Association between Gene Polymorphisms and SNP-SNP Interactions of the Matrix Metalloproteinase 2 Signaling Pathway and the Risk of Vascular Senescence

Objective This study aimed to explore the association of single nucleotide polymorphisms(SNP)in the matrix metalloproteinase 2(MMP-2)signaling pathway and the risk of vascular senescence(VS).Methods In this cross-sectional study,between May and November 2022,peripheral venous blood of151 VS patients(case group)and 233 volunteers(control group)were collected.Fourteen SNPs were identified in five genes encoding the components of the MMP-2 signaling pathway,assessed through carotid-femoral pulse wave velocity(cf PWV),and analyzed using multivariate logistic regression.The multigene influence on the risk of VS was assessed using multifactor dimensionality reduction(MDR)and generalized multifactor dimensionality regression(GMDR)modeling.Results Within the multivariate logistic regression models,four SNPs were screened to have significant associations with VS:chemokine(C-C motif)ligand 2(CCL2)rs4586,MMP2 rs14070,MMP2rs7201,and MMP2 rs1053605.Carriers of the T/C genotype of MMP2 rs14070 had a 2.17-fold increased risk of developing VS compared with those of the C/C genotype,and those of the T/T genotype had a19.375-fold increased risk.CCL2 rs4586 and MMP-2 rs14070 exhibited the most significant interactions.Conclusion CCL2 rs4586,MMP-2 rs14070,MMP-2 rs7201,and MMP-2 rs1053605 polymorphisms were significantly associated with the risk of VS.

Prevalent false positives of azoospermia factor a （AZFa） microdeletions caused by single-nucleotide polymorphism rs72609647 in the sY84 screening of male infertility

Multiplex polymerase chain reaction （PCR） has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology （EAA） and the European Molecular Genetics Quality Network （EMQN） have recommended the use of sY84 and sY86 markers for the detection of azoospermia factor a （AZFa） microdeletion during DNA testing for male infertility. In this study, a large-scale analysis of AZF microdeletion in a total of 630 Chinese males, including healthy semen donors （n=200）, infertile males with normal sperm count （n=226） and patients with either nonobstructive azoospermia or severe oligozoospermia （n=204）, was performed. A series of nine sequence-tagged site （STS） markers from the AZF region of the Y chromosome was used to detect microdeletions. All primers were designed based on the recommendations of the National Center for Biotechnology Information. An unusually high incidence （73/630, 11.6%） of sY84-absent but sY86-present genotypes was observed in the AZFa microdeletion screening. Sequencing the sY84-flanking region revealed a total of 73 patients with sY84-absent but sY86-present genotypes have a T-to-G transversion at the fifth base from the 5＇ end of the reverse sY84 primer. These prevalent false positives, which were not only observed in infertile men, but also observed in donors, resulted from a single-nucleotide polymorphism （SNP） named rs72609647 in the targeting sequence of the reverse sY84 primer. Our study suggests that a pre-screening of existence of rs72609647 polymorphism can prevent the frequent false positive results of AZFa microdeletions detection in the infertile Chinese males. Given the SNP rs72609647 was recently found in a deep sequencing of a Chinese individual, the current EAA and EMQN standards may need to be scrutinized among different populations to avoid the potential genetic variations in the primer binding sequences.

Single-nucleotide polymorphisms of HLA and Polygonum multiflorum-induced liver injury in the Han Chinese population

BACKGROUND Polygonum multiflorum is one of the leading causes of herb-induced liver injury in China.HLA-B*35:01 is reported to be a potential biomarker of Polygonum multiflorum-induced liver injury(PM-DILI).However,little is known about the relationship between single-nucleotide polymorphisms(SNPs) and PM-DILI.AIM To identify SNPs that indicate susceptibility to PM-DILI METHODS We conducted a systematic study enrolling 382 participants from four independent hospitals,including 73 PM-DILI patients,118 patients with other drug-induced liver injury(other-DILI) and 191 healthy controls.Whole-exome sequencing was performed for 8 PM-DILI patients and 8 healthy controls who were randomly selected from the above subjects.Nineteen SNPs that showed high frequencies in the 8 PM-DILI patients were selected as candidate SNPs and then screened in 65 PM-DILI patients,118 other-DILI patients and 183 healthy controls using the MassARRAY system.HLA-B high-resolution genotyping was performed for the 73 PM-DILI and 118 other-DILI patients.The Han-MHC database was selected as a population control for HLA-B analysis.P <6.25 x 103 after Bolferroni correction was considered significant.RESULTS The frequencies of rslll686806 in the HLA-A gene,rs1055348 in the HLA-B gene,and rs202047044 in the HLA-DRB1 gene were significantly higher in the PM-DILI group than in the control group [27.2% vs 11.6%,P=1.72×105,odds ratio(OR)=3.96,95% confidence interval(Cl):2.21-7.14;42.5% vs 8.6%,P=1.72×10-19 OR=13.62,95% CI:7.16-25.9;22.9% vs 8.1%,P=4.64×106,OR=4.1,95% CI:2.25-7.47].Only rs1055348 showed a significantly higher frequency in the PM-DILI group than in the other-DILI group(42.5% vs 13.6%,P=1.84×10-10,OR=10.06,95% Cl:5.06-20.0),which suggested that it is a specific risk factor for PM-DILI.rs1055348 may become a tag for HLA-B*35:01 with 100% sensitivity and 97.7% specificity in the PM-DILI group and 100% sensitivity and 98.1% specificity in the other-DILI group.Furthermore,HLA-B*35:01 was confirmed to be associated with PM-DILI with a frequency of 41.1% in the PM-DILI group compared with 11.9%(P=4.30×10-11,OR=11.11,95% CI:5.57-22.19) in the other-DILI group and 2.7%(P=6.22×10-166,OR=62.62,95% Cl:35.91-109.20) in the Han-MHC database.CONCLUSION rslll686806,rs1055348,and rs202047044 are associated with PM-DILI,of which,rs1055348 is specific to PM-DILI.As a tag for HLA-B*35:01,rs1055348 may become an alternative predictive biomarker of PM-DILI.

PLASMA RESISTIN LEVELS AND SINGLE-NUCLEOTIDE POLYMORPHISMS IN RESISTIN GENE 5 FLANKING REGION IN PATIENTS WITH STROKE

Objective To analyze the role of resistin in insulin resistance (IR) through investigating the variation of plasma resistin levels and single-nucleotide polymorphisms (SNPs) in resistin gene 5’ flanking region in stroke patients.Methods In 103 atherothrombotic cerebral infarction (ACI) patients, 85 lacunar infarction (LI) patients, 70 intracerebral hemorrhage (ICH) patients, and 86 healthy controls, plasma resistin and insulin levels were measured by ELISA , SNPs in resistin gene 5’ flanking region were detected by PCR and direct DNA sequencing. The subjects’ body height and weight, the body mass index, quantitative insulin sensitivity check index (QUICKI), blood pressure, and the concentration of fasting plasma glucose, triglyceride, total cholesterol, creatinine, low-density lipoprotein, and high-density lipoprotein were also determined. Results QUICKI was significantly lower in the ACI and ICH patients (0.316±0.037 and 0.309±0.032, respectively) than that in the controls (0.342±0.043, P<0.001), while plasma resistin level was significantly higher in the ACI and ICH patients (6.36±3.79 and 7.15±4.27 ng/mL, respectively) than that in the controls (5.28±2.56 ng/mL, P<0.05), but such difference was not observed in the LI patients compared with controls. There was a statistically negative correlation between plasma resistin level with QUICKI (r=-0.228, P<0.001). The distributions of allele and genotype frequencies of resistin gene -420C>G and -537A>C SNPs were not significantly different among the different groups, and those SNPs were not correlated with other clinical and biochemical parameters.Conclusions Plasma resistin is associated with stroke by participating in the development of IR. The SNPs in resistin gene 5’ flanking region has no impact on the plasma resistin level.

Association of Bovine Fatty Acid Desaturase 2 Gene Single-Nucleotide Polymorphisms with Intramuscular Fatty Acid Composition in Japanese Black Steers

Beef from Japanese Black cattle (JBK), is popular in Japan and valued for its highly marbled fat content. In JBK, genes affecting oleic acid content in meat have been studied mainly to lower the fat melting point and improve tenderness;however, there has been no direct correlation demonstrated between beef taste and oleic acid. To investigate genes affecting other fatty acids other than oleic acid, polymorphisms of the fatty acid desaturase 2 (FADS2) gene were genotyped and associations with fatty acid profile in JBK beef were investigated. Amplifications of 5’-flanking regions, 12 exons, and 3’-untranslated regions of the FADS2 gene in three Japanese and five Western cattle breeds via PCR, were amplified, sequenced and SNPs were identified using specific TaqMan genotyping assay. Fatty acid composition of intramuscular adipose tissue of the Trapezius muscle was analyzed in JBK steers. Six of the 15 identified SNPs are novel and have never been registered in any public bovine SNP database. A non-synonymous SNP (rs211580559;C > T;294 Ala > Val) in exon 7 was examined in order to evaluate its association with fatty acid profiles. The data showed that highly significant association existed between rs211580559 and C18:2 (n-6) composition, and accounted for 22.3% of the variation. There were no significant relationships between rs2115-80559 and the other fatty acids. It was concluded that rs211580559 of the FADS2 gene may be a useful selection marker for reducing unfavorable volatiles generated from linoleic acid in JBK beef during the cooking process.

Single Nucleotide Polymorphisms (SNPs) Discovery and Linkage Disequilib-rium (LD) in Forest Trees

With completion of the Populus genome sequencing project and the availability of many expressed sequence tags （ESTs） databases in forest trees, attention is now rapidly shifting towards the study of individual genetic variation in natural populations. The most abundant form of genetic variation in many eukaryotic species is represented by single nucleotide polymorphisms （SNPs）, which can account for heritable inter-individual differences in complex phenotypes. Unlike humans, the linkage disequilibrium （LD） rapidly decays within candidate genes in forest trees. Thus, SNPs-based candidate gene association studies are considered to be a most effective approach to dissect the complex quantitative traits in forest trees. The present study demonstrates that LD mapping can be used to identify alleles associated with quantitative traits and suggests that this new approach could be particularly useful for performing breeding programs in forest trees. In this review, we will describe the fundamentals, patterns of SNPs distribution and frequency, summarize recent advances in SNPs discovery and LD and comment on the application of LD in the dissection of complex quantitative traits in forest tress. We also put forward the outlook for future SNPs-based association analysis of quantitative traits in forest trees.

Single Nucleotide Polymorphisms (SNPs) of URAT1 (rs7932775) and ABCG2 (rs3825016) on Chronic Kidney Disease Patients with Hyperuricemia

Background: More and more chronic kidney disease (CKD) patients are accompanied with hyperuricaemia. As is known, hyperuricaemia is an independent hazard of both cardiovascular diseases (CVD) and chronic kidney diseases. We aim at identifying Single Nucleotide Polymorphism (SNP) difference of hURAT1 (rs7932775) and ABCG2 (rs3825016) on CKD patient with hyperuricemia and/or gout. Methods: All forty-two CKD patients were divided into two groups: hyperuricemia, and control group. 24 hours urine sample and serum were prepared for testing biochemistry parameters. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is used to analyze hURAT1 and ABCG2 single nucleotide polymorphisms in different groups. Results: 17 patients have CT SNP of hURAT1 (rs7932775) and 13 patients have CT SNP of ABCG2 (rs3825016) in hyperuricemia group, while only 5 persons and 6 persons have the same mutations in control group respectively. 7 patients have CT SNP of both hURAT1 (rs7932775) and ABCG2 (rs3825016) in hyperuricemia group, while only 2 persons have the same mutations in control group. CT mutation rates of hURAT1 (rs7932775) and ABCG2 (rs3825016) in hyperuricemia group were 60.7% (17/28) and 50% (13/28) respectively, higher than that of control group (35.7% (5/14) and 42.8% (6/14)). What is more, Double SNP mutations in both hURAT1 (rs7932775) and ABCG2 (rs3825016) in hyperuricemia group were 25% (7/28), higher than that of control group (14.2%, 2/14). Conclusion: There are higher mutation rates of CT SNP in hURAT1 (rs7932775) and/or ABCG2 (rs3825016) in hyperuricemia group. We can conclude that hyperuricemia is a high risk factor in progress of CKD, which is necessary to take measures of decreasing serum uric acid to delay CKD progress.

Digital Barcode Development for Single Nuclotide Polymorphism (SNP) Identification of Suzhong Swine Individuals

Suzhong swine is a hybrid breed derived from Taihu sows and Landraee boars. To identify Suzhong swine individuals and trace the source of pork products, single nucleotide polymorphisms （SNPs） identification of Suzhong swine individuals was studied. A total of 29 pairs of primers were designed and sev- en pairs of primers were used for identification of Suzhong swine individuals. The products amplified by seven pairs of primers could be directly sequenced, with clean sequencing map background and no ambiguity in sequence read. Totally 52 SNPs loci were amplified by seven pairs of primers, and 41 SNPs loci were reserved for identification of Suzhong swine individuals through correlation analysis and heterezygosity filtration （ H ≥0.1 ）. Meantime, the digital barcodes for SNP identification of 96 individuals of Suzhong swine derived from seven boars and 12 sows were developed, which well distinguished 96 individuals of Suzhong swine. Theoretically, 41SNPs amplified by seven pairs of primers could be used for identification of 5.0 × 10^6 pig individuals. Therefore, digital barcode devel- opment method for SNP identification of Suzbong swine individuals can be used for individual identification of Suzhong swine in scale pig farm and meat product traceability.

Development of organelle single nucleotide polymorphism (SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis (Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitochondria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga Pyropia yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria)inheritance in P.yezoensis,the wild type RZ(maternal strain)was crossed with the red mutant HT(paternal strain)and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT)were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP)loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.

Maternal TMPRSS6 Gene Polymorphism rs855791SNP in Women with Preeclampsia

Introduction: Preeclampsia can lead to several maternal and perinatal adverse effects. There are few published data on the association between transmembrane serine protease 6 (TMPRSS6) gene polymorphism and preeclampsia. Objective: To assess the association between TMPRSS6 gene polymorphism rs855791SNP in women with preeclampsia compared with healthy pregnant women. Method: A case-control study (60 women in each arm) was conducted at Saad Abuaela Maternity Hospital in Khartoum, Sudan. Sociodemographic and clinical data were gathered through a questionnaire. The participant was genotype for TMPRSS6 gene rs855791SNP using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP). The results were confirmed by DNA sequencing. Result: There was no significant difference in the median of age, parity, and body mass index. The distribution of the genotypes and alleles of TMPRSS6 rs855791 was consistent with the HWE. The overall TMPRSS6 rs855791 polymorphism was not significantly associated with preeclampsia. However, the proportion of heterozygotes (TC) was considerably higher in the women with preeclampsia (46.7%) than in the control group (23.3%) (p = 0.001;OR = 2.71;95% CI = 1.21 - 6.07). The proportion of homozygotes (TT) and T alleles was not significantly different between women with preeclampsia and the control group. Conclusion: The overall TMPRSS6 rs855791 polymorphism was not significantly associated with preeclampsia and healthy control.

基于多重PCR靶向测序的烟草1.8K SNP育种液相芯片的开发与应用

本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。

Association of-238G/A and -857C/T Polymorphisms of Tumor Necrosis Factor-Alpha Gene Promoter Region With Outcomes of Hepatitis B Virus Infection

Objectives To determine whether -238G/A and -857C/T polymorphisms of tumor necrosis factor-alpha （TNF-o0 gene promoter were associated with outcomes of hepatitis B virus infection. Methods A total of 246 HBV self-limited infected subjects and 443 chronic hepatitis B （HB） patients were recruited in this case-control study. TNF-α-238G/A and -857C/T gene promoter polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP）. Results The frequency of TNF-α-238 GG （90.7%） in chronic HB group was significantly lower than that （95.1%） in self-limited group （P=0.041）. The frequency of TNF-oc-857 CC （79.7%） in chronic HB patients was significantly higher than that （70.9%） in self-limited infected subjects （P=0.021）. Multiple logistic regression analysis revealed that both TNF-oc-238GA and -857CC were independently associated with chronic HB. Conclusions TNF-α promoter variants are likely to play a substantial role in influencing the outcomes of HBV infection.

Associations of Gonadotropin-Releasing Hormone Receptor (GnRHR) and Neuropeptide Y(NPY) Genes’Polymorphisms with Egg-Laying Traits in Wenchang Chicken

Single nucleotide polymorphisms （SNP） of chicken gonadotropin-releasing hormone receptor （GnRHR） and neuropeptide Y （NPY） were selected to identify the genotypes of Wenchang （Chinese indigenous breed） chicken with restricton fragment length polymorphisms. The associations of the SNPs with the total egg production （NE）, average days of continual laying （ADCL）, and number of double-yolked eggs （DYE） traits were analyzed. The frequency of restriction enzyme A/a alleles in the population was for GnRHR 0.69 （Bpu1102 Ⅰ A） and 0.31 （Bpu1102 Ⅰ a） and for NPY 0.46 （Dra Ⅰ B） and 0.54 （Dra Ⅰ b）. Trait data from a total of 120 hens, which were purebred introduced from Hainan Province, China from one generation were recorded. Two significant effects of genes＇ marker were found： for GnRHR and number of eggs （dominant; t= 2.67, df= 116） and NPY and number of eggs （additive; t= 1.97, df= 116）. The current research supports the effects of GnRHR and NPY genes on egg-laying traits of chickens.

A case-control study about the association between vascular endothelial growth inhibitor gene polymorphisms and breast cancer risk in female patients in Northeast China

Objective： The inhibition of the neovascularization in tumors is a potential therapeutic target of cancer. Vascular endothelial growth inhibitor （VEGI） is a member of the TNF superfamily which has the ability to suppress the formation of new vessels in tumors. In order to study the association between VEGI gene polymorphisms and breast cancer risk, a case-control study was conducted in Chinese Han women in Northeast China. Methods： Our study involved 708 female breast cancer patients and 685 healthy volunteers. Four SNPs of VEGI gene were analyzed through the polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method. The association between VEGI gene polymorphisms and breast cancer risk was analyzed in our study. The relation between VEGI gene variants and clinical features of breast cancer including lymph node （LN） metastasis, esl^ogen receptor （ER）, progestrogen receptor （PR）, tumor protein 53 （1953）, human epidermal growth factor receptor 2 （Her-2） and triple negative （ER-/PR-/Her-2-） status was analyzed as well. Results： We found that the CT genotype and T allele of rs6478106 were more frequent in patients than in controls. There was also a statistical difference in the distribution of Crs6478106Grs4263839 haplotype between patients and controls. In addition, SNP rs6478106 and rs4979462 were related with the Her-2 status. Conclusions： Our results suggest that VEGI gene variants may be related to the breast cancer risk and the clinical features of breast cancer in Chinese Han women in Northeast China.

Expression analysis,single nucleotide polymorphisms within SIRT4 and SIRT7 genes and their association with body size and meat quality traits in Qinchuan cattle

Silent information regulator 2 （Sir2） proteins, or sirtuins, are nicotine adenine dinucleotide （NAD）-dependent deacetylases that connect metabolism with longevity in lower organisms. In mammals, there are seven Sir2 homologs, namely, silent information regulators （SIRT1-7）. SIRT4 and SIRT7 genes play a crucial role in regulating lipid metabolism, cellular growth and metabolism. This suggests that they are potential candidate genes for affecting body size and meat quality traits in animals. Hence, this study aimed to detect genetic variations of both SIRT4 and SIRT7 bovine genes in Qinchuan cattle, and to evaluate the effect of these variations on economically important body size and meat quality traits. Expression analysis using quantitative real-time PCR （qPCR） indicated that SIRT4 and SIRT7 were broadly expressed in all thirteen studied tissues. The expression of SIRT4 was higher in liver, muscle, and in subcutaneous fat tissue. In the case of SIRT7, the expression was higher in lung, abomasum, and subcutaneous fat. Using DNAsequencing, a total of three single nucleotide polymorphisms （SNPs） were identified within SIRT4 and SIRT7 genes in 468 Qinchuan cattle. These included one novel SNP within 3＇ untranslated regions （UTR） of SIRT4 （SNP1： g. 13915A〉G） and two novel synonymous substitutions in SIRT7 （SNP2： g.3587C〉T and SNP3： g.3793T〉C）. Statistical analyses indicated that all three SNPs could significantly influence some body size and meat quality traits in Qinchuan cattle. These novel findings will provide a background for application of bovine SIRT4 and SIRT7 genes in the selection program of Chinese cattle.

LBP and CD14 polymorphisms correlate with increased colorectal carcinoma risk in Han Chinese

AIM: To explore the associations of polymorphisms of lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) with the colorectal carcinoma (CRC) risk in Han Chinese. METHODS: Polymorphisms of LBP (rs1739654, rs223 2596, rs2232618), CD14 (rs77083413, rs4914), TLR-4 (rs5030719), IL-6 (rs13306435) and TNF-α (rs35131721) were genotyped in 479 cases of sporadic colorectal carcinoma and 486 healthy controls of Han Chinese in a case-control study. Single-nucleotide polymorphisms (SNPs) between cases and controls were analyzed by unconditional logistic regression. RESULTS: GA and GG genotypes of LBP rs2232596 were associated with a significantly increased risk ofCRC [odds ratio (OR) = 1.51, 95% confidence interval (CI) 1.15-1.99, P = 0.003; OR = 2.49, 95% CI 1.16-5.38, P = 0.016, respectively]. A similar association was also observed for the CG genotype of CD14 rs4914 (OR= 1.69, 95% CI 1.20-2.36, P = 0.002). In addition, a combination of polymorphisms in LBP rs2232596 and CD14 rs4914 led to a 3.4-fold increased risk of CRC (OR = 3.44, 95% CI 1.94-6.10, P = 0.000). CONCLUSION: This study highlights the LBP rs2232596 and CD14 rs4914 polymorphisms as biomarkers for elevated CRC susceptibility in the Chinese Han population.

Association between PPARG genetic polymorphisms and ischemic stroke risk in a northern Chinese Han population: a case-control study

Two common polymorphisms of the peroxisome proliferator-activated receptor gamma(PPARG) gene, rs1801282 and rs3856806, may be important candidate gene loci affecting the susceptibility to ischemic stroke. This case-control study sought to identify the relationship between these two single-nucleotide polymorphisms and ischemic stroke risk in a northern Chinese Han population. A total of 910 ischemic stroke participants were recruited from the First Hospital of China Medical University, Shenyang, China as a case group, of whom 895 completed the study. The 883 healthy controls were recruited from the Health Check Center of the First Hospital of China Medical University, Shenyang, China. All participants or family members provided informed consent. The study protocol was approved by the Ethics Committee of the First Hospital of China Medical University, China on February 20, 2012(approval No. 2012-38-1). The protocol was registered with the Chinese Clinical Trial Registry(registration number: ChiCTR-COC-17013559). Plasma genomic DNA was extracted from all participants and analyzed for rs1801282 and rs3856806 single nucleotide polymorphisms using a SNaPshot Multiplex sequencing assay. Odds ratios(ORs) and 95% confidence intervals(CIs) were calculated using unconditional logistic regression to estimate the association between ischemic stroke and a particular genotype. Results demonstrated that the G allele frequency of the PPARG gene rs1801282 locus was significantly higher in the case group than in the control group(P < 0.001). Individuals carrying the G allele had a 1.844 fold increased risk of ischemic stroke(OR = 1.844, 95% CI: 1.286–2.645, P < 0.001). Individuals carrying the rs3856806 T allele had a 1.366 fold increased risk of ischemic stroke(OR = 1.366, 95% CI: 1.077–1.733, P = 0.010). The distribution frequencies of the PPARG gene haplotypes rs1801282-rs3856806 in the control and case groups were determined. The frequency of distribution in the G-T haplotype case group was significantly higher than that in the control group. The risk of ischemic stroke increased to 2.953 times in individuals carrying the G-T haplotype(OR = 2.953, 95% CI: 2.082–4.190, P < 0.001). The rs1801282 G allele and rs3856806 T allele had a multiplicative interaction(OR = 3.404, 95% CI: 1.631–7.102, P < 0.001) and additive interaction(RERI = 41.705, 95% CI: 14.586–68.824, AP = 0.860;95% CI: 0.779–0.940;S = 8.170, 95% CI: 3.772–17.697) on ischemic stroke risk, showing a synergistic effect. Of all ischemic stroke cases, 86% were attributed to the interaction of the G allele of rs1801282 and the T allele of rs3856806. The effect of the PPARG rs1801282 G allele on ischemic stroke risk was enhanced in the presence of the rs3856806 T allele(OR = 8.001 vs. 1.844). The effect of the rs3856806 T allele on ischemic stroke risk was also enhanced in the presence of the rs1801282 G allele(OR = 2.546 vs. 1.366). Our results confirmed that the G allele of the PPARG gene rs1801282 locus and the T allele of the rs3856806 locus may be independent risk factors for ischemic stroke in the Han population of northern China, with a synergistic effect between the two alleles.

Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR（AS-PCR） approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3（UCP3）gene. AS-PCR is a competitive PCR method which is based on positioning the 3＇ base of a PCR primer to match one single nucleotide polymorphism（SNP） allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3＇ end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T（rs1800849） polymorphisrn of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism（RFLP）, the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.

Relationship between nerve injury-induced protein gene 2 polymorphism and stroke in Chinese Han population

The aim of present study was to investigate the relationship between nerve injury-induced protein 2 （NINJ2） gene polymorphism and stroke in Chinese Han population. Fifty-two patients with large-artery atherosclerosis （LAA） infarction, 85 patients with small-artery occlusion lacunar （SAO） infarction, 50 patients with intracerebral hemorrhage （ICH） and 66 controls were included. Genotypes and alleles frequencies of the two single nucleotide polymorphisms （SNPs） of NINJ2 among different groups were analyzed and compared. In regard to rs12425791, the frequencies of the AG and AA＋AG genotypes of the LAA and SAO groups were significantly higher than those in the control group; the frequency of the A allele of the SAO group was significantly higher than that of the control group. In regard to rs11833579, there were not any significant differences between the case and the control groups. The SNP rs12425791 is significantly associated with ischemic stroke, and the A allele increases the susceptibility to stroke. The SNP rs11833579 is not significantly associated with stroke.

A single nucleotide polymorphism in SPA TA17 may be a genetic risk factor for Japanese patients with meiotic arrest

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, 10 novel genes involved in human spermatogenesis were identified by microarray analysis of human testicular tissue. One of these is spermatogenesis-associated 17 （SPATA17）. To investigate whether defects in the SPATA17 gene are associated with azoospermia due to meiotic arrest, a mutational analysis was conducted, in which the SPATA17 coding regions of 18 Japanese patients with this condition were sequenced. A statistical analysis was carried out that included 18 patients with meiotic arrest, 20 patients with Sertoli-cell-only syndrome （SCOS） and 96 healthy control men. No mutations were found in SPA TA17. However, three coding single nucleotide polymorphisms （cSNPs： SNP 1-SNP3） were detected in the patients with meiotic arrest. No significant differences in the genotype or allele frequencies of SNP1 and SNP2 were found between patients with meiotic arrest and the others. However, the frequency of the SNP3 allele was significantly elevated in the meiotic arrest group （P 〈 0.05）. This study suggests that SPATA17 may play a critical role in human spermatogenesis, especially in meiosis.