用Amp－FLP技术研究中国汉族群体D4S95和DXS52位点的遗传多态性

为研究中国汉族群体Ｄ４Ｓ９５和ＤＸＳ５２位点的遗传多态性，应用改进的Ｄ４Ｓ９５和ＤＸＳ５２位点的扩增片段长度多态性（Ａｍｐ－ＦＬＰ）分析技术，检测了中国汉族个体共２２２名。结果分别为，（１）Ｄ４Ｓ９５位点：在１０８名无关个体中发现了７个等位基因（片段长度为９１０～１１５０ｂｐ），１８种基因型，杂合性为０．７６，个人鉴别力为０．８７１（２）ＤＸＳ５２位点：在１１４名无关个体中（男性７０人，女性４４人）发现了１４个等位基因（片段长度为６９５～２４００ｂｐ），在４４名女性个体中发现了２２种基因型，杂合性为０．７７．个人鉴别力为男性０．８９，女性０．９３１检测了两个家系两代３口之家的两位点的基因型，证明该两位点的基因按Ｍｅｎｄｅｌ定律遗传，该技术在法医科学鉴定中具有重要的应用价值。     

中国人群DXS102座位多态性鉴定及其应用

目的探讨中国人群中ＤＸＳ１０２座位的多态分布。方法应用ＰＣＲ扩增片段长度多态性（Ａｍｐ－ＦＬＰ）研究了无亲缘关系的２３４条Ｘ染色体。结果ＤＸＳ１０２座位等位片段有８个，核心单元ＡＣ二核苷酸重复数为１３～２１，频率分布在０．０１３～０．１５６之间，杂合度观察值和无偏估测值分别为０．８７和０．８０，多态信息含量（ＰＩＣ）０．８０，女性基因型数为２２个，男性基因型数为８个，该座位多态分布符合Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ平衡定律。ＤＸＳ１０２座位在中国人群和欧洲人群的分布有明显的种族差异，在中国人群中发现了两个新的等位片段。应用ＤＸＳ１０２座位的短串联重复序列多态性对一接受基因治疗的血友病Ｂ家系进行分析和携带者筛查。结论ＤＸＳ１０２座位连锁分析有望成为一种有效的血友病Ｂ基因诊断的方法。     

用Amp—FLP技术研究中国汉族群体D4S95和DXS52位点的遗传多态性

为研究汉族群体Ｄ４Ｓ９５和ＤＸＳ５２位点的遗传多态性，应用改进的Ｄ４Ｓ９５和ＤＸＳ５２位点的增片段欧态性分析技术，检测了中国汉族个体共２２２名。结果分别为，（１）Ｄ４Ｓ９５位点：在１０８名无关个体中发了７个等位基因，１８种基因型，杂合性为０．７６，个人鉴别为０．８７；（２）ＤＸＳ５２位：在１１４名无关个体中发现了１４个等位基因，在４４名女性个体中发现了２２种基因型，杂合性为０．７７。     

联合应用STR连锁分析与多重等位基因特异PCR进行PKU基因诊断

应用ＰＣＲ结合银染色显色法分析了苯丙氨酸羟化酶基因内含子３中１个四核苷酸（ＴＣＴＡ）重复序列的多态性，结果表明：在５２例正常人和２３个ＰＫＵ家系中检测到９种等位基因片段，从２２４～２５６ｂｐ连续分布，其中２２４ｂｐ等位片段第１次在中国人群中被检测到，多态信息量（ＰＩＣ）分别为０．６５４和０．７３０。同时分析了ＳＴＲ多态性与多重等位基因特异ＰＣＲ（ＭＡＳＰＣＲ）在ＰＫＵ基因诊断中的联合应用，结果表明     

中国汉族群体c—Ha—ras基因3‘端—VNTR位点遗传多态性的初步研究

为了探讨中国汉族ｃ－Ｈａ－ｒａｓ基因３′端ＶＮＴＲ位点遗传多态性，用ＡＦＬＰ－ＰＡＧＥ法对１１２例无亲缘关系的健康个体进行了ｃ－Ｈａ－ｒａｓ基因３′ＶＮＴＲ位点分析，共检出２１个等位基因，其片段大小分布范围在６３５～２６５１ｂｐ之间，基因频率分布在０．００４５～０．５９３８，父权排除率（Ｐｅ）＝０．４４４５；杂合度（Ｈ）＝０．６２５，个体识别力（Ｄｐ）＝０．８４４０，共发现３４种基因型，经Ｘ＾２检     

中国汉族群体c-Ha-ras基因3′端-VNTR位点遗传多态性的初步研究

为了探讨中国汉族ｃ－Ｈａ－ｒａｓ基因３′端ＶＮＴＲ位点遗传多态性，用ＡＦＬＰ－ＰＡＧＥ法对１１２例无亲缘关系的健康个体进行了ｃ－Ｈａ－ｒａｓ基因３′端ＶＮＴＲ位点分析，共检出２１个等位基因，其片段大小分布范围在６３５～２６５１ｂｐ之间，基因频率分布在０．００４５～０．５９３８。父权排除率（Ｐｅ）＝０．４４４５；杂合度（Ｈ）＝０．６２５；个体识别力（Ｄｐ）＝０．８４４０。共发现３４种基因型，经χ２检验（χ２＝２３７．１１，ｄｆ＝２１０，Ｐ＝０．１０２５），符合Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ平衡。家系调查表明这一位点的等位基因按孟德尔规律遗传。本遗传标记系统在人类学、法医学和医学遗传学的研究中有重要意义。     

我国Marfan综合征基因连锁标记的研究

为了探索对Ｍａｒｆａｎ综合征进行产前诊断和症状前诊断的方法，应用１５号染色体ＦＢＮ１基因内两个多态位点ＴａｑⅠ限制性片段长度多态性（ＲＦＬＰ）和（ＴＡＡＡＡ）ｎ扩增片段长度多态性（Ａｍｐ－ＦＬＰ）为遗传标记，在正常人群中检出前者有５．０ｋｂ（＋）和６．０ｋｂ（－）两种等位基因，基因频率各为２７％和７３％；后者有１５０ｎｔ（１）和１６０ｎｔ（２）两种等位基因，基因频率分别为３１％和６９％，未见白种人中的罕见变异型。两个患病家系的单倍型分离分析表明，致病基因均与－，２单倍型连锁，提示中国人群中Ｍａｒｆａｎ综合征也与ＦＢＮ１基因连锁。以该基因内的多态位点为遗传标记，可对该病的一些家系成员进行产前或症状前诊断。     

汉坦病毒S基因的分段表达及其表达产物的鉴定

用含ＰＲＰＬ启动子的原核表达载体ＰＧＥＸ－４Ｔ系统，构建了含汉坦病毒７６－１１８株Ｓ基因全长片段（１．３ｋｂ）及部分片段（１．１ｋｂ和０．７ｋｂ）的表达载体ｐＧＥＸ－ＨａｎＳ，并大肠杆菌中分别进行了表达，用１８析具有不同特异性的抗汉坦病毒ｍＡｂ以ＥＬＩＳＡ夹心法对上述表达产物进行了检测，结果表明，重组基因组全长片段的表达产物（ＮＰ）与其中１３株抗汉坦病毒组特异性和Ｉ型特异性ＮＰ的ｍＡｂ均可发生反应     

D13S301位点Amp—FLP分析及在Wilson病基因诊断中的应用

Ｗｉｌｓｏｎ病（ＷＤ）基因内的Ｄ１３Ｓ３０１位点为二核苷酸重复序列（ｄＧ－ｄＴ）ｎ。为了探讨该病的产前诊断方法，应用聚合酶链反应方法对４１名无关中国汉族个体和４个ＷＤ家系Ｄ１３Ｓ３０１位点的多态性进行检测，用银染聚丙烯酰胺变性胶分析扩增片段长度多态性共发现８个等位片段，杂合率为７５％，多态信息量（ＰＩＣ）为０．７８。表明Ｄ１３Ｓ３０１位点ＰＩＣ高，可以作为中国汉人的ＷＤ基因的遗传标记对ＷＤ家系进行基因诊断。     

用SSLP连锁分析进行成人多囊肾病快速基因诊断的研究

目的为了探索一种简便、快速的成人多囊肾病（ａｄｕｌｔｐｏｌｙｃｙｓｔｉｃｋｉｄｎｅｙｄｉｓｅａｓｅ，ＡＰＫＤ）基因诊断方法。方法应用ＰＣＲ技术结合ＳＲＳ－ＳＳＬＰ，对成人多囊肾病进行研究。结果ＰＣＲ扩增ＰＫＤ１近端微卫星序列ＳＭ７，观察了中国人群ＳＭ７的多态性，在６７名非血缘关系正常人中发现７种等位片段，杂合子频率５２．２％，ＰＩＣ值０．６２。以ＳＭ７为遗传标记，对２个ＡＰＫＤ家系进行ＳＳＬＰ连锁分析，和３′ＨＶＲ／ＰｖｕⅡＲＦＬＰ连锁分析进行对照，结果二者相符。结论ＳＭ７在中国人中具有较高的多态性，用ＳＳＬＰ连锁分析进行成人多囊肾病的基因诊断切实可行。     

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by PCR-based analysis of the BclI/intron 18 and St14 VNTR polymorphisms

We have undertaken this study to identify the usefulness of polymerase chain reaction (PCR)-based analysis of DNA polymorphisms in the BclI/intron 18 and St14 variable number of tandem repeats (VNTR) loci for carrier detection and prenatal diagnosis of hemophilia A in the Korean population. We have analyzed these polymorphisms in members of 105 unrelated Korean families with severe hemophilia A. The observed heterozygosity rates for the BclI/intron 18 and St14 VNTR polymorphisms were 21.0% and 71.3%, respectively. The BclI/intron 18 polymorphism was less informative in Koreans when compared with Caucasians and Japanese. The allele frequencies for St14 VNTR in Koreans were different from those in Caucasians. Compared with Caucasians, there was a markedly higher occurrence of low molecular weight alleles in Koreans. The observed heterozygosity for the St14 VNTR polymorphism in combination with the BclI/intron 18 polymorphism was 81.9%. These two polymorphisms were applied to determine the carrier status of 107 women from 65 unrelated families, and to assess fetal status in 37 pregnancies. So far, we have experienced one case of misdiagnosis of carriership. Our study demonstrated that the PCR-based analysis of the BclI/intron 18 and St14 VNTR polymorphisms was useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. Received: March 2, 2000 / Accepted: March 30, 2000     

联合运用St14(DXS52)位点VNTR和 FⅧ基因内的(CA)n重复多态性诊断甲型血友病

目的　提高甲型血友病 (hemophilia A,HA)家系成员基因诊断及产前基因诊断的准确性和可诊断率。方法　采用 St14 (DXS5 2 )位点的可变串联重复序列和 F 基因第 13内含子的 (CA) n重复多态性连锁分析对 HA家系进行间接基因诊断。结果　单用上述 2个多态位点中的 1个对 9个 HA家系进行连锁分析 ,可诊断率均为 6 6 .7% ,联合 2个多态位点 ,可诊断率则提高到 88.9% ,完成了 4个家系的产前基因诊断 ,并监测到 1例单用 St14位点的可变串联重复序列多态连锁分析可能发生的产前诊断的误诊。结论联合采用上述 2个多态位点可以对近 90 %的 HA家系作出快速、准确的基因诊断和产前基因诊断。     

10个甲型血友病家系FⅧ基因突变分析

目的 分析10个家系中甲型血友病(hemophilia A,HA)患者及女性疑似携带者FⅧ基因突变,并指导产前诊断.方法 应用聚合酶链反应(polymerase chin reaction,PCR)、变性高效液相色谱技术(denaturing high performance liquid chromatogramphy,DHPLC)和DNA测序技术对10个家系中8例HA患者、12名女性疑似携带者的FⅧ基因进行突变检测,并应用St14(DXS 52)、13(CA)n、EX18/BclⅠ3个遗传标记位点对HA家系进行连锁分析.产前诊断检测已知突变位点.针对未见报道的新突变,用限制性内切酶进行分析,同时与100名表型正常的无关个体进行比较,排除多态性.结果 (1)在10个家系中发现5例错义突变、3例移码突变、2例无义突变和2个单核苷酸多态性(single nucleotide polymorphism,SNP)位点.错义突变c.878A＞G、c.1015A＞G和c.6870G＞T,移码突变c.1282delA、c3072_3073insT和c.4880_4881insA以及SNP位点c.5000 G＞A均为国际血友病网站和人类突变数据库未记载的突变或多态.在100名正常人中未检测到错义突变c.878A＞G、c.1015A＞G和c.6870G＞T.(2)在12名女性疑似携带者中,基困水平确诊9例为HA携带者,3名为正常人.(3)遗传连锁分析为4个家系提供了X风险染色体的有效信息.(4)产前诊断结果显示2例胎儿正常、1例HA携带者和1例HA患者.结论 发现c.878 A＞G、c.1015A＞G、c.6870G＞T、c.1282delA、c.3072 3073insT及c.4880_4881insA共6种能引起甲型血友病的新突变;PCR、DHPLC和DNA测序技术可有效检测甲型血友病患者基因突变,而联合限制性内切酶分析和遗传连锁分析能快速筛查HA携带者,进行有效的产前诊断.

Abstract：

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

Cystic fibrosis mutations in French Canadians: Three CFTR mutations are relatively frequent in a Quebec population with an elevated incidence of cystic fibrosis

The French-Canadian population in the Saguenay-Lac St. Jean region of northeastern Quebec has an elevated frequency of cystic fibrosis (CF). The average incidence of cystic fibrosis was 1 in 891 births and the prevalence of CF carriers was estimated to be 1 in 15. We tested for 10 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 133 French-Canadian CF families from Quebec. Ninety-one families were from the Saguenay-Lac St. Jean region and 42 families were referred from other regions of Quebec. We detected the CFTR mutation in 93 and 92% of the CF chromosomes in the Saguenay-Lac St. Jean and the major-urban Quebec families, respectively. The two groups of French-Canadian families were significantly different for the proportions of CFTR mutations. The three most common mutations in the Saguenay-Lac St. Jean families were ΔF508 (58%), 621 + 1G → T (23%), and A455E (8%); and in the major-urban Quebec families were ΔF508 (71%), 711 + 1G → T (9%), and 621 + 1G → T (5%). These results provide evidence for the role of founder effect in the elevated incidence of cystic fibrosis in the Saguenay-Lac St. Jean population.     

应用长距离PCR技术对60例重型血友病甲进行Ⅷ因子基因倒位的基因诊断

目的对天津60例重型血友病甲(hemophiliaA,HA)患者作出有无FⅧ基因倒位的基因诊断.方法外周血提取DNA,通过长距离PCR(long distance-polymerase chain     

血友病甲基因分析技术的改进及其在产前诊断中的应用

目的对血友病甲基因分析技术进行改进并应用于携带者检查和产前诊断。方法长距离聚合酶链反应方法直接检测凝血因子Ⅷ第22内含子倒位,对非倒位家系用FⅧ基因内限制酶切位点XbaⅠ、HindⅢ、二核苷酸重复序列多态性位点STR13和STR22,以及基因外可变数目串联重复序列DXS52(St14)位点进行基因连锁分析。结果52个家系共检出71位携带者。21个家系为第22内含子倒位,28个家系经连锁分析得到明确诊断,3个家系无法诊断,可诊断家系占94.2%。为18个家系做胎儿产前诊断,其中10例诊断为血友病甲胎儿;诊断7例正常男胎和1例携带者女胎,随访1年发育正常。结论应用长距离聚合酶链反应和多位点基因连锁分析技术可以快速有效地进行血友病甲携带者检查和产前诊断。     

Two Novel Low-Density Lipoprotein Receptor Gene Mutations (E397X and 347delGCC) in St. Petersburg Familial Hypercholesterolemia

Familial hypercholesterolemia (FH), a monogenic disease known to be caused by low-density lipoprotein receptor (LDLR) gene mutations, results in the development of premature atherosclerosis and coronary artery disease in affected individuals. The spectrum of LDLR gene mutations in Russia is poorly known. Using polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis, followed by DNA sequencing, we have screened selected exons of the LDLR gene in 80 unrelated St. Petersburg FH patients for the presence of mutations. Two new LDLR gene mutations, 347delGCC and E397X, were characterized among individuals with familial hypercholesterolemia in St. Petersburg. The carriers of both mutations possessed highly elevated blood serum cholesterol. Cosegregation of E397X mutation and LDLR gene RFLP haplotypes with hyperlipidemia was demonstrated by family study. Both mutations seem to be specific to Slavic patients.     

Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: Association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease

The CFTR intron 8 variable length polythymidine tract modulates the cystic fibrosis (CF) phenotype associated with the mutation R117H. To explore whether other mutations reside on multiple intron 8 backgrounds with discernible impacts on phenotype, we developed an allele-specific PCR assay to characterize this locus. Our approach types samples rapidly without the use of radioisotopes. Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789+5 G>A, 3849+10kb C>T), and/or located at hypermutable CpG loci (R117H, 3849+10kb C>T, R553X, R334W, S945L and R75Q). R117H was detected in cis with each of three alleles (5T, 7T, 9T) at the intron 8 locus. The novel R117H-9T association was detected in a 10-month old African-American male with borderline-to-mildly elevated sweat chloride values (˜50–66 mEq/L). All other mutations studied were associated with 7T except 3849+10kb C>T, which was detected on both 7T and 9T backgrounds, but not 5T. Three individuals with a ΔF508/3849+10kb C>T genotype were 9T,9T and had pancreatic sufficiency and normal sweat chloride values, whereas 15 others who carried 3849+10kb C>T on a 7T background had variable pancreatic function (sufficient, n = 12, insufficient, n = 3), and variable sweat chloride values (normal, n = 12, elevated, n = 3). Surprisingly, when not associated with known CFTR mutations, 5T was detected with elevated frequency among individuals with sinopulmonary disease of ill-defined etiology, but with some characteristics of variant CF. In summary, the 5T allele was not found in cis with CF-causing mutations besides R117H, but an elevated 5T allele frequency in variant CF patients suggests 5T may be associated with disease in some situations. Hum Mutat 10:108–115, 1997. © 1997 Wiley-Liss, Inc.