贵州四个民族人群线粒体DNA编码区的多态性

目的研究贵州土家族、侗族、仡佬族和彝族人群线粒体DNA（mtDNA）编码区的核苷酸多态性。方法采用PCR-RFLP技术和DNA测序法对贵州4个群体145例样本mtDNA编码区的8个SNP基因座及COⅡ/tRNAlys基因间9 bp缺失进行多态性分析。结果贵州4个民族群体的9 bp缺失频率依次为土家族18.4%,侗族29.7%,仡佬族25%,彝族16.7%,平均缺失频率为22.8%;在8个SNP基因座中,A10398G、C10400T突变在4个群体中较普遍;A663G、C5178A和G12406A突变在部分民族群体中也有较高的频率;共检测出14种单倍型,其中仡佬族11种,土家族10种,侗族8种,彝族6种。结论贵州4个民族群体mtDNA编码区可能存在不同的突变热点,在等位基因和单倍型分布频率上存在一定差异。     

贵州苗族、布依族、侗族人群线粒体DNA多态性研究

目的:调查贵州苗族、布依族、侗族人群mtDNA的群体遗传特点。方法:用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术和DNA测序法,对3个民族男性个体mtDNA上12个SNP进行多态性分析。结果:285名个体共检出47种单倍型和12种单倍群。主要单倍型H32、H36、H10在3个民族间频率分布差异有统计学意义(P0.05);3个民族共有单倍型为11种(72.98%)且在民族间频率分布差异有统计学意义(P0.05),单倍群B(21.05%)和M7(21.05%)频率最高,B在苗族、布依族中分布频率与侗族间差异有统计学意义(P0.05)。结论:贵州苗族、布依族、侗族人群mtDNA单倍型分布相似,单倍群频率及主成分分析表现出南方群体特征。侗族母系遗传结构较苗族和布依族更为复杂,民族间存在基因交流的可能。     

心脏钠离子通道α亚单位基因单核苷酸多态性及其在汉族人群中的分布

实验旨在研究中国汉族人群心脏钠离子通道α亚单位(voltage-gated sodium channel type Ⅴ,SCN5A)基因的单核苷酸多态性(single nucleotide polymorphism,SNP)及其分布。应用荧光标记自动测序法测定120名非亲缘关系中国南方汉族人群的SCN5A基因序列,确定其单核苷酸多态位点及基因型。结果如下,在中国南方汉族人群中共检测到5个SNPs:3个位于编码区,另2个分别位于3’侧翼区和intron23邻接供体剪接位点的区域。各个SNP在基因中呈不均匀分布,其基因频率分别为G87A(A29A)27.5％,A1673G(H588R)10.4％,4245+82A>G 32.8％,C5457T(D1819D)41.3％和G6174A44.9％。其中G87A(A29A),G6174A和4245+82A>G为新发现的SNP。A1673G(H588R)的基因频率在中国南方汉族人群、日本人群和美国人群之间无显著差异(P>0.05)。C5457T(D1819D)在中国南方汉族人群和日本人群中的分布非常接近(P>0.5),但都明显高于美国人群中的分布(均P<0.005)。各SNP在不同性别中的分布无显著差异(均P>0.05)。S1102Y及其余10个国外已经报道的多态位点在本研究中未检测到。各SNP等位基因频率在人群中的分布符合Hardy—Weinberg平衡。结果提示,SCN5A基因SNP具有较大的民族差异。     

浙江汉族人群细胞因子基因多态性分析

为了探讨浙江汉族人群13种细胞因子基因多态性的分布情况, 采用PCR-SSP对100名汉族人群的IL-1α(T/C-889)、IL-1β(C/T-511, T/C+3962)、IL-1R(C/T Pst-I 1970)、IL-1RA(T/C Mspa1-I 1100)、IL-2 (T/G -330, G/T +166)、IL-4 (T/G -1098, T/C -590, T/C -33)、IL-4Rα(G/A +1902)、IL-6 (G/C-174, G/A nt565)、IL-10 (G/A -1082, C/T -819, C/A-592)、IL-12(C/A -1188)、gIFN (A/T UTR 5644)、TGFβ(C/T codon 10, G/C codon 25)、TNFα(G/A -308, G/A -238)等细胞因子基因多态性进行分型。结果显示, (1)在检测的13种细胞因子中, TGF(G codon 25)等位基因频率为1.00, 未检测到TGF(C codon 25)等位基因; (2)细胞因子等位基因频率大于0.95的有IL-1α(C-889)、IL-1β(C +3962)、IL-4 (T –1098)、IL-6 (G-174)、IL-6(G nt565)、IL-10(A –1082)和TNFa (G -238); 频率低于0.05的有IL-1α(T -889)、IL-1β(T +3962)、IL-4(G –1098)、IL-6 (A-174)、IL-6 (A nt565)、IL-10 (G –1082) 和TNFa (A -238); (3) IL-10高表达单倍型GCC频率为0.05, 低表达单倍型ATA频率为0.735; TGF β高表达单倍型TG频率为0.49, 低表达单倍型CC频率为0; TNF a高表达单倍型AG和AA频率为0.055, 低表达单倍型GG和GA频率为0.945。结果表明, 浙江汉族人群细胞因子基因多态性较为丰富, 具有地区性遗传特征。     

一组中国河南汉族群体血管紧张素转换酶基因多态性与血清酶活性研究

为研究中国汉族群体血管紧张素转换酶(angiotensin-1 converting enzyme,ACE)活性、基因多态性分布及相互关系,用分光光度法检测496例汉族个体血清酶活性,PCR后的限制性酶切片段长度多态性(PCR-RFLP)法检测血管紧张素转换酶基因启动子区A-5466C、T-3892C、A-240T及编码区T1237C、G2215A、G2350A共6个SNP位点,以及第16内含子的A／u片段插入／缺失(I／D)和3’端4656(CT)。共8个多态位点的分布,同时用最大期望值(expectationmaximization,EM)算法估计基因连锁不平衡状态和单倍型结构。结果发现,上述8个多态存在于常见的9个单倍型中,其中两种最常见的单倍型为A(A-T-A-T-G-I-A-3)和B(C-C-T-C-A-D-G-2),A和B在每个位点都不相同。最大简约法分析提示本群体可被分为3个进化簇,Ⅲ簇最有可能由Ⅰ簇和Ⅱ簇产生。ACE基因各位点多态性在个体中的分布与血清中ACE活性有关,组成单倍型A的各等位位点与血清中ACE低活性有关,单倍型B的各等位位点与血清中ACE高活性有关。研究结果提示,本群体中ACE基因存在连锁不平衡,有两种主要的单倍型,单倍型B可能与导致ACE升高的数量性状(QTL)关联,但确定具体的数量性状位点还需绘制精细物理图谱。     

一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。     

牛HTR1B基因的分子遗传特性研究

用PCR-SSCP技术研究了涉及肉牛和奶牛共计7品种HTR1B基因的编码区和3′侧翼区的多态性,以期为牛性情的标记辅助选择积累数据。扩增得到4个片段, 有3个片段存在(SSCP)多态性。对不同的SSCP带型对应片段进行测序, 共发现6个SNP多态位点(G205T、C507T、C546G、C744T、G816A和G942A)。各遗传群体内G205T、C744T、G816A和G942A 位点均处于Hardy-Weinberg平衡, 而C507T和C546G位点只有鲁西牛处于Hardy-Weinberg平衡。奶牛205T等位基因频率显著高于其他肉牛品种(χ2 = 6.87)。奶牛G205T位点多态信息含量为0.25, 其余各位点在不同群体内均小于0.10, 说明牛HTR1B基因较保守。     

葡萄糖转运体9(GLUT9)基因启动子区的rs13124007(G/C)及rs6850166(G/A)位点的单核苷酸多态性(SNP)与汉族女性痛风相关性研究

目的:探讨葡萄糖转运体9(GLUT9)基因启动子区的rs13124007(C/G)及rs6850166(A/G)位点的单核苷酸多态性(SNP)与中国汉族女性人群痛风易感性之间的相关性.方法:选取185例痛风患者和300例正常对照者,提取基因组DNA,采用聚合酶链式反应(PCR技术),特异性扩增GLUT9基因所需要的目的片段,对扩增的目的片段进行测序后,比较痛风组和正常对照组的基因型频率及等位基因频率分布情况.结果:女性痛风组中GLUT9基因的启动子区rs13124007和rs6805116两个位点的基因型频率分布与正常对照组相比,统计学上无明显的差异(X2=0.906,P=0.636;X2=3.335,P=0.189),rs13124007 SNP位点的C等位基因频率和rs6850166SNP位点的A的等位基因频率与正常对照组相比也无明显的统计学差异(X2=0.506,P=0.477;X=3.268,P=0.071).结论:葡萄糖转运体9(GLUT9)基因启动子区的rs 13124007(C/G)及rs6850166(A/G)位点的单核苷酸多态性(SNP)与中国汉族女性人群痛风易感性无明显的相关性.     

云南汉族酒精依赖综合征与COMT遗传多态性的关联研究

目的:研究儿茶酚胺氧位甲基转移酶(COMT)的不同基因型及等位基因频率在云南汉族酒精依赖综合征患者组和健康对照组的分布差异。方法:应用聚合酶链式反应-限制性片段长度多态性分析法,对COMT基因的rs2075507、rs737865、rs4680、rs165599四个基因位点进行特异性扩增,限制性内切酶酶切分型。结果:上述4个候选基因中,COMT基因rs737865位点C/C基因型频率在健康对照组较酒依赖组高,其基因型分布在两组中有差异,且具有统计学意义(P<0.05)其余3个位点统计学分析均无显著性差异(P>0.05)。单倍型分析:上述四个候选基因构建出12种主要单倍型(每种单倍型在对照组和酒依赖组中的频率至少有一个大于1%),单倍型A-C-A-A有可能是云南汉族酒精依赖发生的一种危险因子(OR:2.865,P=0.003347)连锁不平衡分析显示:云南汉族人群中,COMT基因的rs2075507和rs737865之间存在着强连锁(D>0.8)结论:在云南汉族人群中,COMT基因rs2075507、rs4680和rs165599位点与酒依赖无关联性,rs737865C/C基因型可能是酒精依赖的保护因子,可能降低嗜酒的发生率。单倍型A-C-A-A有可能是云南汉族酒精依赖发生的一种危险因子云南汉族人群中,COMT基因的rs2075507和rs737865之间存在着强连锁。     

天津地区人群hMLH1和hMSH2基因多态性与散发性结直肠癌易感性的关系

为探讨错配修复基因hMLH1和hMSH2单核苷酸多态性(Single nucleotide polymorphism,SNP)与散发性结直肠癌(Sporadic colorectal caner,SCRC)发病易感性之间的关系,文章采用聚合酶链式反应-变性高效液相色谱方法和序列分析技术,检测了天津地区600例SCRC患者和600例健康对照个体hMLH1394G/C、hMSH2943-1G/A、hMSH21917T/G和hMSH22783C/A的基因型频率分布。结果显示:SCRC患者组hMSH22783C/A3种基因型C/C、C/A、A/A频率(90%、9%、1%)与对照组(95%、4.8%、0.2%)相比差异具有统计学意义(χ2=11.91,P0.01)。与hMSH22783C/C基因型相比,C/A和A/A基因型能增加SCRC发病风险(OR值分别为1.77和11.94,95%CI分别为1.03～3.03和1.38～103.2)。多态性位点联合分析显示,SCRC组与对照组单倍型分布差异有统计学意义(χ2=38.38,P0.01);与394G/943-1G/2783C单倍型相比,394G/943-1G/2783A单倍型显著增加SCRC的发病风险(OR=2.18,95%CI:1.40～3.40)。结果提示hMSH22783C/A多态性可能成为预测SCRC发病风险的独立因素,394G/943-1G/2783A单倍型可能增加SCRC的发病风险。     

我国3个民族13个SNPs位点多态性及遗传学关系的比较

采用荧光标记复合扩增毛细管电泳技术, 基于等位基因特异性PCR原理, 通过正交实验法建立了荧光标记复合扩增片段长度差异等位基因特异性SNPs分型体系, 该体系可以根据产物长度和产物峰的数量一次完成13个SNPs分型, 纯合子为单一产物峰, 杂合子为长度相差4 bp的两个产物峰。采用该体系对我国辽宁地区汉族、内蒙古地区蒙古族和广西地区壮族3个民族13个SNPs位点多态性进行群体调查, 获得了3个民族13个SNPs等位基因分布频率, 比较了3个民族等位基因的差异, 并对其遗传学关系进行了研究。结果显示: 3个民族13个SNPs的等位基因分布具有多态性, 多个SNPs等位基因分布具有显著性差异(P≤0.01), 抽样调查结果符合Hardy-Weinberg平衡; 辽南地区汉族人群与内蒙古蒙古族人群的亲缘关系更为接近, 与广西壮族之间的亲缘关系相对较远。     

Correlation Between Cerebral Infarction and ALOX5AP Gene Expression

Linkage/linkage-disequilibrium analysis studies, based on positional information and gene function, indicated that ALOX5AP gene was an independent risk factor of cerebral infarction in humans; however, this needs to be verified among different populations. Herein, we verified whether ALOX5AP was a risk factor of cerebral infarction in the Chinese Han population. For this purpose, 547 cerebral infarction patients were enrolled as the case group; the control group comprised 794 healthy, age-matched individuals unrelated to case group and had no history of cerebral infarction/transient ischemic attack. Regarding single-nucleotide polymorphism (SNP) selection and ALOX5AP genotyping, we selected four SNP loci (SG13S25, SG13S114, SG13S89, and SG13S32) and determined allelic frequencies. Genotyping of SG13S114 and SG13S32 adopted a method of combining real-time quantitative PCR and allele-specific PCR. A linkage-disequilibrium analysis of ALOX5AP was also performed. We found that the allelic frequencies of SG13S25 and SG13S89 were below 5 % and those of SG13S114 and SG13S32 were above 5 %. We did not find any differences between the case and control groups regarding allele, allele types, and haplotype gene frequencies of two SNP loci. The results indicate that the two genetic polymorphisms of ALOX5AP, SG13S114 and SG13S32, are not associated with cerebral infarction in Chinese Han population.     

新疆维吾尔族和汉族亚甲基四氢叶酸还原酶基因多态性研究

目的:研究新疆维吾尔族、汉族两民族亚甲基四氢叶酸还原酶(Methyleneterahyofolate reductase MTHFR)基因多态性的分布情况,获取新疆维吾尔族与汉族MTHFR 1298位群体遗传学数据。方法:应用PCR-RFLP技术检测新疆维吾尔族及汉族MTHFR1298位多态性位点基因频率及基因型频率。结果:新疆维吾尔族、汉族MTHFR 1298位C等位基因分布频率分别为12%、23%,P<0.05有统计学差异性,且新疆维吾尔族MTHFR 1298位C等位基因分布频率与现有报道的少数民族贵州苗族、布依族具有统计学差异。结论:MTHFR 1298位多态性在不同民族具有差异性:MTHFR 1298位多态性在新疆维吾尔族和汉族有民族差异;新疆新疆维吾尔族MTHFR 1298位C等位基因频率与贵州苗族、布依族少数民族之间具有民族差异性。     

Polymorphism of the STR-locus of Y chromosomes in Eastern Slavs in three populations from Belorussia, Russia and the Ukraine

Allelic polymorphism of five microsatellite loci of the human Y chromosome (DYS19, DYS390, DYS391, DYS392, and DYS393) was analyzed in samples of male populations from Ukraine, Russia, and Belarus (152 subjects in total). The allelic diversity indices (Dg) were determined for all loci; they varied from 0.23 to 0.72. The mean values of this parameter in the Ukrainian, Russian, and Belarussian populations were 0.45, 0.47, and 0.52, respectively. A total of 53 different haplotypes were found in 152 subjects from three populations. The most frequent haplotype was found in 14.5% of the subjects, whereas 35 haplotypes (23%) were each found in only one person. The haplotypic diversity index (Dhp) was 0.94. The genetic distances between the populations studied and some populations of Western and Central Europe were estimated. These data were used to construct a phylogram (tree) of genetic similarity between the populations, which demonstrated that the three Eastern Slavic populations are genetically close to one another and remote from Western European populations.     

Association between chloroplast and mitochondrial lineages in oaks

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.      

The phylogeography of the asian sockeye salmon <Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>, inferred from the data on the variability of mitochondrial SNP loci: Analysis of scenarios for post-glacial expansion of the species over the asian coast of the pacific ocean

The variability of three single nucleotide polymorphism (SNP) loci in the mitochondrial DNA (mtSNP) is analyzed for sockeye salmon populations over a major part of the species range, from Chukotka to the Kuril Islands. Two basic mtSNP haplotypes, GCC and GTT, have been revealed in 20 sockeye samples from 15 lake–river systems on the Asian coast of the Pacific Ocean. In most of the samples, the ratio of the haplotypes is approximately equal. The GTT haplotype dominates the populations from the Kuril Islands (except Shumshu Island); only the GCC haplotype has been found in the sample from the Commander Islands. This geographic pattern of haplotype distribution was presumably caused by the historico-demographic events related to the formation of the Asian sockeye range in the Middle–Late Pleistocene, viz., fragmentation of the species range and subsequent secondary contact between previously diverged populations. These data provide a basis to consider different scenarios for the formation of the modern diversity of sockeye mtSNP haplotypes, all of which suggest multiple expansions of the species to Asian waters during the periods of oceanic transgression after the Pleistocene glaciations.     

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.     

Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA

Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single-nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosarcoma tissues and patient-matched blood. SNP calls were then generated by Affymetrix® GeneChip® DNA Analysis Software. In two osteosarcoma cases, using unamplified DNA, we identified 793 and 1070 SNP loci with allelic imbalance, respectively. In a parallel experiment with amplified DNA, 78% and 83% of these SNP loci with allelic imbalance was detected. The average false-positive rate is 13.8%. Furthermore, using the Affymetrix® GeneChip® Chromosome Copy Number Tool to analyze the SNP array data, we were able to detect identical chromosomal regions with gain or loss in both amplified and unamplified DNA at cytoband resolution.     

Polymorphism of STR Loci of the Y Chromosome in Three Populations of Eastern Slavs from Belarus,Russia, and Ukraine

Allelic polymorphism of five microsatellite loci of the human Y chromosome (DYS19, DYS390, DYS391, DYS392, and DYS393) was analyzed in samples of male populations from Ukraine, Russia, and Belarus (152 subjects in total). The allelic diversity indices (D _g) were determined for all loci; they varied from 0.23 to 0.72. The mean values of this parameter in the Ukrainian, Russian, and Belarussian populations were 0.45, 0.47, and 0.52, respectively. A total of 53 different haplotypes were found in 152 subjects from three populations. The most frequent haplotype was found in 14.5% of the subjects, whereas 35 haplotypes (23%) were each found in only one person. The haplotypic diversity index (D _hp) was 0.94. The genetic distances between the populations studied and some populations of Western and Central Europe were estimated. These data were used to construct a phylogram (tree) of genetic similarity between the populations, which demonstrated that the three Eastern Slavic populations are genetically close to one another and remote from Western European populations.