21羟化酶缺乏症高风险胎儿的产前诊断分析

目的 对1例21羟化酶缺乏症（21OHD）先证者家系CYP21A2基因缺陷高风险胎儿进行产前分子诊断.方法 提取先证者及其父母的外周血DNA,分段扩增CYP21A2基因全长并测序,确定先证者及父母基因型.采用亲子鉴定试验排除母体DNA污染.针对先证者突变位点,扩增羊水DNA并测序.结果 测序结果显示先证者存在IVS2-13A〉G纯合突变,父母均为携带者.比较STR位点未见羊水DNA受母体DNA污染迹象.胎儿为该突变杂合子,判断胎儿为携带者.培养后羊水检测结果与培养前一致.胎儿娩出后发育良好,证实了产前诊断结果.结论 建立了21OHD产前分子诊断方法,并成功应用于1例21OHD高风险胎儿的产前诊断分析.     

CYP17基因的新的突变导致二例17α羟化酶与17,20裂链酶联合缺乏

目的：分析2例无血缘关系的17α羟化酶与17，20裂链酶联合缺乏征患者的CYP17基因序列。方法：外周血中提取基因组DNA。设计引物行PCR反应（包括8个外显子及内、外显子交界处和启动子）并对PCR产物进行序列测定。结果：在2例患者的CYP17基因中，共发现了3种基因突变，且均位于第6外显子。例1有2种突变：其一是1103C〉A．即第368编码子由CCT→CAT，编码的氨基酸由脯氨酸转变为组氨酸（Pro368His）；其二是插入缺失突变，985缺失TAC插入AA（985delTACinsAA）。例2是一纯合子突变：1135T〉C，即第379编码子由TCC转变为CCC，编码的氨基酸由丝氨酸转变为脯氨酸（Ser379Pro）。结论：发现了3种突变。其中985delTACinsAA是一种新的突变形式。3种突变均发生在第6外显子。提示该区域与P450C17酶活性密切相关。     

家庭性高胆固醇血症LDL—R基因点突变的初步研究

目的：建立一种快速、高效地检测LDL－R基因点突变的方法用于对家族性高胆固醇血症患进行分析。方法：采用同一种程序和反应体系分别扩增家族性高胆固醇血症LDL－R基因包括18外显子和启动子在内的21个片段，琼脂糖电泳检测PCR产物，SSCP分析和PCR产物直接测序检测点突变。结果与结论：本方法可以快速、简便、有效地扩增LDL－R基因包括启动子在内的21个片段，PCR产物直接测序初步发现一家族性高胆固醇血症患存在点突变。     

2型糖尿病患者胰岛素受体胰岛素结合哉基因突变的研究

目的：研究胰岛素受体结合域基因突变与胰岛素受体（INSR）功能异常的关系。方法：采用聚合酶链反应－单链构象多态性（PCR－SSCP）分析法和核酸银染法,对70例2型糖尿病患者INSR结合域基因第2,3,6三个外显子（exon）及部分相邻内含子进行基因突变检测。结果：在对外显子6的检测中发现有9例突变,突变又可分为B,C两种不同的SSCP带型。其中B型7例,C型2例,C型突变经测序结果,与外显子6的3′端毗邻的内含子的第43位A→C突变。在外显子3中发现了2例突变。为同一带,在外显子2中尚未发现突变带型。结论：INSR结合域基因的突变在2糖尿病中出现的频率较低。C型突变对INSR结合域的影响及其在2型糖尿病发病中的作用尚待进一步研究。     

促甲状腺素受体基因突变一个家系研究

目的：探讨1个甲状腺发育不良先天性甲状腺功能减退症(先天性甲减)患者家系促甲状腺素受体(TSHR)基因突变遗传规律。方法：调查包括先证者3代家系成员共计13人，检测血清甲状腺激素。用TKM法提取基因组DNA，PCR扩增TSHR基因第10外显子序列，对PCR产物正反向测序，测序结果同Genbank人TSHR基因序列对比。结果：先证者TSHR基因同时存在2个位点纯合子型点突变R450H／D727E，家系12人甲状腺功能均正常，6人为R450H／D727E杂合子型点突变，其中5人血清sTSH轻度升高。结论：R450H／D727E纯合子导致先证者甲状腺发育不良及先天性甲减，R450H／D727E杂合子甲状腺功能正常仅sTSH轻度升高。     

RET基因Cys634Arg突变致多发性内分泌腺瘤2A型2例家系分析

目的 总结2个多发性内分泌腺瘤2A型(MEN2A)家系的临床特征及RET基因的突变类型.方法 分析2例MEN2A先证者及其家系成员的临床表现;提取其外周血基因组DNA,应用PCR技术对RET基因的第10、11外显子进行扩增并双向测序.结果 2个家系的先证者及5名家庭成员均存在RET基因第11外显子的Cys634Arg点突变,且临床均表现为甲状腺髓样癌和嗜铬细胞瘤.其中,家系2合并有垂体无功能腺瘤.结论 该MEN2A家系的RET基因是Cys634Arg突变;其临床表现主要为甲状腺髓样癌及肾上腺嗜铬细胞瘤.     

唐山市苯丙酮尿症患儿筛查及PAH基因突变情况分析

摘要：目的　分析唐山市苯丙酮尿症（PKU）患儿筛查结果及苯丙氨酸羟化酶（PAH）基因突变的情况。方法　选取2015年1月—2018年12月唐山市新生儿303 777例,通过茚三酮免疫荧光法检测新生儿足跟血中苯丙氨酸（PA）含量。再利用聚合酶链反应（PCR）和基因测序的方法对筛查出的PKU患儿PAH基因进行检测。结果　303 777例新生儿初步筛查共发现609例可疑阳性,召回其中411例（67.49%）进行复查,确诊42例（13.8/10万）。42例PKU患者的PAH基因测序显示,在84条染色体上共检测到62个（73.81%）12种突变,其中错义突变8种,无义突变2种,缺失突变1种,剪接突变1种。患者PAH基因突变分布在第2、3、6、7、9外显子上,其中第7外显子最多（35个,56.45%）,其次为第3外显子（14个,22.58%）。最常见的突变基因为Exon7-R243Q（18个,29.03%）和Exon3-R111X（10个,16.13%）、Exon7-R261Q（10个,16.13%）。筛查中发现1例典型PKU患儿,该患儿在PAH基因外显子区域同时发现2处杂合突变：c.208-210delTCT（缺失突变）和c.964G＞A（鸟嘌呤＞腺嘌呤）。结论　唐山市新生儿PKU发病率略高于全国,PAH基因突变以错义突变为主,第7外显子是唐山市患儿PAH基因高频突变位点。     

多发性脂囊瘤一家系临床调查及分子发病机制研究

目的研究一多发性脂囊瘤(SM)家系KRT17基因的突变情况。方法用聚合酶链反应(PCR)方法扩增家系中2例患者(先证者、先证者父亲)、家系中的2名健康者和与该家系无关的50名健康者的基因组DNA角蛋白K17的外显子1,PCR产物直接进行测序以检测突变。结果在家系中2例患者的角蛋白K17基因第94位密码子由CGC突变为TGC,导致K171A区杂合错义突变R94C,即第94位精氨酸被半胱氨酸取代。而该家系中的2名健康者及与该家系无关的50名健康者未发现此突变。结论在一中国人SM家系中检测到K171A区R94C突变。     

肺癌患者O6-甲基鸟嘌呤-DNA甲基转移酶基因多态性研究

目的 检测人类O^6－甲基鸟嘌呤－DNA甲基转移酶（MGMT）基因5个外显子的单核苷酸多态（SNPs）及其与肺癌的关系。采用聚合酶链式反应（PCR）－单链构象多态性（SSCP）－DNA直接测序法对100例正常人和60例肺癌患者外周血白细胞进行MGMT基因SNPs研究。结果 在正常人群和肺癌患者中检测到MGMT基因的第3、4外子上存在着SNPs，即第3外显子上第250位碱基C被T所取代，导致错义突变（CTT→TTT，使Leu84Pphe）；第4外显子第280位碱基C被T取代，导致同义突变（TTC→TTT，均编码Phe）。这两个位点的SNPs在两组人群间的检出率差异无显著性（P＞0．05）。结论 MGMT基因第3、4外显子上存在着SNPs，其侈态改变与肺癌形成的关系较弱。     

塞替派诱发人支气管上皮细胞恶性转化过程中的基因突变（二）

目的 观察抗癌药物塞替派诱发永生化人支气管上皮细胞恶性转化过程中相关基因的突变并分析其意义。方法 以塞替派作为致癌原诱导永生化人支气管上皮细胞（BEAS－2B）,获得转化细胞（BEAS－TE）和克隆化多倍体癌前转化细胞（BEAS－STE）。采用PCR－SSCP法检测上述3种细胞p53、p16和Ki-ras 3种基因是否出现点突变,进一步测序确定其突变情况。结果 SSCP结果阳性的有BEAS－TE细胞p53第7外显子,BEAS－STE细胞p53第8外显子以及这二种细胞的p16基因第1外显子;Ki-ras基因第1外显子的结果仅为可疑阳性。测序证明,p53、Ki-ras基因存在多位点的碱基突变,而p16基因仅为单位点的碱基突变。结论 塞替哌可诱发人支气管上皮细胞p53、Ki-ras多位点的碱基突变和p16的单位点突变。分析前者为塞替派诱导细胞转化过程中发生的重要分子事件,后者是次要分子事件。     

Reliability of the omeprazole hydroxylation index for CYP2C19 phenotyping: possible effect of age, liver disease and length of therapy

AIMS: To evaluate the reliability of the omeprazole hydroxylation index as a marker for polymorphic CYP2C19 activity in a Japanese population of healthy young subjects (n = 78) and patients with peptic ulcer (n = 72). METHODS: Healthy subjects were administered a single dose of omeprazole (20 mg), whereas patients received 20 mg daily for at least 1 week. The ratio of the serum concentration of omeprazole to hydroxyomeprazole at 3 h postdose was determined and used as a measure of CYP2C19 activity. The CYP2C19 wild type (wt) gene and four mutant alleles associated with the poor metaboliser phenotype of (S)-mephenytoin, CYP2C19*2 in exon 5, CYP2C19*3 in exon 4, CYP2C19m4 in exon 9, and CYP2C19m3 in the initial codon were analysed. RESULTS: In the healthy volunteer study there was complete concordance between genotype and phenotype. However, eight of the patients who had the EM genotype had a high value for their hydroxylation index, and were classified as phenotypic PMs. No CYP2C19m4 and CYP2C19m3 mutations were detected in the eight mismatched patients. They were all genotypic heterozygous EMs, elderly (> or = 65 years) and/or had hepatic disease. Therefore, impaired CYP2C19 activity combined with partial saturation of omeprazole metabolism during multiple dosing may have contributed to the discrepancy between CYP2C19 genotyping and phenotyping. CONCLUSION: Although omeprazole has been used instead of mephenytoin as a probe for polymorphic CYP2C19, it does not appear to be reliable enough for clinical application in Japanese patients.     

细胞色素P450 1B1基因多态性与子宫内膜癌易感性研究

目的研究细胞色素P4501B1基因外显子2密码子119（G-T）、外显子3密码子432（C-G）多态性与子宫内膜癌遗传易感性的关系。方法采用等位基因特异性聚合酶链反应（AS-PCR）法对72例子宫内膜癌患者和80例对照者进行CYP1B1基因密码子119（G-T）、密码子432（C-G）突变分析,用SP免疫组化法进一步研究雌激素受体（ER）、孕激素受体（PR）的表达是否受CYP1B1基因多态性的影响。结果CYP1B1基因密码子119、432中等位基因G、T和C、G在子宫内膜癌组和对照组分布的差异有显著性（χ^2=16.817,P=0.000;χ^2=9.133,P=0.003）,其中等位基因T、G使患子宫内膜癌危险性分别增加了3.052和2.213倍。CYP1B1基因密码子119G/T各基因型分布两组间有显著性差异（χ^2=18.267,P〈0.01）,纯合突变T/T、G/G基因型、杂合突变G/T、C/G基因型与野生G/G、C/C基因型相比,患子宫内膜癌的危险度分别提高了4.258、3.871倍和3.235、2.214倍。此外,119T/T、G/T基因型、432G/G、C/G基因型者ER阳性表达率高于119G/G、432C/C野生基因型,有显著性差异（χ^2=6.750,P=0.034;χ^2=6.977,P=0.031）。结论CYP1B1基因密码子119、432突变等位基因与子宫内膜癌的发生有一定关联,突变基因型增加了子宫内膜癌的发病风险,且与ER的表达相关。     

Analysis of point mutation in exon 2 of CYP2E1 gene in renal cell/urothelial cancer patients in comparison with control population

OBJECTIVE: Genetic polymorphisms of human cytochrome P450s have been implicated to be of importance for susceptibility to different cancers. Recently, a point mutation was found in the exon 2 of the CYP2E1 gene (CYP2E1*2) [Hu et al. 1997]. In order to evaluate a possible link between the point mutation in exon 2 of the CYP2E1 gene and the susceptibility to renal cell/urothelial cancer, we developed a screening method based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). MATERIAL: DNA of peripheral white blood cells was isolated from 158 renal cell/urothelial cancer patients as well as from 150 controls. METHOD: Primers for PCR were designed by the Primer 3 release 0.1 program. The PCR yield a product of 215 base pairs (bp), which was digested with the restriction enzyme Hha I. The DNA fragments were separated on a 3% agarose gel stained with ethidium bromide. Restriction enzyme digestion of the PCR product obtained from the wild-type DNA resulted in the appearance of a 66 bp, a 43 bp, a 40 bp, a 39 bp and a 28 bp DNA fragment. In contrast to the wild-type, the digestion of the PCR product from DNA carrying the point mutation resulted in the loss of the 39 bp and 40 bp fragments and the appearance of an additional 79 bp fragment. Therefore, the loss of one Hha I restriction site caused by a single nucleotide exchange is suitable for the identification of the point mutation in exon 2 of CYP2E1 gene. RESULTS: However, we could not detect any point mutation in any of the 158 renal cell/urothelial cancer patients or the 150 controls. The distribution of the point mutation in exon 2 of CYP2E1 gene did not show any difference in renal cell/urothelial cancer patients and controls. CONCLUSION: This might indicate a lack of association between this CYP2E polymorphism (CYP2E1*2) and renal cell/urothelial cancer.     

儿童侵袭性纤维瘤中β-catenin 的表达和基因突变分析及与复发相关性实验研究

目的：分析儿童侵袭性纤维瘤病β-catenin 的表达和突变状态,并探讨其与肿瘤复发的相关性。方法搜集该院2007年6月—2013年5月期间手术留取侵袭性纤维瘤标本,应用免疫组化法对32例侵袭性纤维瘤病患儿（21例复发和11原发性发病例）和15例对照组进行分析。在β-catenin 的3号外显子的体细胞点突变进行聚合酶链反应产物的测序。结果在侵袭性纤维瘤病样本中检测β-catenin 的核表达为94％（30／32）,而对照组为13％（2／15）（P ＜0．001）。在侵袭性纤维瘤病样本中B 连环蛋白基因3号外显子突变率为78％（25／32）（19／21复发病例,6／11原发发病例;P ＝0．032）。在复发病例主要发生突变的密码子是45（S45F）,而原发病例中41号密码子（T41A）是最常见的突变（P ＝0．002）。结论在β-catenin 基因中,3号外显子的改变,特别是 S45F 突变,可以作为儿童患者复发的危险因素和可能被用作预后因素。     

A new variant CYP2D6 allele (CYP2D6*21) with a single base insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype.

Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.     

Characterization of human CYP2G genes: widespread loss-of-function mutations and genetic polymorphism

CYP2G1 is an abundant, olfactory mucosa-specific cytochrome P450 enzyme active in the metabolism of sex steroids and xenobiotic substrates in mammalian animals. Two different human CYP2G genes, CYP2GP1 and CYP2GP2, were characterized in the present study. Polymorphisms in these genes were also studied. CYP2GP1 contained a single nucleotide deletion in exon 2 (deltaC) and a 2.4-kb deletion between exons 3 and 7 (deltaE4-6), whereas CYP2GP2 contained a nonsense mutation in exon 1 and another in exon 3. The coding region sequences in exons 1-3 and 7-9 of the two genes were 96.7% identical. Both genes were localized to human chromosome 19, and Southern blot analysis of human genomic DNA did not detect any additional copies of the CYP2G gene. The occurrence of these loss-of-function mutations was analysed by polymerase chain reaction-based genotyping in more than 200 individuals. The deltaE4-6 deletion in CYP2GP1 was detected in 94% of subjects (either homozygous or heterozygous), and an allele which does not contain this deletion was detected in 11.6% of individuals. The nonsense mutation in CYP2GP2 exon 3 was detected in 86% of individuals (either homozygous or heterozygous); however, a potentially functional CYP2GP2 allele based on the absence of the nonsense mutation in exon 3 was also detected in 31% of individuals. These results indicate that a functional CYP2G allele is rare in humans. Analysis of the allelic distribution in different ethnic groups suggested that a functional CYP2G allele, if present, is more likely to be found in Black and Hispanic subjects.     

湖南地区238 例非小细胞肺癌EGFR 基因突变状态分析

目的：评价湖南地区非小细胞肺癌患者EGFR基因突变状态及其与临床特征之间的关系。方法收集2012年1月至2013年6月中南大学湘雅医学院附属肿瘤医院手术切除的非小细胞肺癌石蜡包埋组织238例,采用PCR扩增结合直接基因测序的方法,对组织DNA中EGFR基因第18-21外显子突变进行检测。结果在238例非小细胞肺癌中,EGFR突变的检出率为35.3％（84/238）。其中,第18、19、20、21号外显子突变占总突变的比例分别为2.4％（2/84）、67.9％（57/84）、3.6％（3/84）、26.2％（22/84）。19号外显子的突变均为第746-753密码子的碱基缺失,以Del E746-A750为主;21号外显子突变类型以L858R为主。女性EGFR基因的突变率显著高于男性,不吸烟者显著高于吸烟者（P〈0.05）。而EGFR基因的突变率与非小细胞肺癌患者的年龄、临床分期和淋巴结转移均无相关性（P〉0.05）。结论湖南地区的非小细胞肺癌EGFR基因突变以19、21外显子为主,且19外显子的突变率高于21外显子,多见于女性、腺癌、不吸烟患者。     

Single nucleotide polymorphisms of the human cyp2a13 gene: evidence for a null allele.

Human CYP2A13 is believed to be important in the metabolic activation of tobacco-specific nitrosamines in the respiratory tract; therefore, genetic polymorphisms of the CYP2A13 gene may be associated with interindividual differences in the risks of tobacco-related tumorigenesis. Our earlier studies identified a frequent single nucleotide polymorphism in CYP2A13 exon 5, Arg257Cys, which led to an approximate 50% decrease in metabolic activities. In the present study, three additional coding region mutations (Arg25Gln, Arg101Stop, and Asp158Glu) and several mutations in the introns and flanking regions were identified in a Chinese patient population. Of particular interest is the Arg101Stop mutation, which was due to a C > T change in exon 2. Thus, individuals homozygous for this nonsense mutation would not have a functional CYP2A13 protein and, therefore, might have reduced sensitivity to xenobiotic toxicity resulting from CYP2A13-mediated metabolic activation in the respiratory tract. The frequencies of the coding region mutations were further examined using random samples of white, black, Hispanic, and Asian newborns from New York. The frequency of the Arg25Gln mutation in Asian newborns (9.6%) was very similar to that found in the Chinese population (10.9%). On the other hand, the Arg101Stop mutation was not detected in 136 newborn samples examined (23 white, 21 black, 19 Hispanic, and 73 Asian), suggesting that this mutation may be unique for the Chinese patient population. Haplotype analysis indicated that the Arg25Gln and Arg257Cys mutations are parts of a common haplotype. However, an additional haplotype that consists of the 25Gln but not the 257Cys allele was also identified.