多等位基因PCR-SSO反相杂交法及其在HLA-DR_4亚型结构分析中的应用

本文介绍一种适合于多等位基因定型和结构分析的反相顺序特异性寡核苷酸探针杂交方法。将化学合成的多种探针分别经脱氧核苷末端转移酶(TdT)催化,于3′端加上100个以上碱基的Poly(dT)尾,然后将各探针分别以斑点固定于同一张尼龙膜上。用PCR法对该基因位点进行扩增,扩增的同时加入α-^(32)P-dCTP以直接参入放射标记,然后将PCR产物与膜固定探针杂交,以四甲基氯化铵根据探针长度统一洗涤温度。该方法克服了以往对同一份样品进行多个等位基因检测时需要进行多次探针标记和杂交的缺点。我们用该方法对中国人群MHC-Ⅱ类DR4多等位基因进行了研究。

Y-DNA多态与人类进化

作为父系遗传的 Y染色体 ,绝大部分为非重组区 ,单体群保存完整并且易于鉴定和使用 ,因而其多态性是研究人类起源和迁徙的理想工具。本文对

人线粒体DNA与Y染色体DNA多态性的研究进展

人mtDNA为母系遗传 ,Y DNA为父系遗传 ,且mtDNA分子和Y染色体特异区在减数分裂时均不发生重组 ,这就积累较多的突变 ,记录了人类的进化史 ,因此mtDNA和Y DNA的多态性 ,是探索人类起源、进化和迁移规律有价值的工具。本文介绍了近年来人mtDNA的D环区、V区和Y染色体DNA的双等位基因。

人类Y-DNA多态性研究概述

Y-DNA为父系遗传,Y染色体特异区在减数分裂时不发生重组,这就积累较多的突变,记录了人类的进化史,因此Y-DNA的多态性,是探索人类起源、进化和迁移规律有价值的工具。本文就Y染色体DNA的双等位基因、多等位基因多态性的研究进展做一综述。

Characterization of Genetic Polymorphism of Novel MHC B-LBⅡ Alleles in Chinese Indigenous Chickens

Genetic polymorphism of the major histocompatibility complex （MHC） B-LBⅡ gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB Ⅱ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB Ⅱsequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LBⅡ alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LBⅡ gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB Ⅱ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%±0.94% and 14.64%±2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the β1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB Ⅱ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

A Recursive Algorithm for Offspring's Genotype Frequency of Selfing Population on Multiple Alleles with Limited Loci

This paper studies the offspring＇s genotype frequency of the selfing population on multiple alleles with limited loci.A recursive algorithm is given for it.It is discovered that the genotype frequency of homozygous gene of limited loci increases by generations.Relative increment reduces by generations and the genotype frequency tends to a definite value finally.The genotype frequency of limited loci with hybrid gene tends to 0 finally.But it is possibility that the genotype frequency increases in previous generations then reduces later.It is found that the number of the hybrid gene are more,the speeds tending to 0 are quicker.

Genetic predisposition to inflammation:a new risk factor of Alzheimer's disease

Inflammation has been shown to play an important role in the progression of Alzheimer＇s disease （AD）. Recent epidemical study indicates that the incidence of AD in some populations is substantially influenced by the gene polymorphisms of the inflammation mediators. Meanwhile, an ensured risk factor, the ApoE ε4 allele is also reported to directly promote inflammation. Accordingly, it appears that an individual genetic background has partly determined his predisposition for AD by the extent of the inflammation response to the chronic stimulus by β-amyloid peptide （Aβ） deposits and other antigen stressor in the elderly. Hence we present a hypothesis that the inflammation genotypes may contribute to AD susceptibility. This may provide a new orientation both for future identification of individuals at risk and for personalized medication.

Association of polymorphic alleles of CTLA4 with inflammatory bowel disease in the Japanese

AIM： To examine an association between the cytotoxic Tlymphocyte antigen 4 （CTLA4） gene that plays a role in downregulation of T-cell activation and inflammatory bowel disease consisting of ulcerative colitis （UC） and Crohn＇s disease （CD） in the Japanese.METHODS： We studied 108 patients with UC, 79 patients with CD, and 200 sex-matched healthy controls, with respect to three single nucleotide polymorphisms （SNPs） in CTLA4, such as C-318T in the promoter region, A＋49 Gin exon 1 and G＋6230A in the 3＇ untranslated region （3＇-UTR） by a PCR-restriction fragment length polymorphism method, and to an （AT）, repeat polymorphism in 3＇-UTR by fragment analysis with fluorescence-labeling on denaturing sequence gels. Frequency of alleles and genotypes and their distribution were compared statistically between patients and controls and among subgroups of patients, using X^2 and Fisher exact tests.RESULTS： The frequency of ＂A/A＂ genotype at the G＋6230A SNP site was statistically lower in UC patients than in controls （3.7% vs 11.0%, P = 0.047, odds ratio （OR） = 0.311）. Moreover, the frequency of ＂G/G＂ genotype at the A＋49G SNP site was significantly higher in CD patients with fistula （48.6%） than those without it （26.2%）（P = 0.0388, OR=2.67）.CONCLUSION： The results suggest that CTLA4 located at 2q33 is a determinant of UC and responsible for fistula formation in CD in the Japanese.

Microsatellite analysis of variation among wild, domesticated, and genetically improved populations of blunt snout bream （Megalobrama amblycephala）

In the present study, the genetic diversity of one selected strain （Pujiang No. 1）, two domesticated populations （GA and HX） and four wild populations （LZ, YN, SS and JL） of blunt snout bream （Megalobrama amblycephala） was analyzed using 17 microsatellite markers. The results showed that an average of 4.88-7.65 number of alleles （A）; an average of 3.20-5.33 effective alleles （Ne）; average observed beterozygosity （Ho） of 0.6985-0.9044; average expected beterozygosity （He） of 0.6501--0.7805; and the average polymorphism information content （PIC） at 0.5706-0.7226. Pairwise FST value between populations ranged from 0.0307-0.1451, and Nei＇s standard genetic distance between populations was 0.0938-0.4524. The expected heterozygosities in the domesticated populations （GA and HX） were significantly lower than those found in three wild populations （LZ, SS and JL）, but no difference was detected when compared with the wild YN population. Likewise, no difference was found between the four wild populations or two domesticated populations. The expected heterozygosity in Pujiang No. 1 was higher than the two domesticated populations and lower than the four wild populations. Regarding pairwise Fsr value between populations, permutation test P-values were significant between the GA, HX and PJ populations, but not between the four wild populations. These results showed that the expected beterozygosity in the selected strain of blunt snout bream, after seven generations of selective breeding, was lower than that of wild populations, but this strain retains higher levels of genetic diversity than domesticated populations. The genetic differences and differentiation amongst wild populations, domesticated populations and the genetically improved strain of blunt snout bream will provide important conservation criteria and guide the utilization of germplasm resources.

THE ASSOCIATION OF POLYMORPHISMS AT A VNTR LOCUS 3’TO THE APOLIPOPROTEIN B GENE WITH CORONARY HEART DISEASE IN CHINESE POPULATION

The polymorphisms of variable number of tandem repeats (VNTR) 3’to the apolipoprotein B (apo B) gene were investigated using polymerase chain reaction (PCR) in a sample of 103 patients with documented coronary heart disease (CHD) and 100 healthy individuals selected from Chinese Han nationality.Twelve segregating alleles (3’β29 -51) were observed in the pooled total of 203 subjects. The most common allele was 3’β 37. followed by 3’β39 with frequencies of 0. 362 and 0. 296, respectively. This model of allele distribution was coincident with the results form different ethnic groups, but the relative frequencies of alleles were different. In comparison with the allele frequencies between the patients and controls,alleles bigger than 3’β39 (3’VNTR-B) were significantly more common among the patients than among the controls (P<0. 001). Moreover. in the CHD group patients with plasma levels of TC≥3.88 mmol/L,LDL-C≥2. 59 mmol/L and HDL-C<l. 16mmol/L had significantly higher frequencies of 3’ VNTR-B allele (P<0. 01). Therefore,it is suggested that 3’ VNTR-B allele might be involved in the development of coronary atherosclerosis, presumably through their influences on lipid metabolism.This study supported by “8. 5” grant from Ministry of PublicHealth.

Analysis of Genetic Diversity and Differentiation of Seven Stocks of Litopenaeus vannamei Using Microsatellite Markers

Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatellite loci were polymorphic, with polymorphism information content(PIC) values ranging from 0.593 to 0.952. Totally 92 alleles were identified, and the number of alleles(Na) and effective alleles(Ne) varied between 4 and 21 and 2.7 and 14.6, respectively. Observed heterozygosity(Ho) values were lower than the expected heterozygosity(He) values(0.526–0.754), which indicated that the seven stocks possessed a rich genetic diversity. Thirty-seven tests were detected for reasonable significant deviation from Hardy-Weinberg equilibrium. Fis values were positive at five loci, suggesting that there was a relatively high degree of inbreeding within stocks. Pairwise Fst values ranged from 0.0225 to 0.151, and most of the stock pairs were moderately differentiated. Genetic distance and cluster analysis using UPGMA revealed a close genetic relationship of L. vannamei between Pop2 and Pop3. AMOVA indicated that the genetic variation among stocks(11.3%) was much lower than that within stocks(88.7%). Although the seven stocks had a certain degree of genetic differentiation and a rich genetic diversity, there is an increasing risk of decreased performance due to inbreeding in subsequent generations.

Genetic Diversity Analysis of Guangxi Donglan Black-bone Chicken

[Objective] This study aimed to understand the genetic diversity and breed conservation effect, and to provide reference for future germplasm resources protection and breeding researches of Guangxi Donglan black-bone chicken. [Method] The wing venous blood of Donglan Black-bone chicken（silky feather and contour feather） was collected, and the total DNA was extracted. The 18 microsatellite loci were amplified by PCR technology, and the obtained gene fragments were scanned. The number of alleles（Na）, number of effective alleles（Ne）, heterozygosity（H） and polymorphism information content（PIC） were determined using Pop Gen 32 biological software to do cluster analysis of Guangxi indigenous chicken breeds. [Result] The18 microsatellite loci of Donglan black-bone chicken were found to be highly polymorphic. A total of 98 alleles were detected out from 80 Donglan black-bone chicken samples. The averages of Na, Ne, PIC and H per locus were 5.44, 2.78, 0.5382 and 0.596 3, respectively. A total of 93 and 98 alleles were detected out from silky- and contour-feather Donglan black-bone chickens, respectively. The averages of Ne per locus were 2.62 and 2.76, of PIC per locus were 0.508 8 and 0.539 8,of H per locus were 0.624 7 and 0.599 1, respectively. The results of cluster analysis showed that there was greater genetic distance between Donglan black-bone chicken and any of other Guangxi indigenous chicken breeds, including three yellow chicken, Xiayan chicken and Nandan Yao chicken, which was consistent with the breed evolution, geographical distribution and breeding history of Donglan black-bone chicken. [Conclusion] There are greater genetic variation and abundant genetic diversity in the conserved population of Donglan black-bone chicken, suggesting abundant genetic diversity and great breeding potential of Donglan black-bone chicken.

Genetic Variation Assessed with Microsatellites in Mass Selection Lines of the Pacific Oyster (Crassostrea gigas) in China

Abstract Four successive mass selection lines of the Pacific oyster, Crassostrea gigas, selected for faster growth in breeding pro- grams in China were examined at ten polymorphic microsatellite loci to assess the level of allelic diversity and estimate the effective population size. These data were compared with those of their base population. The results showed that the genetic variation of the four generations were maintained at high levels with an average allelic richness of 18.8-20.6, and a mean expected heterozygosity of 0.902-0.921. They were not reduced compared with those of their base population. Estimated effective population sizes based on temporal variances in microsatellite frequencies were smaller to that of sex ratio-corrected broodstock count estimates. Using a rela- tively large number ofbroodstock and keeping an equal sex ratio in the broodstock each generation may have contributed to retaining the original genetic diversity and maintaining relatively large effective population size. The results obtained in this study showed that the genetic variation was not affected greatly by mass selection progress and high genetic variation still existed in the mass selection lines, suggesting that there is still potential for increasing the gains in future generations of C. gigas. The present study provided im- portant information for future genetic improvement by selective breeding, and for the design of suitable management guidelines for genetic breeding of C. gigas.

Nur-related receptor 1 gene polymorphisms and alcohol dependence in Mexican Americans

AIM： To investigate the association of polymorphisms of nur-related receptor 1 （Nurrl） and development of alcohol dependence in Mexican Americans. METHODS： Peripheral blood samples were collected from 374 alcoholic and 346 nonalcoholic Mexican Amer- icans; these two groups were sex- and age-matched. Sample DNA was extracted and genomic DNA was amplified by polymerase chain reaction. The -2922（C） 2-3 polymerase chain reaction products were digested with Sau96I, alleles of 1345（G/C）, and -1198（C/G） in the regulatory region as well as Ex＋132 （G/T/A/C） and Ex＋715（T/-） in exon 3 were studied by sequencing. RESULTS： The C2/C2, C2/C3, C3/C3 genotype distribu- tion of -2922（C） 2-3 was 34.4%, 38.2% and 27.5% in the nonalcoholic group compared to 23.3%, 51.2% and 25.4% in the alcoholic group （P = 0.001）. The C/C, C/G ,G/G genotype distribution of -1198（C/6） was 23.5%, 46.1% and 30.3% in the nonalcoholic group compared to 13.9%, 50.9% and 35.3% in the alcoholic group （P = 0.007）. However, the -1345 （G/C）, Ex3＋132（G/T/A/C） and Ex3＋715（T/-） alleles were not polymorphic in Mex- ican Americans, and all those studied had G/G, G/G and T/T genotype for these three alleles, respectively. The -2922（C） 2-3 did not show allele level difference be- tween alcoholic and nonalcoholic individuals, but -1198 （C/G） showed a significant allele frequency difference between alcoholic （39.3%） and nonalcoholic （46.6%） populations （P = 0.005）. Excluding obese individuals, significant differences were found at both genotypic and allelic levels for the -2922（C） 2-3 polymorphism （P = 0.000 and P = 0.049） and the -1198 （C/G） polymor- phism （P = 0.008 and P = 0.032） between nonobese alcoholics and nonobese controls. Excluding smokers, a significant difference was found only at the genotypic level for the -2922（C） 2-3 polymorphism （P = 0.037） between nonsmoking alcoholics and nonsmoking con- trols, but only at the allelic level for the -1198（C/G） polymorphism （P = 0.034）. CONCLUSION： Polymorphisms in the regulatory region of Nurrl are implicated in pathogenesis of alcohol de- pendence and the Nurrl/dopamine signaling pathway might be important for this dependence development in Mexican Americans.

Genetic Biodiversity in Buffalo Population of Iraq Using Microsatellites Markers

Genetic structure of Iraqi buffalo population, in the South, Middle and North area of the country was characterized, using six microsatellite markers （ETHI52, ETH02, ETH225, CSSM060, BM1706 and INRA005）. Seventy alleles were detected across the six loci. Total number of alleles per locus （TNA） varied from 3 （INRA005 locus） to 16 （ETH 152 locus）. The mean number of allele （MNA） across the six loci in Iraqi indigenous buffalo was 11.4. The locus ETHI52 was the most polymorphic marker according to its number of allele （16）, the expected heterozygosity （0.86） and polymorphism information content （0.80） number of alleles （3）, expected Heterozygosity （0.1-0.2） and polymorphism information content （0.1-0.2）. Results showed that these markers were suitable in population genetics researches. It was concluded that a high degree of genetic diversity exist in the Iraqi buffalo populations.

Ku80 gene G-1401T promoter polymorphism and risk of gastric cancer

AIM:To evaluate the possible relationship between the Ku80 gene polymorphism and the risk of gastric cancer in China.METHODS:In this hospital-based case-control study of gastric cancer in Jiangsu Province,China,we investigated the association of the Ku80 G-1401T (rs828907) polymorphism with gastric cancer risk.A total of 241 patients with gastric cancer and 273 age-and sexmatched control subjects were genotyped and analyzed by polymerase chain reaction-restriction fragment length polymorphism.RESULTS:The frequencies of genotypes GG,GT and TT were 65.6%,22.8% and 11.6% in gastric cancer cases,respectively,and 75.8%,17.6% and 6.6% in controls,respectively.There were significant differences between gastric cancer and control groups in the distribution of their genotypes (P=0.03) and allelic frequencies (P=0.002) in the Ku80 promoter G-1401T polymorphism.CONCLUSION:The T allele of Ku80 G-1401T may be associated with the development of gastric cancer.

Evaluation of SNPs in miR-196-a2, miR-27a and miR-146a as risk factors of colorectal cancer

AIM: To investigate whether selected single nucleotide polymorphisms (SNPs) in miR-196a2, miR-27a and miR146a genes are associated with sporadic colorectal cancer (CRC).METHODS: In order to investigate the effect of these SNPs in CRC, we performed a case-control study of 197 cases of sporadic CRC and 212 cancer-free controls originating from the Central-European Caucasian population using TaqMan Real-Time polymerase chain reaction and allelic discrimination analysis. RESULTS: The genotype and allele frequencies of SNPs were compared between the cases and the controls. None of the performed analysis showed any statistically significant results. CONCLUSION: Our data suggest a lack of association between rs11614913, rs895819 and rs2910164 and colorectal cancer risk in the Central-European Caucasian population, a population with an extremely high incidence of sporadic colorectal cancer.

-449 C>G polymorphism of NFKB1 gene,coding nuclear factor-kappa-B,is associated with the susceptibility to ulcerative colitis

AIM:To clarify the association between a polymorphism-449 C>G(rs72696119) in 5'-UTR of NFKB1 with ulcerative colitis(UC).METHODS:The studied population comprised 639 subjects,including patients with UC(UC cases,n = 174) and subjects without UC(controls,n = 465).We employed polymerase chain reaction-single strand conformation polymorphism to detect the gene polymorphism.RESULTS:The rs72696119 G allele frequencies in controls and UC cases were 33.4% and 38.5%,respectively(P = 0.10).Genotype frequency of the GG homozygote in UC cases was significantly higher than that in controls(P = 0.017),and the GG homozygote was significantly associated with susceptibility to UC [odds ratio(OR),1.88;95%CI,1.13-3.14].In male subjects,the GG homozygote was associated with an increased risk for UC(OR,3.10;95%CI,1.47-6.54;P = 0.0053),whereas this association was not found in female subjects.In addition,the GG homozygote was significantly associated with the risk of non-continuous disease(OR,2.06;95%CI,1.12-3.79;P = 0.029),not having total colitis(OR,2.40;95%CI,1.09-3.80,P = 0.040),disease which developed before 20 years of age(OR,2.80;95%CI,1.07-7.32,P = 0.041),no hospitalization(OR,2.28;95%CI,1.29-4.05;P = 0.0090) and with a maximum of 8 or less on the UCDAI score(OR,2.45;95%CI,1.23-4.93;P = 0.022).CONCLUSION:Our results provide evidence that NFKB1 polymorphism rs72696119 was significantly associated with the development of UC.This polymorphism influences the susceptibility to and pathophysiological features of UC.

Genetic Association Study of KCNB1 Gene with the Susceptibility of Hypertension Related LVH （Left Ventricular Hypertrophy） Patients in Malaysia

LVH （Left ventricular hypertrophy） is an independent risk factor for the development of heart failure, cardiac arrhythmias and stroke. A recent genome-wide association study reported the involvement of a candidate gene namely KCNBI in mechanism for development of LVH in hypertension. This study aimed to replicate the finding by investigating the genetic association of KCNBI gene among the hypertensive LVH patients from Malaysia. We genotyped a SNP （single nucleotide polymorphism） located in KCNBI namely, rs6063397 among 200 subjects consisting of61 LVH and 139 non LVH patients using Sanger sequencing method. Statistical analysis revealed no significant association between the L VH susceptibility between the allele and genotype frequencies （P = 0.2719 and 0.4768, respectively）. This finding suggests that KCNBI may not playa role in LVH susceptibility in hypertensive patients in Southeast Asian populations.

Coagulation factor Ⅶ gene polymorphisms are not associated with the occurrence or the survival of hepatocellular carcinoma:a report of 37 cases

Objective: Coagulation factor VII(FVII) triggers the extrinsic pathway of blood coagulation. In our previous study, we showed that FVII plays an important role in tumorigenesis of hepatocellular carcinoma(HCC). However, the role of FVII polymorphism in HCC is still unknown. The present study aimed to investigate the relationship between HCC carcinogenesis and single nucleotide polymorphism of FVII.Methods: Thirty-seven HCC patients and 30 healthy donors were recruited in this study. Four common FVII gene polymorphisms– a decanucleotide insertion at position –323(–323 ins10-bp), a G to T substitution at position –401(–401 G/T), a G to A substitution at position –402(–402 G/A), and a T to C substitution at position –122(–122 T/C) – were analyzed by sequencing or commercialized assays using genomic DNA isolated from blood samples. Clinicopathological parameters between control and HCC subjects were compared according to the specific genotypes.Results: The most common nucleotide variation was –402 G/A. However, no statistically significant difference was observed between healthy controls and HCC subjects for all four polymorphisms in terms of genotype distribution and allele frequencies,indicating that these polymorphisms may not affect HCC tumorigenesis. Furthermore, no association was found between–402 G/A polymorphisms and tumor stage, recurrence, and overall survival.Conclusions: Our results indicate that FVII polymorphisms may not be a key factor that clinically impact tumorigenesis and outcomes of HCC, although further investigations should be conducted to confirm our findings.