Cloning, sequencing and identification of single nucleotide polymorphisms of partial sequence on the porcine CACNA1S gene

CACNA1S gene encodes the α1 subunit of the calcium channel. The mutation of CACNA1S gene can cause hypokalemic periodic paralysis (HypoKPP) and maliglant hyperthermia synarome (MHS) in hu-man beings. Current research on CACNA1S was mainly in human being and model animal, but rarely in livestock and poultry. In this study, Yorkshire pigs (23), Pietrain pigs (30), Jinhua pigs (115) and the second generation (126) of crossbred of Jinhua and Pietrain were used. Primers were designed ac-cording to the sequence of human CACNA1S gene and PCR was carried out using pig genome DNA. PCR products were sequenced and compared with that of human, and then single nucleotide poly-morphisms (SNPs) were investigated by PCR-SSCP, while PCR-RFLP tests were performed to validate the mutations. Results indicated: (1) the 5211 bp DNA fragments of porcine CACNA1S gene were ac-quired (GenBank accession number: DQ767693 ) and the identity of the exon region was 82.6% be-tween human and pig; (2) fifty-seven mutations were found within the cloned sequences, among which 24 were in exon region; (3) the results of PCR-RFLP were in accordance with that of PCR-SSCP. Ac-cording to the EST of porcine CACNA1S gene published in GenBank (Bx914582, Bx666997), 8 of the 11 SNPs identified in the present study were consistent with the base difference between two EST frag-ments.     

Association of single nucleotide polymorphism in melatonin receptor 1A gene with egg production traits in Yangzhou geese

In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5? regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34‐week egg‐laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real‐time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.     

鸡视网膜母细胞瘤基因1(RB1)多态性与体重性状的相关性

为探讨鸡视网膜母细胞瘤基因1(Retinoblastoma1,RB1)多态性对体重性状的影响,文章以东北农业大学高、低脂双向选择品系肉鸡为实验材料,采用MALDI-TOF-MS、PCR-RFLP方法进行基因多态性检测和个体基因型分析,共获得27个SNP位点的基因型数据。采用滑动窗口法构建单倍型,进而利用单位点和单倍型分别与鸡体重性状进行关联分析。结合单位点和单倍型分析结果,确定了RB1基因上4个显著影响1周龄体重的SNP位点,2个显著影响1、3周龄体重的SNP位点。研究结果表明RB1基因是影响鸡早期体重性状的重要候选基因。     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Association of single nucleotide polymorphisms in the endothelial differentiation sphingolipid G-protein-coupled receptor 1 gene with marbling in Japanese Black beef cattle

Marbling defined by the amount and distribution of intramuscular fat, so-called Shimofuri , is an economically important trait of beef cattle in Japan. The endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, involved in blood vessel formation, has been previously shown to be expressed at different levels in musculus longissimus muscle between low-marbled and high-marbled steer groups. It is located within the genomic region of a quantitative trait locus for marbling, and thus was considered as a positionally functional candidate for the gene responsible for marbling. In this study, two single nucleotide polymorphisms (SNPs) in the 5' untranslated region (UTR) and the 3' UTR of EDG1 , referred to as c. - 312A>G and c.*446G>A , respectively, were detected between the two steer groups. The two SNPs were associated with the predicted breeding value for beef marbling standard number by analyses using a population of Japanese Black beef cattle. The effect of genotypes at each of the SNPs on the predicted breeding value for subcutaneous fat thickness was not statistically significant ( P  >   0.05). Reporter gene assays revealed no significant differences in gene expression between alleles at each of the SNPs. These findings suggest that EDG1 SNPs, although they may not be regarded as a causal mutation, may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle.     

Association between TL1A gene polymorphisms and systemic lupus erythematosus in a Chinese Han population

Our previous studies showed elevated tumor necrosis factor-like ligand 1 aberrance (TL1A) expression in systemic lupus erythematosus (SLE). However, TL1A polymorphisms with SLE susceptibility remain to be elucidated. In addition, we made meta-analysis to evaluate the relationship of TL1A polymorphisms and autoimmune diseases owing to inconsistent results. The present research was carried out by 404 SLE, 150 primary Sjogren's syndrome (pSS) patients, and 574 healthy individuals. Three TL1A polymorphisms (rs3810936, rs6478109, rs7848647) were genotyped using TaqMan genotyping assay. Then, the meta-analysis was performed by collecting the present case-control study and previously published research. Results showed that genotypes of rs3810936, rs7848647 were different between SLE patients and healthy controls, whereas no significant association was observed in the three polymorphisms and pSS patients. Genotypes distribution of rs6478109, rs7848647 were strongly related to lupus nephritis within SLE (p = 0.004, p = 0.011), respectively. Moreover, combined meta-analysis consisted of ten comparative research involving 4,305 patients and 5,600 controls. An association between autoimmune diseases and rs6478109 polymorphism was found. Our findings indicate that gene polymorphisms (rs3810936, rs7848647) of TL1A might correlate with lupus.     

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Association of SULT1A1 and UGT1A1 polymorphisms with breast cancer risk and phenotypes in Russian women

Estrogens are critical for breast cancer initiation and development. Sulfotransferase 1A1 (SULT1A1) and UDP-glucuronosyltransferase 1A1 (UGT1A1) conjugate and inactivate both estrogens and their metabolites, thus preventing estrogen-mediated mitosis and mutagenesis. SULT1A1 and UGT1A1 are both polymorphic, and different alleles encode functionally different allozymes. We hypothesize that low-activity alleles SULT1A1*2 and UGT1A1*28 are associated with higher risk for breast cancer and more severe breast tumor phenotypes. We performed a case-control study, which included 119 women of Russian ancestry with breast cancer and 121 age-matched Russian female controls. We used PCR followed by pyrosequencing to determine the SULT1A1 and UGT1A1 genotypes. Allele UGT1A1*28 was present at a higher frequency than the wild-type UGT1A1*1 allele in breast cancer patients as compared to controls (P = 0.002, OR = 1.79, CI 1.23–2.63). Consistently, the frequency of genotypes that contain allele UGT1A1*28 in the homozygous or the heterozygous state was greater in breast cancer patients as compared with the frequency of the wild-type UGT1A1*1/*1 genotype (P = 0.003, OR = 4.00, CI 1.49–11.11 and P = 0.014, OR = 2.04, CI 1.14–3.57, respectively). Individuals carrying allele UGT1A1*28 in the homo-or heterozygous state had larger breast tumors (>2 cm) as compared to the group with high-activity genotypes (P = 0.011, IR = 3.44, CI 1.42–8.36). No association was observed between any of the SULT1A1 genotypes and breast cancer risk or phenotypes. Our data suggest that UGT1A1, but not SULT1A1, genotypes are important for breast cancer risk and phenotype in Russian women. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 2, pp. 263–270. The article was translated by the authors.     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

Genetic association of single nucleotide polymorphisms in dystrobrevin binding protein 1 gene with schizophrenia in a Malaysian population

Dystrobrevin binding protein 1 (DTNBP1) gene is pivotal in regulating the glutamatergic system. Genetic variants of the DTNBP1 affect cognition and thus may be particularly relevant to schizophrenia. We therefore evaluated the association of six single nucleotide polymorphisms (SNPs) with schizophrenia in a Malaysian population (171 cases; 171 controls). Associations between these six SNPs and schizophrenia were tested in two stages. Association signals with p < 0.05 and minor allele frequency > 0.05 in stage 1 were followed by genotyping the SNPs in a replication phase (stage 2). Genotyping was performed with sequenced specific primer (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). In our sample, we found significant associations between rs2619522 (allele p = 0.002, OR = 1.902, 95%CI = 1.266 – 2.859; genotype p = 0.002) and rs2619528 (allele p = 0.008, OR = 1.606, 95%CI = 1.130 – 2.281; genotype p = 6.18 × 10⁻⁵) and schizophrenia. Given that these two SNPs may be associated with the pathophysiology of schizophrenia, further studies on the other DTNBP1 variants are warranted.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR = 5.58, 95% CI = 1.32-23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

湖北汉族人群载脂蛋白A5遗传多态性分析

采用聚合酶链反应-限制性片断长度多态性(polymerase chain reaction restriction-fragment length polymorphism, PCR-RFLP)对257例湖北健康汉族人群APOA5 -1131T>C及56C>G基因多态性进行鉴定。结果发现: 湖北汉族人群中ApoA5 -1131T>C存在TT、TC、CC基因型, 3种基因型的频率分别为50.9%、32.9%及16.2%; 56C>G位点存在CC、CG基因型, 257名研究对象中, G等位基因分布频率小于5%; 各基因型频率和等位基因频率在不同种族和地域间分布存在显著性差异。结论: 湖北汉族人群中ApoA5基因-1131T>C位点存在单核苷酸多态性(single nucleotide polymorphism, SNP), 56C>G在该人群中应视为一个突变位点而不是多态性位点     

Relationships among calpastatin single nucleotide polymorphisms,calpastatin expression and tenderness in pork longissimus1

Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphisms (SNP) in calpastatin were identified and used to genotype a population (n = 1042) of Duroc–Landrace–Yorkshire swine for association with longissimus lumborum slice shear force (SSF) measured at days 7 and 14 postmortem. Three genetic markers residing in the calpastatin gene were significantly associated with SSF (P < 0.0005). Haplotypes constructed from markers in the calpastatin gene were significantly associated with SSF (F‐ratio = 3.93; P‐value = 0.002). The levels of normalized mRNA expression of calpastatin in the longissimus lumborum of 162 animals also were evaluated by real‐time RT‐PCR and were associated with the genotype of the most significant marker for SSF (P < 0.02). This evidence suggests that the causative variation alters expression of calpastatin, thus affecting tenderness. In summary, these data provide evidence of several significant, publicly available SNP markers associated with SSF that may be useful to the swine industry for marker assisted selection of animals that have more tender meat.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Abstract

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR?=?5.58, 95% CI?=?1.32–23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

Association of cytotoxic T lymphocyte-associated antigen 4 gene single nucleotide polymorphism with type 1 diabetes mellitus in Madurai population of Southern India

Type 1 diabetes mellitus formerly called juvenile diabetes, is an organ specific T-cell mediated autoimmune disease characterized by the progressive loss of function of the insulin producing beta-cells of the islets of Langerhans. Cytotoxic T lymphocyte-associated antigen 4 gene (CTLA-4) has been proposed as a candidate gene for conferring susceptibility to autoimmunity. Association of CTLA-4 gene polymorphism is well established in autoimmune endocrinopathies across world population. The present study was conducted to investigate the association of CTLA-4 exon 1 49A/G polymorphism with TIDM in Madurai, a city in Southern India. Fifty three clinically proven T1DM patients and 53 control subjects with no history of autoimmune disease were recruited for the study. Genomic DNA was extracted from peripheral blood. CTLA-4 exon 1 49 A/G polymorphism was assessed using PCR-RFLP methods. Our findings revealed a significant association of CTLA-4 exon 1 49 A/G polymorphism with T1DM in Madurai population.     

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.     

Paraoxonase 1 gene polymorphisms, paraoxonase/arylesterase activities and oxidized low-density lipoprotein levels in patients with migraine

The vascular endothelial dysfunction has been implicated in the pathogenesis of migraine. Oxidized low‐density lipoprotein (ox‐LDL) may impair endothelial function. Paraoxonase‐1 (PON‐1) prevents oxidative modification of LDL cholesterol (LDL‐C). So we investigated serum PON‐1 and arylesterase (ARE) activities, PON‐1 55 L/M and 192Q/R polymorphisms and the serum lipid profile in patients with migraine. Biochemical parameters and PON‐1 polymorphism analyses were assessed in 104 patients with migraine and 86 healthy subjects. Ox‐LDL was detected by ELISA, and polymorphisms were determined using PCR–restriction fragment length polymorphism analysis. Patients with migraine had lower PON‐1 and ARE activities (p < 0·001, for both) and higher ox‐LDL and LDL‐C levels (p < 0·001, for both) and ox‐LDL: LDL‐C ratio (p < 0·005) than the controls. The genotype distribution and the allele frequencies for PON‐1 55 L/M and 192Q/R polymorphisms were not different among the study populations. The results of our current study indicate that migrainous patients have decreased serum PON‐1 and ARE activities and increased serum ox‐LDL levels, which may have a clinical importance in the treatment of migraine. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of public single nucleotide polymorphisms in commercial pig populations

More than 5500 pig single nucleotide polymorphisms (SNPs) were recently identified and deposited in the public domain. To test the usefulness of these public SNPs, 109 SNPs were analysed for polymorphism within six commercial pig populations. A functional polymerase chain reaction (PCR) assay was obtained for 103 SNPs and it was possible to validate c. 59% by PCR-restriction fragment length polymorphism. Furthermore, polymorphism was found using a relatively limited number of genomic DNA samples, indicating that these polymorphisms are segregating at a useful frequency in these populations. The high percentage of validated markers demonstrates the utility of these public pig SNPs to identify loci responsible for economically important traits in commercial pig populations.     

Association between polymorphisms in AXIN1 gene and atrial septal defect

Abstract

Context: AXIN1 is a central component of Wnt signalling pathway which is essential for embryonic development.

Objective: To investigate whether polymorphisms of AXIN1 contribute to ASD susceptibility.

Materials and methods: Three tag SNPs (rs12921862, rs370681 and rs1805105) in AXIN1 were genotyped in 208 ASD patients and 302 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a Chinese population.

Results: Significantly increased ASD risk was observed to be associated with the A allele of rs12921862 (p?<?0.0001, OR?=?3.096, 95% CI?=?2.037–4.717). Increased ASD risk was observed to be associated with rs370681 in a codominant (p?=?0.043, OR?=?1.52, 95% CI?=?1.04–2.22) and overdominant model (p?=?0.016, OR?=?1.57, 95% CI?=?1.08–2.27).

Conclusion: rs12921862 and rs370681 may contribute to ASD susceptibility.