A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Influence of host genetic polymorphisms involved in immune response and their role in the development of Chikungunya disease: a review

Chikungunya virus (CHIKV) is transmitted by the bite of infected mosquitoes and can cause significant pathogenicity in humans. Moreover, its importance has increased in the Americas since 2013. The primary vectors for viral delivery are the mosquito species Aedes aegypti and Aedes albopictus. Several factors, including host genetic variations and immune response against CHIKV, influence the outcomes of Chikungunya disease. This work aimed to gather information about different single nucleotide polymorphisms (SNPs) in genes that influence the host immune response during an infection by CHIKV. The viral characteristics, disease epidemiology, clinical manifestations, and immune response against CHIKV are also addressed. The main immune molecules related to this arboviral disease elucidated in this review are TLR3/7/8, DC-SIGN, HLA-DRB1/HLA-DQB1, TNF, IL1RN, OAS2/3, and CRP. Advances in knowledge about the genetic basis of the immune response during CHIKV infection are essential for expanding the understanding of disease pathophysiology, providing new genetic markers for prognosis, and identifying molecular targets for the development of new drug treatments.

Effect of MTTP -493G/T, I128T, Q95H and Q244E polymorphisms on hepatic steatosis in patients with chronic hepatitis

Abstract Background: Chronic hepatitis C is characterized by a progressive deterioration of liver function and is involved in metabolic complications, such as hepatic steatosis. Objective: The aim of this study was to investigate the role of host and viral characteristics associated with -493G/T (rs1800591), I128T (rs3816873), Q95H (rs61733139), and Q244E (rs17599091) Single Nucleotide Polymorphisms (SNPs) in the Microsomal Triglyceride Transfer Protein (MTTP) gene on hepatic steatosis in chronic hepatitis C. Methods: SNPs were genotyped by PCR-RFLP and analyzed in combination with host and viral characteristics by multiple logistic regression in different genetic models of inheritance. Results: The authors analyzed 236 patients with chronic hepatitis C, and 53% had hepatic steatosis. The mutated allele frequencies were > 5%, and the genotypes were in Hardy-Weinberg equilibrium (p ≥ 0.05). It was observed that patients with HCV genotype 3 infection (OR = 2.74, 95% CI 1.24‒6.06, p = 0.013), female sex (OR = 2.28, 95% CI 1.21‒4.28, p = 0.011) and moderate- and high-intensity liver inflammatory activity (A2-A3) (OR = 3.61, 95% CI 1.86‒7.01, p < 0.001) alone exhibited a higher risk of steatosis. The results of multiple logistic regression analysis for interaction showed that for the -493G/T SNP, when the GT/TT genotype (dominant model) and the GT genotype (codominant model) were each combined with HCV genotype 3 infection, an 11.51-fold (95% CI 2.08‒63.59, p = 0.005) and a 15.69-fold (95% CI 2.46‒99.85, p = 0.004) increased risk of steatosis, respectively, was observed. For the I128T SNP, when both the IT/TT genotype (dominant model) and the IT genotype (codominant model) were combined with HCV genotype 3 infection, an 8.51-fold (95% CI 1.59‒45.54, p = 0.012) and an 8.40 fold (95% CI 1.51‒46.91, p = 0.015) increased risk of steatosis, respectively, was observed. Conclusion: The present study showed that the viral genotype combined with the -493G/T and I128T SNPs in the MTTP gene influences hepatic steatosis.

Application of a new GC rich region sequence-specific polymerase chain reaction (SS-PCR) method for detection of C729T SNP / 西安交通大学学报(医学版)

Objective: To develop a sequence-specific polymerase chain reaction (SS-PCR) method for detection of single nucleotide polymorphism (SNP) in GC rich region and analyze the C729T SNP in the exon 3 of estrogen receptor α gene of endometrial cancer (EC). Methods: We selected 22 EC and 42 controls. A new GC rich region SS-PCR was developed. The two internal specific primers, identical to the two single strands of double strand DNA, were designed, and the 3' end of the primer coincided with the SNP locus. The PCR extension was controlled by the 3' end primer and the SNP was determined according to the bands of the extension product. The specificity of the extension reaction was increased by introducing a mismatched base at the third position upstream in the 3' end of the SNP specific primer. This method was used to confirm C729T genotypes in the exon 3 SNP, followed by direct sequencing. Results: The results showed that the SS-PCR was appropriate for genotyping C729T SNP of exon 3 in human ERα gene. Conclusion: A GC rich region SS-PCR method developed can be successfully applied in C729T SNP determination in the exon 3 of estrogen receptor α gene of endometrial cancer.

Epidemiology Study on P53 (Rs1614984) C>T Mutation in Cigarette Smokers

ABSTRACT Epidemiology data have established that smoking is a prime threat for the cancers, largely lung cancer. Single-nucleotide polymorphisms (SNPs),P53 SNPs have been found to be associated with the predisposition of different cancers. Their decreased expression is reported in breast and lung cancer patients. p53 (rs1614984) had been reported to be linked with the SNPs found associated with breast cancer. The primary aim of this study to determine the association of p53 variant rs1614984 with the cigarette smokers and smoking related cancers in smokers. Among the smokers, 38% were found with CC genotype, 55% were heterozygous CT and 7% were TT, respectively. The homozygous TT genotype was seen in lower percentage of smokers (7%) when compared to non-smokers (8%) whereas; Significant difference was not observed when encompassed by CC, CT and TT genotypes (χ2 = 4.892, p=0.087). However, CC vs CT genotype showed a significant difference between smokers and non-smokers (p=0.031, OR 1.447 (1.035-2.025) and the dominant model CC vs CT+TT was also significantly different among smoker and non-smokers (p=0.047, OR 1.39 (1.004-1.924). Furthermore, smokers are at the risk of developing variety of diseases including lung cancer. Our finding suggests a higher percentage of heterozygous CT genotype in smokers when compared to non-smokers. Therefore, this finding gives a clue that the transition mutation of C>T (rs1614984) may leads to the lung diseases including cancer in smokers. However, there will be a need of more extensive and elaborated study to set down the aspect of p53(rs1614984) C>T in lung cancer among smokers.

Association of BCL11A genetic variant (rs11886868) with severity in β-thalassaemia major & sickle cell anaemia.

Background & objectives: The amount of foetal haemoglobin that persists in adulthood affects the clinical severity of haemoglobinopathies including β-thalassaemia major and sickle cell anaemia (SCA). The present study was undertaken to analyse β-thalassaemia as well as SCA patients for the single nucleotide polymorphism (SNP), rs11886868 (T/C) in BCL11A gene and to evaluate the association between this polymorphism and severity of β-thalassaemia major and SCA. Methods: A total of 620 samples (420 β-thalassaemia major and 200 SCA cases) were analysed before blood transfusion using basic screening tests like complete blood analysis and osmotic fragility and further confirmed by high performance liquid chromatography (HPLC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot blot techniques. All patients were transfusion dependent. Patients with β-thalassaemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. β-thalassaemia as well as SCA patients were analysed for the SNP, rs11886868 (T/C) in BCL11A gene and association between this polymorphism and severity of β-thalassaemia major as well as SCA was evaluated. Results: There was a significant difference in genotypic and allelic frequencies of BCL11A gene polymorphism between mild and moderate and mild and severe cases in both the groups. A significant (P<0.001) difference was observed in the mean HbF levels between the three genotypes in different severity groups. HbF levels were found to be high in CC genotype bearing individuals followed by TC and TT in β-thalassaemia major as well as SCA. Interpretation & conclusions: This study confirms that the T/C variant (rs11886868) of the BCL11A gene causing downregulation of BCL11A gene expression in adult erythroid precursors results in the induction of HbF and ameliorates the severity of β-thalassaemia as well as SCA.

Association of single nucleotide polymorphisms of ABCB1, OPRM1 and COMT with pain perception in cancer patients / 华中科技大学学报（医学）（英德文版）

Pain perception is influenced by multiple factors. The single nucleotide polymorphisms (SNPs) of some genes were found associated with pain perception. This study aimed to examine the association of the genotypes of ABCB1 C3435T, OPRM1 A118G and COMT V108/158M (valine 108/158 methionine) with pain perception in cancer patients. We genotyped 146 cancer pain patients and 139 cancer patients without pain for ABCB1 C3435T (rs1045642), OPRM1 A118G (rs1799971) and COMT V108/158M (rs4680) by the fluorescent dye-terminator cycle sequencing method, and compared the genotype distribution between groups with different pain intensities by chi-square test and pain scores between groups with different genotypes by non-parametric test. The results showed that in these cancer patients, the frequency of variant T allele of ABCB1 C3435T was 40.5%; that of G allele of OPRM1 A118G was 38.5% and that of A allele of COMT V108/158M was 23.3%. No significant difference in the genotype distribution of ABCB1 C3435T (rs1045642) and OPRM1 A118G (rs1799971) was observed between cancer pain group and control group (P=0.364 and 0.578); however, significant difference occurred in the genotype distribution of COMT V108/158M (rs4680) between the two groups (P=0.001). And the difference could not be explained by any other confounding factors. Moreover, we found that the genotypes of COMT V108/158M and ABCB1 C3435T were associated with the intensities of pain in cancer patients. In conclusion, our results indicate that the SNPs of COMT V108/158M and ABCB1 C3435T significantly influence the pain perception in Chinese cancer patients.

Association of Single Nucleotide Polymorphisms in the MD-2 Gene Promoter Region With Der p 2 Allergy

PURPOSE: Sensitization to house dust mite (Dermatophagoides pteronyssinus) is a considerable risk factor for the progression of allergic disease. The group 2 allergen from Dermatophagoides pteronyssinus, Der p 2, is considered a major one in patients with specific immunoglobulin E (IgE) to Der p 2. Der p 2 has structural homology with myeloid differentiation 2 (MD-2), which is involved in the lipopolysaccharide-binding component of the Toll-like receptor 4 signaling pathway and the development of inflammation. The aim of this study was to examine the genetic association of single nucleotide polymorphisms (SNPs) in the promoter region of MD-2 with Der p 2-sensitive allergy. METHODS: We investigated associations between cohort's characteristics, including 280 allergic and 80 healthy subjects by examining total IgE, eosinophils, D. pteronyssinus-specific IgE, Der p 2-specific IgE, the number of IgE-producing B cells induced by Der p 2, and the odds ratio of allergic symptoms. RESULTS: Based on the 1,000 genome project data, the minor allele frequencies of the rs1809441 and rs1809442 are 0.467 and 0.474, respectively. However, the correlation of linkage disequilibrium (LD) between these 2 SNPs is D'=1, the genotype frequencies of the 2 MD-2 (LY96) SNPs (rs1809441 and rs1809442) that are located nearby were significantly different between allergic and health subjects: the TT genotype of rs1809441 and the GG genotype of rs1809442 were more frequent in allergic subjects than in healthy subjects (16.1% vs 2.5% in both genotypes). The allergic patients with these genotypes exhibited significantly higher levels of D. pteronyssinus-specific IgE and Der p 2-specific Ig E, and a larger number of Der p 2-activated B cells. In addition, these 2 SNPs in the MD-2 promoter region were significantly associated with the prevalence of nasal, skin, and asthmatic allergic symptoms. CONCLUSIONS: Our results indicated that 2 SNPs in the MD-2 promoter region were significantly associated with Der p 2-specific Ig E, and thereby suggest that these SNPs may play a major role in susceptibility to Der p 2-triggered immune responses in a Taiwanese population.

CDKAL1, KCNQ1, and IGF2BP2 are Identified as Type 2 Diabetes Susceptibility Genes in a Regional Chinese Population.

Aims: Diabetes has become a major public health problem in China. Recent advances in genetic studies have identified numerous susceptibility loci for type 2 diabetes (T2D). Current models to detect risk of T2D are largely based on studies from European populations; this study aims to replicate those efforts in a Chinese population from the Ningbo region in order to diversify and fortify risk models. Methodology: We successfully genotyped 11 single nucleotide polymorphisms (SNPs) in 222 subjects with T2D and 140 subjects with normal glucose regulation in a population from the Ningbo region of China. Additive and dominant models were used to analyze the associations between SNPs and T2D. Results: Adjusting for age, triglycerides, total cholesterol, low density lipoprotein, and BMI in the dominant model, we identified three SNPs which were associated T2D: CDKAL1 (OR=2.29 [95%CI=1.25-4.19]), KCNQ1 (4.22 [1.79-9.99]), and IGF2BP2 (1.76 [1.06-2.94]). No significant association was found between T2D and SNPs from KCNJ11, PPARG, TCF7L2, SALC30A8, CDKN2B, HHEX, HNF1β, and WFS1. Conclusion: Our data indicates that in this population, CDKAL1, KCNQ1, and IGF2BP2 are T2D susceptibility genes.

Association between IRF6 gene SNPs and non-syndromic cleft lip with or without cleft palate in Xinjiang Uyghur population / 实用口腔医学杂志

Objective:To study the association between rs2235371,rs2013162,rs2235377 SNPs in interferon regulatory factor 6 (IRF6)gene and non-syndromic cleft lip with or without cleft palate(NSCL/P)in Xinjiang Uyghur population.Methods:100 Uyghur NSCL/P patients from Xinjiang were included in the case group and 60 Uyghur inpatients with upper respiratory tract infection were se-lected in the control group.Next,generation sequencing was used,DNA sequencing results were compared with the information on the genome database and genetic analysis were made.Results:There were no significant differences in the frequency distribution of both genotypes and alles when the cases were campared with the controls at the rs2235371,rs2013162 and rs2235377 loci(P >0.05). Above three loci were located in the same block,rs2235371 and rs2235377 loci presents the strong linkage disequilibrium(r2 =0.949, D'=0.974).Possible haplotypes were:CCT >CAT >TAC,and there was no significant difference between the cases and controls in haplotype distribution(P >0.05).Conclusion:Polymorphisms of rs2235371,rs2013162 and rs2235377 in IRF6 gene may be associ-ated with NSCL/P in Xinjiang Uygur people.

Association of gene polymorphisms of rapsyn exons with myasthenia gravis / 中国免疫学杂志

Objective:To investigate the association of single nucleotide polymorphisms (SNPs) of receptor-associated protein at the synapse ( rapsyn ) with myasthenia gravis ( MG ).Methods: The genomic DNA was extracted from peripheral blood cells , sampled from 132 patients with MG and 153 control individuals.The 8 exons of rapsyn gene were amplified by PCR ,then the products of PCR sequenced directly.Each sequence was compared with wild-type rapsyn gene , and the association between mutation and clinical symptoms of MG analysed.Results:No mutation was found in the exons 1,2,4,5,6,7,and 8 of rapsyn gene both in MG patients and control group compared with the wild-type rapsyn gene.However,a new SNP,L222R[CTG>CGG(2)] or T665G,was found in exon-3.The allele and genotype frequencies of SNP L 222R met Hardy-Weinberg genetic equilibrium (P>0.05),indicating the group repre-sentativeness.The allele frequencies of G were not statistically different between patient and control groups ( P>0.05 ).There were differences in the 3 genotypes TT , TG and GG between patient ( 42.4% vs 48.5% vs 9.1%) and control ( 49.0% vs 33.3% vs 17.6%) groups ( P<0.05 ).The genotype frequencies of GG were statistically higher in control group than that in patient group , showing a recessive model of inheritance.Conclusion: The SNPs in the rapsyn gene are associated with MG in this study.L222R ( T665 G) is a new SNP found and allele G might be a protective factor for MG.

DNA sequence variation of drought-response candidate genes in Austrocedrus chilensis

Background: Austrocedrus chilensis (D. Don) Pic. Ser. et Bizzarri commonly known as Patagonian cypress is a member of the Cupressaceae family, characterized by a high adaptive potential for growing in marginal areas and good timber quality. The species grows over a wide area and under a wide range of rainfall. This study assessed adaptive genetic variation at SNP level in candidate genes involved in response to drought stress. Results: A total of 18 single nucleotide polymorphisms (SNPs) were found among 1,428 bp. Average nucleotide diversity value (π = 0.00312) was similar to those previously reported in other Cupressaceae. The Fst average among genes and populations was 0.163 and the lowest differentiation was observed in continuous and humid populations. A number of neutrality tests were applied to find evidence of positive selection in our candidate gene set, but only AcAQP2 gene in Pedregoso and San Ramón populations revealed significant departures from neutrality with positive values suggesting balancing selection. Conclusions: In this study we report the levels of nucleotide diversity searched in some drought stress candidate genes in Austrocedrus chilensis and the selective factors that may be acting on this species.

SNPs discovery and linkage disequilibrium analysis of BSG in Chinese Han population / 中南大学学报(医学版)

Objective:To identify BSG single nucleotide polymorphisms (SNPs) in Chinese Han population. Methods:Peripheral blood samples were collected from 48 unrelated healthy Chinese Han subjects. Sequences at the BSG locus, including the promoter region, all exons and exon-intron boundaries were ampliifed, sequenced and followed by Hardy-Weinberg equilibrium test and linkage disequilibrium (LD) analysis. Results:A total of 19 SNPs were identiifed, 2 of which two were novel. Genotype distributions of all SNPs were consistent with Hardy-Weinberg equilibrium. Four haplotype blocks were constructed throughout the gene locus, and 9 haplotype tag SNPs (htSNPs) were inferred. Distribution of SNPs was in accordance with the neutrality theory in Chinese Han population. Conclusion:For the ifrst time, systematic identiifcation of BSG SNPs in the Chinese Han population has been done, and 9 htSNPs are identified. Our study has provided basis for further genetic association studies for related diseases as wel as pharmacogenetics study for drug response in Chinese Han population.

Association of ALOX5AP with ischemic stroke in eastern Chinese / 世界急诊医学杂志（英文）

BACKGROUND: 5-lipoxygenase protein (ALOX5AP) has been recognized as a susceptibility gene for stroke and coronary artery diseases. The present study was to explore the role of this gene in the eastern Chinese patients with ischemic stroke.METHODS: Using a case-control design, we studied 658 patients with ischemic stroke and 704 unrelated population-based controls who were age- and sex-matched. The 658 patients were classified by the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Two single-nucleotide polymorphisms (SNPs) covering ALOX5AP were genotyped.RESULTS: The genotype frequencies of TG of the SNPs rs17222919 located in the promoter of the ALOX5AP gene were significantly higher in patients with ischemic stroke than in controls (OR*=1.34, 95％CI*=1.02-1.75), especially in patients with ischemic stroke caused by small-artery occlusion (SAO) (OR*=1.40, 95％CI*=1.02-1.93). Meanwhile, the genotype frequencies of TG and TG/GG were higher in female patients than in the controls. After specification, the genotype frequencies of TG and TG/GG were higher in the patients than in controls with hypertension. The genotype frequencies of AG and AG/GG of the SNPs rs9579646 located in the intron of the ALOX5AP gene were higher in the controls than in the patients. After specification, the genotype frequencies of TG were higher in the controls than patients without hypertension.CONCLUSION: The present study suggests that sequence variants in the ALOX5AP gene are significantly associated with ischemic stroke.

Characterization of the DGAT1 gene in the Indian buffalo (Bubalus bubalis)

The positional candidate diacylglycerol O-acyltransferase (DGAT1) gene affecting milk fat percentage is reported in Indian buffaloes (Bubalus bubalis). A comparison with Chinese buffalo (Bubalus bubalis) revealed eight exonic single nucleotide polymorphisms (SNPs), five of which were non-synonymous. A total of 19 SNPs were observed among diverse buffalo breeds in India. A Unique 22 base insertion has been reported in the intron between exon ten and eleven.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.