RET原癌基因C634R突变合并G691S、R982C多态性致多发内分泌腺瘤病2A型的家系研究并文献复习

目的 对一个多发内分泌腺瘤病2A型(MEN2A)家系患者的临床特点及RET原癌基因突变情况进行分析.方法 收集先证者及其家系成员相关临床资料,提取1名先证者及10名家系成员的外周血基因组DNA,对RET基因所有外显子进行PCR扩增,对扩增产物进行测序分析.结果 家系中3位已发病患者的临床表现不同,但其RET原癌基因均同时存在3个错义突变,分别是位于11号外显子的C634R、G691S和位于18号外显子的R982C,而家系中其他成员则不存在上述突变.结论 当C634R与G691S、R982C多态性同时存在时,临床上主要表现为与单纯C634R突变一致的PET蛋白功能异常激活性疾病.     

WFS1基因在低频听力减退家系中的突变筛查

目的 分析低频感音神经性听力减退(LFSNHL)家系的候选基因——WFS1基因的突变。方法 对WFS1基因的全部编码序列设计14对引物，进行PCR扩增反应。应用PCR产物直接测序的方法对LFSNHL。家系中的38名成员进行突变检测及鉴定。结果 WFS1基因的14对引物均有较好的扩增效果，直接测序结果与标准序列比对分析显示所有家系成员在WFS1基因中均未检测到可以引起蛋白质改变的突变。结论 本研究中的LFSNHL中国家系的致病基因并非WFS1基因突变所致。     

实时荧光定量PCR检测应力性骨折新兵外周血TNFRSF10C mRNA表达

目的:检测TNFRSF10C基因在应力性骨折(SF)患者与正常人之间的表达差异。方法:采用1:1配比的病例对照研究,选取入伍新兵SF病例及正常对照各10例,取受试者空腹肘静脉血,采用实时荧光定量PCR检测TNFRSF10C基因表达,结合管家基因GAPDH进行相对定量分析。结果:设计并合成了一组只特异性结合人TNFRSF10C基因的实时荧光定量PCR引物,结果未发现非特异性扩增。10例SF样本中有7例TNFRSF10C基因表达上调,上调1.4倍至6.7倍,另3例SF样本TNFRSF10C基因表达下调,分别为50%、90%和90%。结论:本研究建立的TNFRSF10C基因实时荧光定量PCR检测体系特异性好、可靠。TNFRSF10C基因表达结果为分析SF发病的分子机制及SF早期诊断提供了重要线索。     

DHPLC在X-连锁肾上腺脑白质营养不良分子诊断中的应用(附12例报告)

目的探讨DHPLC在X-连锁肾上腺脑白质营养不良(X-ALD)分子诊断中的应用。方法提取12个X-ALD家系及成员的外周血基因组DNA,分15个片段扩增ABCD1基因的10个外显子,应用DHPLC技术对其进行突变筛查,并对出现异常洗脱峰的PCR产物进行DNA序列测定,证实突变位点的存在。结果12个X-ALD家系存在12种不同的ABCD1基因突变,包括8个错义突变、2个移码突变和2个无义突变,即P534R、G343V、R259W、A141T、R401Q、K276E、Y174C、A314P、fs E471、fs A247、S108X和Q177X。结论DHPLC筛查结合DNA序列测定能快速有效检测出ABCD1基因突变。不同的X-ALD家系有不同的ABCD1基因突变位点,突变类型和表型之间无特殊相关关系。     

7个肾上腺脑白质营养不良家系的基因突变分析

目的对7个肾上腺脑白质营养不良家系进行基因突变分析。方法应用RT-PCR技术，对7位患者的ABcDl基因编码区分4个片段进行扩增并对PCR产物直接测序。应用PCR-限制性酶切或DNA测序等方法分析相应的基因组DNA，进一步确证ABCD1基因的突变位点。结果在7位患者的ABCD1基因上存在6个不同的碱基置换（709C→A、807G→A、1161C→T、2065C→T、2113T→C和2235C→T）、1个碱基缺失（1801delAG）和1个碱基插人（1126insCCATCG），分别造成5个错义突变（A141T、R259W、P560L、L76P和R617C）、2个移码突变（fs I246和fs E471）和1个无义突变（S108X）。结论在中国人肾上腺脑白质营养不良患者中发现4个新的ABCD1基因突变，即S108X、fs I246、R259W和IA76P突变。不同家系具有不同的突变位点，且突变类型和表型之间无特殊的相关性。     

巢式PCR分析电离辐射诱导人外周血线粒体DNA4977bp缺失

目的 探索对不同剂量电离辐射诱导的人外周血线粒体DNA4977bp缺失进行分析的方法。方法 采用3对引物对γ射线诱导的人外周血线粒体DNA缺失进行PCR扩增，建立对人线粒体DNA4977bp缺失进行分析的巢式PCR方法．扩增线粒体DNA缺失区片段，纯化PCR产物进行测序。采集5例正常人外周血进行0～5Gy^60Coγ射线的外照射，利用巢式PCR技术，分析^60Coγ射线照射后外周血有核细胞中线粒体DNA4977bp缺失的发生情况。结果 用建立的巢式PCR方法可在受到照射的外周血样品中检测到mtDNA4977bp缺失。所有外周血样本在照射前均无线粒体DNA4977bp缺失，1～5Gy^60Coγ线照射后均诱导出此缺失。结论 本研究所建立的巢式PCR方法可用来分析电离辐射诱导的人外周血淋巴细胞线粒体DNA4977bp缺失。     

电离辐射致线粒体DNA 4977 bp缺失质粒构建及其鉴定

目的 建立电离辐射诱导的人线粒体DNA（mtDNA）4977bp缺失片段和对照DNA片段的稳定质粒，用于线粒体基因组相关研究。方法 对无mtDNA 4977 bp缺失的正常人外周血进行离体照射10Gy^60Coγ射线，提取细胞总DNA，用巢式PCR扩增mtDNA 4977 bp缺失片段，普通PCR扩增对照ND1基因片段。PCR产物经纯化后构建质粒；提取质粒DNA，DNA样品经酶切、纯化后测序，将测序结果进行BLAST分析。结果 PCR扩增的跨mtDNA 4977 bp缺失的DNA片段和ND1基因片段大小与预期值是吻合的。对制备的质粒DNA样品进行测序，结果 经BLAST分析，两种质粒DNA与人线粒体序列同源性在99％以上。结论 本研究中所构建的质粒是成功的，可以用于人mtDNA 4977 bp缺失的定性或定量研究。     

电离辐射诱导的两种新线粒体DNA缺失的分析鉴定

目的 筛选电离辐射诱导的线粒体DNA(mtDNA)缺失类型,并进行鉴定。方法 用10 Gy ⁶⁰Co γ 射线照射正常人淋巴细胞细胞系,用2对引物的长PCR扩增照射前后样品的mtDNA基因组。发现可能缺失片段后用限制条件的普通PCR进行确认,并对PCR产物进行纯化、克隆、测序和核酸同源性(BLAST)分析。结果 照射前的淋巴细胞系mtDNA样品只扩增出预期全长片段,照射后每对引物还扩增出可能为缺失造成的短片段;进一步限制条件的普通PCR证实了mtDNA缺失的存在。PCR产物经纯化、克隆后测序进行BLAST分析表明两种mtDNA缺失分别为7455 bp (重链nt475～7929)、9225 bp (重链nt7714～369)的mtDNA缺失,均发生在8bp正向重复序列之间。进一步的生物信息学检索得出两种mtDNA缺失为新发现的mtDNA缺失类型。结论 电离辐射诱导出人淋巴细胞mtDNA 7455 bp和9225 bp缺失。     

聚合酶链反应检测嗜肺军团菌的实验研究

 目的探讨并建立聚合酶链反应(PCR)用于检测嗜肺军团菌的方法.方法用PCR方法扩增嗜肺军团菌巨噬细胞感染增强子基因(mip)片段,琼脂糖凝胶电泳检测扩增产物;分析其特异性和敏感性.结果在嗜肺军团菌14个血清型参考株均扩增出特异的340bp片段,而三株非嗜肺军团菌及其他需鉴别诊断的常见病原菌均未扩增出此特异条带.检测灵敏度为2.6 pg/ul基因组DNA.结论依据mip基因建立的嗜肺军团菌PCR检测方法具有高度的敏感性和特异性,是诊断与鉴别诊断嗜肺军团菌感染的一种有效手段.     

常染色体显性遗传先天性板层白内障家系致病基因的排除性定位

目的 定位-个四代常染色体显性遗传先天性板层白内障家系的致病基因.方法 选取在北京同仁医院就诊的河北任丘先天性白内障家系,记录家系遗传史.该家系28例成员(12例患者,16例非患者)进入本研究,12例患者接受全身及眼部检查,以排除存在白内障以外的眼部及全身疾患,16例非患者仅接受眼部检查.28例研究对象均采集外周静脉血5ml,提取基因组DNA,选取在物理距离上与已知非综合征常染色体显性遗传性先天性白内障相关的18个致病基因紧密连锁的微卫星分子标记,基因组聚合酶链式反应(PCR)扩增后进行基因分型.以基因分型的结果为基础,利用等位基因共享分析和基因测序对已知候选基因进行排除定位.结果 该家系遗传特点符合常染色体显性遗传,临床表型为板层先天性白内障;与位于1、2、10、11、12、16、17、21、22染色体上的15个致病基因附近的微卫星位点均不存在等位基因共享,基因测序排除了微卫星杂合度较低(位于3、13,19号染色体)的基因座.结论 该家系存在新的致病基因,进一步确证了先天性白内障具有高度临床和遗传异质性.该家系致病位点的确定有待于进一步研究.     

RHC/c genotyping based on polymorphism in the promoter region of the RHCE gene

Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between RHC/c and RHD were found at positions -468, -304, -58, and -46. On the basis of the nucleotide differences at positions -468 (RHCE vs. RHD) and -292 (RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than either of the methods alone, and therefore, useful and valuable for RHC/c genotyping.     

Demonstration of A antigen and A allele of ABO histo-blood group in nail in a case with the absence of A antigen and anti-A antibody in blood

We report a case with the inconsistency that the red blood cells lacked both A- and B-antigens while the serum showed reactivity with control B-red cells but not with A-red cells. A- and B-antigens were examined by serological blood typing and immunohistochemical staining, and DNA analyses were performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, and hot-stop PCR. A-antigens were demonstrable in the nail of the subject by serological study and immunostaining. DNA analyses showed that the nail retained a small amount of A-allele comparing to that of O-allele. Those genomic analyses of ABO genes were useful for demonstration of A allele in the nail of an individual with the absence of A antigen on red blood cells and the corresponding antibody in serum.     

家族性多发性内分泌腺瘤2A型的基因诊断

目的：提高对多发性内分泌腺瘤2型（multiple endocrine neoplasia 2, MEN2）的诊断和预测诊断水平。方法对临床已诊断首发症状为嗜铬细胞瘤的一个家族内两姐妹患者进行RET（外显子10～16）、VHL（外显子1～3）、SDHD（外显子1～4）、SDHB（外显子1～8）及SDHC（外显子1～6）基因测序。发现两患者均有RET基因634号核酸发生突变,并对其家族其他成员的RET基因进行测序。结果其家族内共有5例患者均有RET基因634号突变（ TGC突变为CGC）,导致其氨基酸编码由半胱氨酸变为精氨酸。其中2例先为临床诊断然后基因诊断,1例先由基因诊断然后临床诊断,2例（均为儿童,分别为6岁及7岁）为基因诊断,目前尚未发病。其中3例行腹腔镜双侧肾上腺肿瘤切除术,病理确诊为双侧嗜铬细胞瘤;1例行甲状腺切除术,病理诊断为甲状腺髓样癌。结论对于多发性内分泌多发肿瘤2A型患者及其家族成员行基因检查不仅可以作为确诊手段,更可以作为无症状者的预测诊断途径。     

肺癌乳头状瘤病毒感染与P53基因突变的研究

应用PCR与原位杂交技术检测了40例原发性肺癌乳头状瘤病毒（HPV）的感染率及与肺癌不同组织类型的关系；应用PCR-RFLP技术分析P53基因第七外显子的扩增与突变，结果显示；肺癌HPV阳性率为55%（22/40例），其中SCLC9/9例，鳞癌8/16例，腺癌5/12例阳性，P53基因第七外显子在HPV阳性的标本中有5/22例扩增，RFLP分析2/5例（SCLC，鳞癌各一例）突变，探讨HPV感染，P53基因与肺癌的关系。     

α粒子诱发转化人支气管上皮细胞BEP2D中p53基因的突变特点

目的 观察α粒子诱发BEP2D细胞转化过程中p53基因的改变。方法 聚合酶链式反应－单链构象多态性分析（PCR－SSCP）。结果 分析发现在细胞转化过程中,p53基因发生重要改变,照射后早期传代细胞中,p53外显子7就发生碱基突变,经酶切分析为249密码子的改变,外显子5／6在照后20代细胞内开始丢失,并经Southern杂交证实,以上改变均在转化过程中持续存在;而外显子8／9未见变化。结论p53基因外显子5、6、7是α粒子对DNA损伤的重要靶位点,在α粒子诱发支气管上皮细胞转化的过程中发挥重要作用。     

Genotyping of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions

The genotyping of ABO blood groups was performed using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from A-transferase were amplified by PCR, and the amplified DNA subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was clearly distinguished by electrophoresis of the PCR products amplified with allele-specific primers. By analyzing the electrophoresis patterns, ABO genotyping was conclusively accomplished. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA =19.8 % and AO = 80.2%; and in the phenotype B group, BB =12.8% and BO=87.2%.     

ABO genotyping by inverse PCR technique

Inverse PCR technique was applied to type three major alleles (A(1), B and O(1)) of the ABO blood group by simultaneously detecting separated allele-determining nucleotides (the 261st base in exon 6 and the 796th and 803rd nucleotides in exon 7) of the ABO gene. A sequence of about 1.7 kb from exons 6 to 7 of each allele was amplified, both termini of the fragment ligated, and allele-typing performed by the inverse PCR-restriction fragment length polymorphism (IP-RFLP) and allele-specific inverse-PCR (ASIP) methods. For intramolecular ligation, primers for the first PCR were designed to have Acc I-restriction sites within the sequences, and both termini of the 1.7-kb fragment were digested with Acc I. Using the IP-RFLP method, the inverse PCR product was digested with Kpn I, Nla III and Dde I, A(1), B, O(1)-standard (O(A)) and O(1)-variant (O(G)) alleles were detected as 365-bp, 272-bp, 193-bp and 128-bp fragments, respectively. By the ASIP method using four allele-specific primers, 222-bp, 124-bp and 232-bp fragments were amplified from A(1), B and O(1) templates, respectively. These techniques would be applicable to detecting separated polymorphic regions of some other genes.     

Evaluation of the paternity probability on an application of minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) to paternity testing

Minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) was applied to a practical case of paternity testing to evaluate the paternity probability. In order to obtain single allele mapping by allele-specific MVR-PCR, three flanking polymorphic sites for each of the MS31A and MS32 loci were investigated and all three individuals were typed as heterozygous for at least one flanking polymorphic site at each locus. Allele-specific MVR-PCR was then performed using genomic DNA. It was confirmed that one allele in the child was identical to that from the mother and the other one in the child was identical to that from the alleged father. Mapped allele codes were also compared with those in the database by dot-matrix analysis, and no identical allele was found although some motifs were shared with Japanese alleles. The paternity index and the probability of paternity exclusion in the case at these two MVR loci were calculated using the presumed values of the allele frequencies. These studies seem to illustrate the practical value of MVR mapping of MS31A and MS32 loci in paternity testing.     

A polymorphism in the alpha2a-adrenoceptor gene and endurance athlete status

PURPOSE: In a case control study, we examined the allelic frequencies and genotype distributions of two restricted fragment length polymorphisms (RFLP) in the alpha-2A-adrenoceptor gene (ADRA2A) and beta-2-adrenoceptor gene (ADRB2) among elite endurance athletes (EEA) and sedentary controls (SC). METHODS: The EEA group included 148 Caucasian male subjects recruited on the basis that they had a VO2max > 74 mL O2 x kg(-1) x min(-1). The SC group comprised 149 unrelated sedentary male subjects, all Caucasians, from the Quebec Family Study. After digestion with the restriction enzymes Dra I (ADRA2A) and Ban I (ADRB2), Southern blotting and hybridization techniques were used to detect the mutations in the two ADR genes, which are encoded on chromosomes 10 (q24-26) and 5 (q31-32), respectively. RESULTS: For the Dra I ADRA2A RFLP, we observed a significant difference in genotype distributions between the two groups (P = 0.037). A higher frequency of the 6.7-kb allele was observed in the EEA group compared with the SC group (P = 0.013). No statistically significant difference was found between groups for the Ban I ADRB2 polymorphic site. Genotype frequencies for both genes in both groups were in Hardy-Weinberg equilibrium. CONCLUSIONS: In summary, we found evidence that ADRA2A gene variability detected with Dra I is weakly associated with elite endurance athlete status, and we conclude that genetic variation in the ADRA2A gene or a locus in close proximity may play a role in being able to sustain the endurance training regimen necessary to attain a high level of maximal aerobic power.