases, particularly lung diseases. In this study, we detected six and four single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR2 and TLR4 genes, respectively. Only SNP 1027C>A of TLR4 was shown to be markedly biased in Western and Oriental pig populations. Hence, the susceptibility of pigs with different genotype at position 1027C>A to Mycoplasma hyopneumoniae (Mhp) infection was investigated, and changes to the expression of TLR2, TLR4, TNF-α and IL-1β were monitored. The results showed that there was no significant difference in susceptibility to Mhp infection between AA and CC individuals despite expression levels for all detected genes of the challenge groups being significantly higher than the corresponding control groups. Furthermore, porcine alveolar macrophages of different genotype were collected and stimulated by lipopolysaccharide. We found that the expression of TLR2, TLR4, TNF-α and IL-1β genes were enhanced to different levels by lipopolysaccharide stimulation. TLR2 and TLR4 gene expressions and their rates of increase of 1027CC pigs were significantly higher than for 1027AC pigs (P?<?0.01), while TNF-α and IL-1β expressions were significantly lower than for 1027AC pigs (P?<?0.01). We predict that allele C at position 1027 of the TLR4 gene contributes to the pig's immune response to gram-negative bacterial infections.     

Effects of DGAT1 gene on meat and carcass fatness quality in Chinese commercial cattle

This study was designed to investigate the candidate single nucleotide polymorphisms (SNPs) in the exon’s region of bovine diacylglycerol O-acyltransferase (DGAT1) gene using bioinformatics and experimental methods. A total of 17 SNPs were screened from public data resources and DNA sequencing. Three SNPs (c.572A>G, c.1241C>T and c.1416T>G) of these candidate SNPs were genotyped by created restriction site-polymerase chain reaction (CRS-PCR) methods. The gene-specific SNP markers and their effects on meat and carcass fatness quality traits were evaluated in Chinese commercial cattle. The c.572A>G and c.1416T>G significantly effected on backfat thickness, longissimus muscle area, marbling score, fat color and Warner-Bratzler shear force. No significant association was detected between the c.1241C>T and measured traits. Results from this study suggested that the SNP markers may be effective for the marker-assisted selection of meat and carcass fatness quality traits, and added new evidence that DGAT1 gene is an important candidate gene for the improvement of meat and carcass fatness quality in beef cattle industry.     

Proopiomelanocortin gene polymorphisms and its association with meat quality traits by ultrasound measurement in Chinese cattle

Ultrasound technology was used to measure live animal meat traits instead of true carcass meat traits for beef production and cattle breeding by an increasing number of institutions. In this study, we analyzed the association between genetic polymorphisms of proopiomelanocortin (POMC) and ultrasound measurement traits in Chinese cattle. Using direct DNA sequencing in 322 individuals of 7 different cattle subpopulation, 7 SNPs were identified for genotyping within 790 bp region of intron 2 and exon 3 of POMC. 6586 T>G in intron 2 and 6769 C>T and 7216 C>T in exon 3 were significantly associated with ultrasound backfat thickness (UBF) (P < 0.05) and ultrasound loin muscle area (ULMA) (P < 0.01) in the total population; 6694 C>T, 6706 T>C, 6796 C>T and 6810 C>T in exon 3 were significantly associated with ULMA (P < 0.0001) in the total population. These results clearly suggest that these SNPs of POMC be benefit for selection of individuals with good quality meat in Chinese cattle breeding program. Following validation in other populations and breeds, these markers could be incorporated into breeding programs to increase the rate of improvement in carcass and meat quality traits.     

Allelic variants of immune response genes in children with infectious complications during the treatment of acute leukemia

Infectious complications that arise during the treatment of children with acute leukemia with chemotherapeutic agents at high doses represent a serious problem in oncohematology. To find genetic conditions that may lead to the development of postchemotherapy infections, the genomes of 12 children with acute leukemia who had severe infectious complications during therapy were examined. At the same time, the coding regions of 17 genes involved in the regulation of the immune response were determined by massive parallel sequencing. The analysis revealed 39 nonsynonymous SNPs that lead to amino acid substitutions, including the following informative genetic markers: PTPN22 c.1858C>T (rs2476601), TLR4 c.896A>G (rs4986790) and TLR4 c.1196C>T (rs4986791), IL7R c.197T>C (rs1494555) and IL7R c.412G>A (rs1494558). The results of massive parallel sequencing were validated by Sanger sequencing. The identification of genetic markers associated with the predisposition to infectious complications may allow one to assess the individual risk of the severe infection development in children with acute leukemia during the treatment with chemotherapeutic agents and to begin the development of personalized approaches to anticancer therapy.     

Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Elevated level of DNA damage was observed in patients with depression. Furthermore, single nucleotide polymorphisms (SNPs) of base excision repair (BER) genes may modulate the risk of this disease. Therefore, the aim of this study was to delineate the association between DNA damage, DNA repair, the presence of polymorphic variants of BER genes, and occurrence of depression. The study was conducted on peripheral blood mononuclear cells of 43 patients diagnosed with depression and 59 controls without mental disorders. Comet assay was used to assess endogenous (oxidative) DNA damage and efficiency of DNA damage repair (DRE). TaqMan probes were employed to genotype 12 SNPs of BER genes. Endogenous DNA damage was higher in the patients than in the controls, but none of the SNPs affected its levels. DRE was significantly higher in the controls and was modulated by BER SNPs, particularly by c.977C>G–hOGG1, c.972G>C–MUTYH, c.2285T>C–PARP1, c.580C>T–XRCC1, c.1196A>G–XRCC1, c.444T>G–APEX1, c.-468T>G–APEX1, or c.*50C>T–LIG3. Our study suggests that both oxidative stress and disorders in DNA damage repair mechanisms contribute to elevated levels of DNA lesions observed in depression. Lower DRE can be partly attributed to the presence of specific SNP variants.     

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.     

Identification of novel SNPs in the Sarda breed goats POU1F1 gene and their association with milk productive performance

The aim of the study was to detect polymorphism in the POU1F1 gene in Sarda breed goat, as well as to establish if SNPs could be associated with milk productive traits. The research was conducted on 129 Sarda breed goats from 4 to 5 years old, multiparous, lactating and in their third to fifth lactation. We report nine exonic and seven non-coding regions SNPs within the Sarda goat POU1F1 gene, namely, Ex 1 61 G>C; Ex 1 108 G>A; Ex 3 C>T; Ex 3 92 C>T; Ex 4 110 A>G; Ex 5 34 G>A resulting in Arg213Lys change; IVS4 641 G>A, IVS4 643 A>C, IVS4 659 G>A, IVS4 677 A>C, IVS4 G699Del, IVS4 709 C>G, Ex 6 17 G>A resulting in Arg228Ser change, Ex 6 58 G>T, Ex 6 172 T>C, 3′UTR 110 T>C. A statistically significant association was found between genotype TT, in position 17 of the exon 6 (3.1 % of frequency), and increased milk yield (P < 0.01) while genotype GT (25.6 % of frequency) was associated with a higher fat content. Genotype TT in position 58 of the exon 6 (3.9 % of frequency) was found to be associated with a higher fat (P < 0.01) and protein content (P < 0.05). Twenty-eight haplotypes were detected, but no significant association between the haplotypes and the milk production traits have been found. Our data, as well as providing new SNPs extending the POU1F1 gene characterization, evidence a relationship between polymorphism and milk production traits in Sarda goat breed.     

Single-nucleotide polymorphisms g.151435C>T and g.173057T>C in PRLR gene regulated by bta-miR-302a are associated with litter size in goats

Single-nucleotide polymorphisms (SNPs) located at microRNA-binding sites (miR-SNPs) can affect the expression of genes. This study aimed to identify the miR-SNPs associated with litter size. Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect SNPs in the caprine prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction, and polymerase chain reaction-restriction fragment length polymorphism. Three novel SNPs (g.151435C>T, g.151454A>G, and g.173057T>C) were identified in the caprine PRLR gene. Statistical results indicated that the g.151435C>T and g.173057T>C SNPs were significantly associated with litter size in Guanzhong and Boer goat breeds. Further analysis revealed that combinative genotype C6 (TTAACC) was better than the others for litter size in both goat breeds. Furthermore, the PRLR g.173057T>C polymorphism was predicted to regulate the binding activity of bta-miR-302a. Luciferase reporter gene assay confirmed that 173057C to T substitution disrupted the binding site for bta-miR-302a, resulting in the reduced levels of luciferase. Taken together, these findings suggested that bta-miR-302a can influence the expression of PRLR protein by binding with 3′untranslated region, resulting in that the g.173057T>C SNP had significant effects on litter size.     

Genetic variants in BMP8B gene are associated with growth traits in Chinese native cattle

As a signaling molecule, bone morphogenetic protein 8B (BMP8B) plays an essential role in bone metabolism and is able to regulate thermogenesis and energy balance, which suggests that BMP8B gene may be a new candidate for growth traits. Here, to characterize the effects of BMP8B gene on growth traits, we first used three Chinese indigenous cattle breeds (n = 845) to detect single nucleotide polymorphisms (SNPs). Five novel SNPs of BMP8B gene (g.− 242C>T, g.2164C>T, g.2639T>C, g.2900C>G and g.10817C>T) were identified by DNA pool sequencing and forced PCR–RFLP. And then we associated the five SNPs with four growth traits (body weight, body length, heart girth, and hucklebone width). Results from association analysis showed that the SNPs 1, 2, and 3 affected growth trait(s) markedly (P < 0.05). Further, 6 combined haplotypes were constructed to guarantee the reliability of analysis results. There were also significant differences in body length, heart girth and body weight between the 6 combined haplotypes (P < 0.05), but not in hucklebone width (P > 0.05). Collectively, our results suggest a modulatory role of BMP8B gene in cattle growth and development, and 3 SNPs could be used as molecular markers in early marker assisted selection (MAS) in beef cattle breeding program.     

Detection of SNPs in the TBC1D1 gene and their association with carcass traits in chicken

TBC1D1 plays an important role in numerous fundamental physiological processes including muscle metabolism, regulation of whole body energy homeostasis and lipid metabolism. The objective of the present study was to identify single nucleotide polymorphisms (SNPs) in chicken TBC1D1 using 128 Erlang mountainous chickens and to determine if these SNPs are associated with carcass traits. The approach consisted of sequencing TBC1D1 using a panel of DNA from different individuals, revealing twenty-two SNPs. Among these SNPs, two polymorphisms (g.69307744C>T and g.69307608T>G) of block 1, four polymorphisms (g.69322320C>T, g.69322314G>A, g.69317290A>G and g.69317276T>C) of block 2 and four polymorphisms of block 3 (g.69349746G>A, g.69349736C>G, g.69349727C>T and g.69349694C>T) exhibited a high degree of linkage disequilibrium in all test populations. An association analysis was performed between the twenty-two SNPs and seven performance traits. SNPs g.69307744C>T, g.69340192G>A and g.69355665T>C were demonstrated to have a strong effect on liveweight (BW), carcass weight (CW), semi-eviscerated weight (SEW) and eviscerated weight (EW) and g.69340070C>T polymorphism was related to BW, SEW and BMW in chicken populations. However, for the other SNPs, there were no significant correlations between different genotypes and carcass traits. Meanwhile, haplotype CT–TG of block 1 and combined genotype AG–TT–AC–CT of block 3 were significantly associated with BW, CW, SEW and EW. Overall, our results provide evidence that polymorphisms in TBC1D1 are associated with carcass traits and would be a useful candidate gene in selection programs for improving carcass traits.     

Polymorphism distribution of RYR1, PRKAG3, HFABP,MYF-5 and MC4R genes in crossbred pigs

This study was designed to screen the crossbred pigs for SNPs in five candidate genes, associated with pork quality traits and to differentiate their genotypes by PCR–RFLP. The results indicated that genotypes of crossbred pigs were NN (90%) and Nn (10%) for RYR1; RR (83%) and QR (17%) for PRKAG3; HH (98%), Hh (1%) and hh (1%) for HFABP; DD (99%) and CD (1%) for MYF-5; and AG (57%), GG (26%) and AA (17%) for MC4R SNPs, respectively. Allelic frequencies for five SNPs {RYR1 (1843C>T), PRKAG3 (c.599G>A), HFABP (c.1322C>T), MYF-5 (c.1205A>C) and MC4R (c.1426A>G)} were 0.95 and 0.05 (N/n), 0.08 and 0.92 (Q/R), 0.99 and 0.01 (H/h), 0.00 and 1.00 (C/D) and 0.45 and 0.55 (A/G), respectively. The effect of RYR1 (1843C>T) SNP was significant on pH₄₅ (P?<?0.05), pH₂₄ (P?<?0.05) and protein % (P?<?0.05). The PRKAG3 (c.599G>A) and MC4R (c.1426A>G) SNP had significant association with dressing percentages. The results revealed that RYR1, PRKAG3 and MC4R SNPs may be used in marker associated selection for pork quality traits in crossbred pigs.     

Polymorphisms in 5��-flanking regions of genes encoding adiponectin, leptin, and resistin are not associated with obesity of Polish children and adolescents

Genes encoding adipokines are important functional candidates for development of obesity. In this study we screened for polymorphism 5??-flanking regions of the adiponectin (ADIPOQ), leptin (LEP) and resistin (RETN) genes in a cohort of Polish obese children and adolescents (n = 243) and a control group of non-obese adults (n = 100). Altogether 13 SNPs (single nucleotide polymorphisms) and 1 InDel (insertion/deletion polymorphism) were found. Among them five polymorphisms, localized in the LEP gene, turned out to be novel, but their distribution was insufficient for association studies. We found no consistent evidence for association between obesity and the SNPs demonstrating minor allele frequency (MAF) above 0.2 (ADIPOQ: ?11377C>G, LEP: ?2548C>T, 19A>G, RETN: ?1300G>A, ?1258C>T, ?420C>G). Comparison of polymorphisms distribution in patients and control group suggested association with ADIPOQ ?11377C>G (Pearson test P = 2.76 × 10^?11), however, we did not observe any effect of this polymorphism on BMI or relative BMI (RBMI) within obese patients (P = 0.41). We conclude that the tested SNPs are not useful markers of childhood and adolescence obesity in Polish population.     

多重等位基因特异性扩增—— 微流控芯片电泳法同时测定多个单核苷酸多态性位点

文章以微流控芯片电泳为检测平台, 建立了一种基于DNA适配器连接介导的多重等位基因特异性扩增同时测定多个单核苷酸多态性(SNP)位点的方法。以白细胞介素1β(IL1B)基因中的7个SNP位点(794C>T、1274C>T、2143T>C、2766T>del、3298G>A、5200G>A和5277C>T)为检测对象, 通过PCR预扩增得一段含该7个待测SNP位点的长片段; 用限制性内切酶MboⅠ将其消化成短片段, 再与DNA适配器(adapter)相连; 以连接产物为模板, 在两管中分别用7条等位基因特异性引物和一条公用引物进行7重等位基因特异性扩增; 最后用微流控芯片电泳法分离等位基因特异性扩增产物, 根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。采用本法成功测定了48名健康中国人的IL1B基因上的7个SNP位点, 与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)和测序法测定结果完全一致。本法结果准确, 可用于同时测定多个SNP位点; 以微流控芯片电泳作为检测平台, 分析速度快, 样品需要量少; 借助于自制筛分凝胶和重复使用芯片, 使得SNP分析成本大大降低。     

A haplotype in the CCR5 gene promoter was associated with the susceptibility to HIV-1 infection in a northern Chinese population

It has been reported that single nucleotide polymorphisms (SNPs) in the promoter of the CCR5 gene are associated with the risk for HIV-1 infection and AIDS progression. Using resequencing, we performed a systematic survey of 78 HIV-1 seropositive individuals and 70 population-matched healthy control individuals from northern China to investigate SNPs of the CCR5 gene promoter and evaluated their effects on HIV-1 infection and the progression of AIDS. Linkage disequilibrium (LD) plots and haplotypes were generated using Haploview software. The association analyses were statistically compared using the Chi-square test with SPSS13.0 software for Windows. Seven SNPs (58755A>G, 58791C>T, 58934G>T, 59029A>G, 59353C>T, 59402A>G and 59653C>T) in the region of the CCR5 gene promoter were evaluated in this study. Among the seven SNPs, the minor allele frequencies of 58755G and 58791T were less than 2%. The differences in frequencies of the other five SNPs were not significant between case and control cohorts (P > 0.05). In the case cohort, the association between these SNPs and clinical features (CD4⁺ T-lymphocyte counts and clinical categories) was not significant (P > 0.05); however, there was a significant association between the haplotype GGTAC and susceptibility to HIV-1 infection (P < 0.05), which is not consistent with other reports studied in different populations. The results suggest that the haplotype GGTAC may have a role in the process of HIV-1 infection in the northern Chinese population.     

Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses

The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26 T > C, g.156 T > C, g.587A > G, g.598C > T, g.1485C > T, g.2115A > G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A > G and g.598C > T residing in the 5′UTR region were novel SNPs identified by this study. The g.2115A > G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.     

Association of IL10 Polymorphisms and Leprosy: A Meta-Analysis

Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal IL10 polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11–1.34) and P-value = 3x10^–5 confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups (OR = 1.2, CI = 1.10–1.31, P-value = 2x10^–5). Also, meta-analysis confirmed association of -592 A allele and leprosy outcome (OR = 1.24, CI = 1.03–1.50, P-value = 0.02). In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r² = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other IL10 polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.     

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.