人蛋氨酸合成酶还原酶A66G基因突变频率的检测

目的 建立一种简便、准确、实用的人蛋氨酸合成酶还原酶(MSR)A66G基因突变频率的检测方法,并了解汉族人MSR基因的分布特点。方法 应用聚合酶链反应(PCR)特异性扩增蛋氨酸合成酶还原酶(MSR)A66G基因序列,扩增产物用限制性内切酶NdeI酶切,聚丙烯酰胺凝胶电泳后,观察酶切位点的限制性片段长度多态性(RFLP)图谱。结果 运用PCR-RFLP法检测了150名汉族人蛋氨酸合成酶还原酶(MSR)A66G基因点突变,其中野生型纯合子频率为25.33％,杂合子频率为52.00％,突变型纯合子频率为22.67％。突变等位基因频率为0.4867。结论 该方法简便、快速、准确,适合于一般实验室检测及大规模的人群调查,汉族人MSR基因的分布与其他地区人群的分布无明显差异。     

Association of apolipoprotein E gene polymorphism with essential hypertension and its complications

The clinical significance of the apolipoprotein E genotype in patients with hypertension has been a subject of debate. We enrolled 94 patients with hypertension and 102 healthy controls in this study and determined their plasma levels of triglyceride, total cholesterol, high- and low-density lipoprotein-cholesterol, apolipoprotein AI, and apolipoprotein B. The apolipoprotein E genotypes were identified by polymerase chain reaction, restriction fragment length polymorphism, and polyacrylamide gel electrophoresis. Apolipoprotein E3/4 genotype and 4 allele frequencies in the hypertensive group were higher than in controls. In hypertensive patients with apolipoprotein E3/4 and E4/4 genotypes, systolic blood pressure was significantly higher than in those with apolipoprotein E2/3 or E3/3 genotypes. Meanwhile, the plasma levels of total cholesterol, low-density lipoprotein-cholesterol, and apolipoprotein B were higher in hypertensive patients with the .4 allele than the 2 or 3 allele. The echographic measurements of carotid artery intimal-medial thickness showed increasing values from 2 to 4 allele carriers in the hypertensive group. Analysis of variance showed that the carotid intimal-medial thickness was significantly greater in hypertensive patients with 4 alleles compared with 2 or 3 alleles. Our data show an association between apolipoprotein E genotype and hypertension and support the hypothesis that the apolipoprotein 4 allele is a susceptibility locus for systolic hypertension and carotid artery atherosclerosis. Received: 23 July 2002 / Accepted: 27 December 2002 Correspondence to Xiaotao Li     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

Identification of a splice mutation at the adenine phosphoribosyltransferase locus in a German family

Summary We examined the molecular basis of adenine phosphoribosyltransferase (APRT) deficiency in homozygous-deficient, identical twin brothers who were born to non-consanguineous German parents. DNA was isolated from blood, and the APRT gene was amplified by PCR, subcloned into M13, and sequenced completely. A single T insertion between bases 1831–1832 or 1832–1833 was identified. This alters the consensus sequence at the exon 4 — intron 4 splice donor site and leads to aberrant splicing. The same mutation has been described previously in two affected brothers from Belgium, and the Indianapolis group has also identified it in two other, unrelated Caucasian patients. Thus, this mutation may be a common cause of APRT deficiency in the Caucasian population.Abbreviations APRT adenine phosphoribosyltransferase - DHA 2,8-dihydroxyadenine - PCR polymerase chain reaction - PP-ribose-P 5-phosphoribosyl-1-pyrophosphate - RFLP restriction fragment length polymorphism     

人CYP3A4第9外显子基因突变频率的检测

目的建立一种简便、准确、实用的人CYP3A4第9外显子基因突变频率的检测方法，并了解汉族人CYP3A4第9外显子的分布特点．方法应用聚合酶链反应（PCR）特异性扩增人CYP3A4第9外显子基因序列，扩增产物用限制性内切酶Hinf Ⅰ酶切，琼脂糖凝胶电泳后，观察酶切位点的限制性片段长度多态性（RFLP）图谱．结果运用PCR—RFLP法检测了92名汉族人CYP3A4第9外显子基因点突变，其中野生型纯合子频率为85．9％，杂合子频率为14．1％，突变型纯合子频率为O．突变等位基因频率为0．0706．结论该方法简便、快速、准确，适合于一般实验室检测及大规模的人群调查，汉族人CYP3A4第9外显子也存在相同的突变位点．     

A family with hydrocephalus as a complication of cerebellar hemangioblastoma: identification of Pro157Leu mutation in the VHL gene

Various mutations in the VHL gene on chromosome 3p25–26 are responsible for von Hippel-Lindau (VHL) syndrome. We report on a Japanese VHL family in which two of the three affected members developed acute occlusive hydrocephalus that necessitated emergency surgery for ventricular shunt or drainage. Direct sequencing and restriction fragment length polymorphism analysis identified a germline missense mutation, Proline-to-Leucine, caused by a C-to-T transition at the second nucleotide of codon 157. Received: September 1, 1999 / Accepted: September 22, 1999     

内皮型一氧化氮合酶基因G894T多态性与青海省汉族妊娠高血压综合征的相关性

目的探讨内皮型一氧化氮合酶(eNOS)G894T基因多态性与青海省汉族妊娠高血压综合征(PIH)的相关性。方法采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)方法对138名妊娠高血压综合征患者和135名正常孕妇e NOS基因G894T多态性进行分型,并测序验证。结果妊娠高血压综合征组基因型频率野生型(GG)、杂合子型(GT)和突变纯合子型(TT)分别为17.4%、82.6%、0;对照组分别为0、95.6%、4.4%,两组基因型频率分布有差异(P0.05)。妊娠高血压综合征组e NOS等位基因频率G、T分别为58.7%、41.3%;对照组分别为47.8%、52.2%,两组比较差异有统计学意义(P0.05),妊娠高血压综合征组G基因频率高于对照组。结论e NOS基因G894T多态性可能与青海省汉族妊娠高血压综合征有关,G等位基因可能为妊娠高血压综合征的易感基因(OR=1.229,95%CI:1.048~1.441),T等位基因可能为妊娠高血压综合征的保护基因。携带GG基因型的孕妇可作为青海省妊娠高血压综合征的易感人群。     

Missense mutation of the β-cardiac myosin heavy-chain gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. We describe a missense mutation of the β-cardiac myosin heavy chain (MHC) gene, a G to T transversion (741 Gly→Trp) identified by direct sequencing of exon 20 in four individuals affected with familial hypertrophic cardiomyopathy. Three individuals with sporadic hypertrophic cardiomyopathy, whose parents are clinically and genetically unaffected, had sequence variations of exon 34 of the α-cardiac MHC gene (a C to T transversion, 1658 Asp→Asp, resulting in FokI site polymorphism), of intron 33 of the α-cardiac MHC gene (a G to A and an A to T transversion), and also of intron 14 of the β-cardiac MHC gene (a C to T transversion in a patient with Noonan syndrome). Including our case, 30 missense mutations of the β-cardiac MHC gene in 49 families have been reported thus far worldwide. Almost all are located in the region of the gene coding for the globular head of the molecule, and only one mutation was found in both Caucasian and Japanese families. Missense mutations of the β-cardiac MHC gene in hypertrophic cardiomyopathy may therefore differ according to race. © 1995 Wiley-Liss, Inc.     

Mitochondrial DNA RFLP and genetical studies of cytoplasmic male sterility in the sunflower (Helianthus annuus)

Fifteen sunflower (Helianthus annuus L.) cytoplasmic male-sterile, and a single male-fertile, cytotypes were studied by both mtDNA (mitochondrial DNA) restriction fragment length polymorphism (RFLP) and genetical analysis of male-fertility restoration patterns. It was found by multivariate analysis that the two methods of identification of cytoplasmic male sterility (CMS) should be of use in sunflower breeding programs. The RFLP study distinguished 13 groups based on differences in mtDNA organization. DNA molecular diversity occurs both within and between the Helianthus species from which the steriles originate. The mitochondrial genes analyzed present specific molecular configurations for each type of sterility studied. The analysis of male-fertility restoration separated the cytotypes into 12 groups. The associations of CMS and inbred restorer lines indicated the presence of specific nuclear genes involved in cytoplasmic male-sterility restoration.     

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi.

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3′ untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.     

汉族人维生素D受体基因TruⅠ酶切位点多态性及其对BsmⅠ酶切位点多态性测定的影响

目的测定汉族人维生素D受体基因TruⅠ酶切位点多态性分布并探讨其对BsmⅠ酶切位点多态性分布测定的影响。方法收集80名健康汉族人外周静脉血标本,提取基因组DNA,用限制性片段多态性长度酶切法测定80名汉族人维生素D受体基因TruⅠ、BsmⅠ酶切位点多态性;换用常规引物再次测定上述标本BsmⅠ酶切位点多态性;分析维生素D受体基因TruⅠ、BsmⅠ酶切位点多态性及两次测定的BsmⅠ位点的一致性。结果测得TruⅠ基因型频率为TT68.7%,Tt26.3%,tt5.0%;同一PCR片段上测得BsmⅠ位点基因型频率为BB6.2%,Bb52.5%,bb41.3%,多态性分布均符合Hardy-Weinberg平衡;换用常规引物测定同批标本BsmⅠ位点多态性,基因型分布为BB20.0%,Bb26.2%,bb53.8%,不符合Hardy-Weinberg平衡（r=13.29,P〈0.01）。与第1次测定相比,有22个标本基因型由Bb型变成BB型或bb型,发生基因型丢失。结论汉族人VDR基因存在TruⅠ多态性,其多态性分布与其它种族不同;TruⅠ酶切位点多态性可引起BsmⅠ位点多态性测定时等位基因的丢失。     

The HLA-A3, Cw6, B47, DR7 extended haplotypes in salt losing 21-hydroxylase deficiency and in the Old Order Amish: identical class I antigens and class II alleles with at least two crossover sites in the class III region

Abstract: The HLA-B47, DR7 haplotype in congenital adrenal hyperplasia (CAH) due to 21–hydroxylase deficiency contains a deletion of most of the active CYP21 gene and the entire adjacent C4B gene. The C4A gene produces a protein which is electrophoretically C4A but anti-genically C4B. In the Old Order Amish, the HLA-B47. DR7 haplotype contains no deletion, but is immunologically identical to the CAH haplotype in both areas flanking the crossover region. We compared some of the genes in the MHC Class II and Class III regions in the Amish and CAH-linked haplotypes to define further the relationships between the two. The complement factor B (Bf) proteins differed, but no Bf RFLPs were identified. The complement factor 2 genes exhibited different BamHI RFLPs. Analyses of the tumor necrosis factor-α genes revealed the same Ncol restriction patterns. The RD genes contained microsatellites of the same size. Portions of the MHC Class II DR and DQ , and Class III CYP21 and C4 alleles were sequenced. The exon 2 sequences of DQ2 and DR7 were identical in the two haplotypes. In the Amish haplotype, both CYP21 and C4 gene pairs were present and functionally normal. The CAH haplotype had two sequence crossovers: from CYP21P to CYP21 in the 7th intron, and from C4A to C4B between codons 1106 (exon 26) and 1157 (exon 28). A model is proposed which accounts for the CAH-linked mutant haplotype arising from a nonmutant homologue via three crossings-over.     

半定量聚合酶链反应在PCR-RFLP酶切法测定基因多态性中的应用

目的探索将半定量聚合酶链反应方法，应用于PCR-RFLP酶切法以提高基因多态性测定精确性和可重复性。方法PCR扩增81例人维生素D受体基因BsmI位点多态性片段，分别在酶量不同的两种酶切体系中测定基因型；PCR产物经半定量后，再次用上述两种体系酶切。考察4次测定的一致性。结果经产物半定量后用PCR-RFLP法测定VDR基因BsmI多态性，不同酶切系统测得基因多态性的一致性明显优于未经半定量的结果。结论应用半定量PCR方法可明显提高PCR-RFLP酶切法的精确性和可重复性。     

泉州地区SLC25A13基因常见突变携带者频率的研究及意义

目的 改良citrin缺陷病致病基因SLC25A13常见突变851del4的诊断方法,并对泉州地区该基因常见突变的人群携带率进行探讨.方法 于450名健康成人中筛查SLC25A13常见突变851del4、1638-1660dup及IVS6+5G＞A的突变携带者,应用改良SLC25A13基因常见突变851del4的诊断方法,论证本诊断方法的可行性.结果 共发现6例851del4、3例1638-1660dup及3例IVS6+5G＞A突变携带者.其总携带者频率为0.027(12/450),本研究采用的新型诊断方法筛查的851del4突变人群携带者经(eneScan方法得到确认.结论 本研究改良的诊断方法对突变85ldel4诊断明确且简单实用,泉州地区SLC25A13常见突变携带者频率略高于台湾等周边地区,在该地区应存在一定数量的citrin缺陷病患者,对于该类代谢遗传病应予重视,以免发生因误诊或延误诊治而导致的不良后果.     

中国汉族人念珠状发患者hHB6基因突变检测

目的 对同患念珠状发的母女俩患者进行分子遗传学分析,确定其致病原因.方法 调查患者家系系谱,取患者和正常对照的头发进行常规的显微观察,获取临床信息.抽提所有参与实验的家系成员的全基因组DNA.PCR扩增人类毛发碱性角蛋白6基因(human hair basic keratin 6 gene,hHB6)的所有外显子和外显子-内含子交界区.采用直接测序法检测突变.用限制性片段长度多态性分析验证突变和在该家系内突变是否与疾病共分离,以及该突变是否在正常人群中存在.结果 在其中一个患者中检测到了hHB6基因的一个杂合突变c.1204G＞A(p.E402K).限制性片段长度多态性分析确证了母女俩患者均携带该突变,而家系中其他人(表型均正常)和150名不相关的正常中国汉族人不携带该突变.结论 在中国汉族人念珠状发患者中检测到了hHB6基因c.1204G＞A(P.E402K)突变,由母亲遗传给其女儿.结果表明毛发角蛋白hHB6在念珠状发发病机制中起关键的作用,初步显示hHB6基因常见c.1204G＞A(P.E402K)突变也是导致中国人患念珠状发的原因.     

Molecular analysis of HLA-DQ A alleles in coeliac disease lack of a unique disease-associated sequence.

Susceptibility to coeliac disease is strongly associated with some HLA class II antigens, encoded by the HLA-D region. Since the HLA-DQ locus seems to be primarily involved, we have analysed by polymerase chain reaction amplification and allele-specific oligonucleotide hybridization the most polymorphic region of the HLA-DQ A1 gene. No difference was observed between the 20 coeliac patients and 20 HLA-D-matched healthy controls who took part in the study. Furthermore, in patients and controls, the restriction fragment length polymorphism analysis of the HLA-DQ A gene using the restriction enzyme BglII did not disclose any specific disease-associated fragment. Our results are not consistent with a unique DQ A coeliac disease-associated sequence, but rather with the hypothesis that some polymorphic residues or allelic hypervariable regions, although found also in the normal population, can predispose to coeliac disease due to their higher frequency in this condition.     

Leber遗传性视神经病变线粒体DNA突变的研究

目的 探讨Leber遗传性视神经病变患者的线粒体DNA突变类型及特点.方法 分别应用等位基因特异性PCR（MSP-PCR）、聚合酶链反应-限制性片段长度多态性（PCR-RFLP）和聚合酶链反应-单链构象多态性（PCR-SSCP）联合DNA测序的方法,对12个家系中21位临床症状疑诊为LHON的患者及其19位无明显眼疾的母系亲属进行线粒体DNA检测.结果 40例受检者中35例发生11778位点突变,2位成员有3460位点突变,有1例发现有4258位点突变（A→G）.结论 11778是LHON患者常见的突变位点,3460突变少见,新发现的突变位点4258可能是新的继发突变或基因多态性.     

宫颈脱落细胞标本中人乳头瘤病毒16、18型E6基因的检测

观察宫颈脱落细胞标本是否能替代宫颈组织标检测HPV16、18型的感染。通过聚合酶链反应－限制性片段多态性分析。比较127例宫颈患者的单、双份宫颈脱落细胞和宫颈病变组织中HPV16、18型E6基因的检出率。发现单、双份宫颈脱落细胞标本中HPV16型E6基因的检出率分别为34．64％和40．73％;HPV18型的检出率为17．32和22．04％,存在差异。组织标本中HPV16、18型的检出率分别为41．73％和22．83％,与双份脱落细胞本的检出率无明显,表明双宫颈脱落细胞标本可替代宫颈组织标本检测患者宫颈组织中HPV16、18型感染情况。     

Use of the RB1 cDNA as a diagnostic probe in retinoblastoma families

Use of an intragenic BamHI restriction fragment length polymorphism within the 5' end of the retinoblastoma gene (RB1) provided improved genetic counselling for five familial and ten non-familial retinoblastoma patients and their relatives. All other polymorphic probes within RB1 were uninformative in three families, and accuracy of diagnosis was improved by use of this polymorphism in two families. In 10/14 informative constitutional DNA-RB tumor DNA pairs, a reduction to homozygosity allowed identification of the RB1 allele at risk to carry a germline RB1 mutation.     

血管紧张素原基因核心启动子区域突变与藏族原发性高血压的关联分析

目的 寻找血管紧张素原(angiotensinogen, AGT)基因核心启动子区域存在的突变,分析该突变在中国西藏人群中的分布以及与原发性高血压的关联.方法 以藏族103例原发性高血压患者和82名健康受试者为研究对象进行病例-对照研究.用聚合酶链反应-单链构象多态性(polymerase chain reaction/single strand conformation polymorphism, PCR/SSCP)分析和自动荧光测序方法,对AGT基因核心启动子区域DNA序列进行突变分析;用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction/restriction fragment length polymorphism, PCR-RFLP)方法分析AGT基因(-6)位点多态性.结果 PCR/SSCP分析发现,AGT基因转录起始位点上游(-20)位存在A→C突变,统计分析显示,藏族正常人群与高血压人群中该位点A等位基因均有较高的发生频率(0.9175,0.9124),突变位点多态性分布无统计学差异(P＞0.8).AGT基因转录起始位点上游(-6)位点存在A→G突变,在藏族正常人群中等位基因A和G分布频率分别为0.780和0.220,原发性高血压群体中它们的分布频率分别为0.626和0.374,两者之间存在差异(P＜0.025).结论 (1)藏族群体中AGT基因(-20)A等位基因有较高的分布频率;(2)AGT基因(-6)G等位基因在藏族高血压患者群体中发生频率较高,可能是藏族原发性高血压的遗传易感因子.