单倍型分析技术研究进展

单倍型是指共存于单条染色体上的一系列遗传变异位点的组合,每条染色体都有自己独特的单倍型。单倍型分析技术作为一种常用的数据分析方法,是寻找单染色体上杂合SNP变异位点的有效方法,也对挖掘致病基因、寻找疾病治疗新方法有重要作用。它主要包括间接推断法和直接实验法。文中介绍了各种单倍型分析方法及应用,尤其详细介绍了单分子稀释法和保留邻近性的转座酶测序法,同时对单倍型分析技术的应用前景进行了展望。     

INCENP基因功能性单倍型可调控启动子活性并影响公牛精液品质

为研究内着丝粒蛋白(Inner centromere protein, INCENP)基因启动子区单核苷酸多态性(SNPs)与精液品质的相关性,本文利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测了250头中国荷斯坦公牛INCENP基因的基因型。在INCENP基因启动子区鉴定出两个SNPs (g.-556 G>T,rs 136823901和g.-692 C>T,rs 211010999),发现了3种单倍型(CG、TT、TG)。分析两个SNP位点的基因型频率和等位基因频率,各SNP及单倍型组合与中国荷斯坦公牛精液品质的相关性,结果表明SNP位点g.-556 G>T GT基因型个体的鲜精活力显著高于GG基因型个体(P<0.05),单倍型组合H1H1(CCGG)、H1H3(CTGT)、H2H3(TTGT)和H3H3(TTTT)个体的鲜精活力和冻精解冻后活力均显著高于H1H2个体(P<0.05)。为进一步研究g.-556 G>T和g.-692 C>T影响精液品质的可能机理,本文将3种单倍型质粒分别转染小鼠睾丸间质细胞(MLTC-1),结果显示含TG单倍型的载体荧光素酶活性最高。由此推测,g.-556 G>T和g.-692 C>T为启动子区功能性突变位点,可通过调节启动子活性来调控INCENP基因表达,进而影响精液品质。     

斑点叉尾GHRH基因3个SNPs位点及其单倍型组合与生长性状的关联分析

研究旨在探讨生长激素释放激素基因（Growth hormone-releasing hormone,GHRH）对斑点叉尾鲖（Ictalurus punctatus）生长性状的影响。采用DNA混池测序法筛选GHRH基因的单核苷酸多态性（Singlenucleotide polymorphisms,SNPs）位点,使用SNaPshot法将筛选到的SNPs多态性位点进行分型,并对这些位点进行连锁不平衡和单倍型分析。结果表明,在GHRH基因内含子区域共检测到4个SNPs位点,并成功地对3个位点进行了分型,3个位点间均不存在强连锁不平衡;3个SNPs位点在176尾斑点叉尾鲖中形成了6种有效单倍型。关联分析表明SNP位点g.6301 GA的AA基因型的体质量显著性地高于AG和GG型（P0.05）,比群体的平均体质量高14%。单倍型组合H1/H4和H1/H5个体的体质量和体长极显著性地高于其他单倍型组合（P0.01）,体质量比群体平均体质量分别高30%和15%,体长比群体平均体长分别高7%和6%。研究为斑点叉尾鲖生长性状分子标记辅助选育和QTL定位提供了参考依据。     

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。     

南海野生斑节对虾的线粒体16S rRNA基因序列单倍型分析

采用聚合酶链式反应(PCR)技术对我国南海海域5个地点(三亚、深圳、阳江、湛江、北海)的野生斑节对虾100个个体的mtDNA 16S rRNA序列进行扩增,扩增产物经纯化后进行测序.用Clustal_X排序软件对所得的100个mtDNA 16S rRNA序列进行比对.通过ARLEQUIN软件对所得100个mtDNA 16S rRNA序列进行比较分析,共检测出28个变异位点,19种单倍型.19单倍型序列的碱基组成显示出较高的A T比例(69.0%).19种单倍型序列的遗传距离在0.002～0.033.根据构建的单倍型进化关系网,5个地点群体中的单倍型呈现一种混杂的分布格局.此外,根据单倍型在5个地点群体出现的频率和单倍型进化关系网,单倍型7是最为原始的单倍型,其他18种单倍型均起源于单倍型7.     

AGT基因单倍型与原发性高血压

选取血管紧张素原（angiotensinogen, AGT）基因启动子区 -217, -152, -20, -6, 内含子1 的+31, 第二外显子T174M（3889）和 T235M（4072）共7个位点,对497例的样本（高血压患者298例,血压正常对照199例） 用PCR-RFLP、和最大期望值（expectation maximization,EM）算法为基础的最大似然法(maximum likelihood estimate,MLE)检测和估算,本群体AGT基因A-6G,C+31T,T235M三位点两两存在完全连锁不平衡(D^,=1);G-217A和G-152A位点,G-152A和3889T位点平衡传递。存在7种单倍型,单倍型H2(-217: A, -152: G, -20: A, -6: G, +31: T, 174: T, 235: M) 在正常血压个体中的频率高于高血压组。研究结果提示AGT基因中H2单倍型可能与控制血压的保护性因素连锁不平衡。此外,本研究结果支持基因剂量效应可能存在于单倍型中,而不与单个位点直接关联。     

猪单个精子单倍型分析方法研究

应用引物延伸预扩增,内嵌引物设计策略进行了ＰＣＲ扩增并结合聚丙烯酰胺凝胶电泳及银染技术,对猪单个精子１２号染色体上４个微卫星位点进行了ＰＣＲ扩增。结果表明,上述技术的应用可以清晰地鉴定出每个精子的单倍型,单精子分型技术的应用为猪高精度遗传作图及需要样本量足够的大遗传现象研究提供了独特的工具。     

福建省马铃薯晚疫病菌线粒体DNA单倍型分析

为明确福建省马铃薯晚疫病菌线粒体DNA单倍型组成和分布情况,采用PCR-RFLP方法分析了2010—2012年从福建省7个地区（福州,长乐,漳州,青口,龙海,霞浦,龙岩）分离的526个马铃薯晚疫病菌株的线粒体DNA单倍型及频率分布。结果表明Ⅰa型为主要单倍型,占72．8％,其次是Ⅱb型,占26．42％,最少的Ⅱa型,占0．76％,没有检测到Ⅰb型。与文献报道历史材料相比较,说明福建省马铃薯晚疫病菌线粒体DNA单倍型的组成发生较大变化,而且福建省马铃薯晚疫病菌线粒体单倍型组成比云南、四川和贵州等地区更为复杂。     

汉族人线粒体DNA单倍型与散发型帕金森病关系的研究

目的：研究汉族人线粒体DNA（mtDNA）单倍型与散发性帕金森病（PD）的关系。方法：用酚／氯仿法从65例PD患者和50名健康者（对照组）的外周静脉血中提取基因组DNA,通过斑点杂交方法对PD患者和对照组的10个单核苷酸多态性（SNP）位点（G1719A,G4580A,C7028T,G8251A,G9055A,A10398G,A12308G,G13366A,C13708T,G16391A）和单倍型进行检测。结果：PD组G1719A突变5例、G9055A突变2例、C13708T突变4例,对照组G1719A突变1例,2组的突变率无显著性差异（P〉0.05）。PD组H单倍型54例（83.1％）,其他11例（16.9％）;对照组H单倍型49例（98.0％）,其他1例（2.0％）。结论：10398G不是汉族人散发性PD发病的易感因子,也没有保护作用;不宜用H、I、J、K、T、U、V、w和x等9个mtDNA单倍型来评估汉族人散发性PD发病风险。     

白色念珠菌分泌型天冬氨酸蛋白酶在口腔扁平苔藓患者唾液中的表达

研究白色念珠菌分泌型天冬氨酸蛋白酶(SAP)家族在扁平苔藓患者唾液中的表达,进而探讨白色念珠菌病与扁平苔藓的关系。选取白色念珠菌携带者、白色念珠菌病患者、携带白色念珠菌的扁平苔藓患者、伴有白色念珠菌病的扁平苔藓患者各20例,念珠菌培养阴性的扁平苔藓患者10例。从唾液标本中直接提取RNA,进行RT-PCR。白色念珠菌携带者唾液中有SAP2、SAP4-6、SAP7的不同表达,携带白色念珠菌的口腔扁平苔藓患者唾液中有SAP2、SAP4-6、SAP7、SAP8、SAP9的不同表达,所有携带者中均未表现SAP1、SAP3和SAP10的阳性扩增。白色念珠菌患病者及伴有白色念珠菌病的口腔扁平苔藓患者唾液中有SAP1-10的不同表达。念珠菌培养阴性的扁平苔藓患者均无SAP1-10的阳性扩增。SAP9与扁平苔藓密切相关。     

Haplotype block and superblock structures of the alpha1-adrenergic receptor genes reveal echoes from the chromosomal past

A significant proportion of the human genome is contained within haplotype blocks across which pairwise linkage disequilibrium (LD) is very high. However, LD is also often high between markers at more remote distances, and within different haplotype blocks. Here, we evaluate the origins of haplotype block structure in the three genes for alpha1 adrenergic receptors (alpha1-AR) in the human genome ( ADRA1A, ADRA1B and ADRA1D) by genotyping dense single-nucleotide polymorphism (SNP) marker maps, and show that LD signals between distant markers are due to the presence of extended haplotype superblocks in individuals with ancient chromosomes which have escaped historic recombination. ARs mediate the physiological effects of epinephrine and norepinephrine, and are targets of many therapeutic drugs. This work has identified haplotype backgrounds of alpha1-AR missense variants, haplotype block structures in US Caucasians and African Americans, and haplotype tag SNPs for each block, and we present strong evidence for ancient haplotype block superstructure at these genes which has been partially disrupted by recombination, and evidence for reinstatement of linkage disequilibrium by subsequent recombination events. ADRA1A is comprised of four haplotype blocks in US Caucasians, while in African Americans Block 1 is split. ADRA1B has four blocks in US Caucasians, but in African Americans only the first two blocks are present. ADRA1D has two blocks in US Caucasians, and the first block is replaced by two smaller blocks in African Americans. For both ADRA1A and ADRA1B, haplotype superstructures may represent a novel, higher-level hierarchy in the human genome, which may reduce redundancy of testing by further aggregation of genotype data.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Th1型和Th2型细胞因子在口腔扁平苔藓患者外周血中的表达及临床意义

目的:研究口腔扁平苔藓(OLP1)患者Th1型和Th2型细胞因子的表达及临床意义。方法:选取2013年1月至2014年3月于我院就诊的21例充血糜烂型及14例光滑型OLP患者为研究对象,18例正常人为对照组,采用密度梯度离心法对各组外周血单个核细胞进行分离,酶联免疫吸附剂测定(ELISA)法对各组外周血单个核细胞中的IL-4和IFN-gamma的表达进行检测,逆转录-聚合酶链反应法对各组血清中IL-4 m RNA和IFN-gamma m RNA的表达进行检测。结果:与正常对照组相比,OLP患者IL-4m RNA及蛋白的表达均增高,而IFN-gamma m RNA及蛋白的表达则降低,差异均有显著统计学意义(均P0.01)。充血糜烂型及光滑型OLP患者组间比较发现,IL-4 m RNA和IFN-gamma m RNA及蛋白的表达差异无统计学意义(P0.05)。结论:OLP发病机制与Th1与Th2的表达失衡有关,为临床治疗提供参考。     

Alteration of Streptococcus salivarius in Buccal Mucosa of Oral Lichen Planus and Controlled Clinical Trial in OLP Treatment

Oral lichen planus (OLP) is a T cell-mediated common chronic inflammatory mucosal disease, with limited therapies available for long-term use. Previous study showed that ratio of genus Streptococcus decreased significantly in OLP patients when compared with controls. Buccal cotton swab samples of 43 OLP patients and 48 healthy individuals were collected for real-time quantitative polymerase chain reaction (RT-PCR) to investigate relative abundance alteration of Streptococcus salivarius in OLP lesions. Bacterial supernatants of S. salivarius ATCC® BAA-2593™ were collected by centrifugation and added to HSC-3 cells, and quantitative analysis of expression of IL-1β, IL-6, IL-8, and TNF-α in the HSC-3 cells was determined by RT-PCR. Then, a randomized, non-blinded, controlled study was conducted. Forty patients with symptomatic OLP were randomly allocated into two groups and received topical treatment of 0.1% triamcinolone acetonide dental paste (group A) and S. salivarius K12 lozenge (group B), respectively, for 4 weeks. Sign scores, visual analogue scale (VAS), and adverse reactions were recorded. Relative abundance of S. salivarius in the OLP group was lower than that of control group (P < 0.05). After treated with 0.1% supernatants of S. salivarius ATCC® BAA-2593™, the expression level of IL-6 in the HSC-3 cells significantly reduced (P < 0.001), while IL-1β, IL-8, and TNF- α showed a decreasing tendency (P > 0.05). There was significant reduction in sign scores and VAS scores in both groups after the 4-week treatment, with no significant difference between two groups. No adverse reaction was observed. S. salivarius might maintain local immune balance by inhibiting the NF-κB pathway. Topical application of Streptococcus salivarius K12 seemed to be effective in treatment of symptomatic OLP, especially with promising potential in long-term use. More detailed clinical studies with long follow-up period and standardized usage/dosage are expected to acquire definite conclusions.