妊娠期肝内胆汁淤积症MDR3基因突变的研究

目的:了解妊娠期肝内胆汁淤积症(ICP)患者MDR3基因外显子突变的分布,探讨此基因在ICP发病机制中的作用。方法:收集30例ICP孕妇,检测其肝功能、血清总胆汁酸(TBA)、γ-谷胺酰转肽酶(γ-GT)水平,同时用聚合酶链反应-单链构象多态性(PCR-SSCP)技术分析ICP患者MDR3基因5个外显子的突变情况。结果:4例ICP孕妇血清γ-GT升高。所有ICP患者及60例正常妊娠妇女PCR均扩增出目的片段,但SSCP分析没有发现异常迁移条带。结论:在MDR3基因突变频率较高的5个外显子中,本地区ICP孕妇未发现有突变。MDR3基因突变可能不是本地区ICP患者发病的主要原因。     

雌激素代谢基因CYP1A2和COMT单核苷酸多态性与妊娠期肝内胆汁淤积症关系的研究

目的:探讨参与雌激素代谢的CYP1A2和COMT基因多态性与成都地区妊娠期肝内胆汁淤积症(ICP)的关系。方法:应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术,对成都地区100例ICP患者(ICP组)和100例正常孕妇(对照组)的CYP1A2基因5-'侧翼区G-2964A多态和COMT基因第4外显子第158号密码子G→A点突变所引起的缬氨酸→蛋氨酸(Val 158 Met)的错义突变多态进行分析。结果:CYP1A2 GA杂合基因型明显增加ICP的发病风险(P=0.029,OR=1.896),COMT含V等位基因的VV和VM基因型也明显增加ICP的发病风险(P=0.027,OR=3.996);两基因组合分析表明同时具有CYP1A2等位基因G和COMT等位基因V的个体发生ICP的风险显著增加(P=0.011,OR=2.852)。结论:CYP1A2和COMT基因单核苷酸多态性与成都地区ICP的易感性有关。     

卵巢早衰患者骨形态发生蛋白15基因突变的研究

目的:通过对卵巢早衰(POF)患者骨形态发生蛋白15(BMP-15)基因编码区的单核苷酸多态性(SNPs)分析,探讨BMP-15突变与POF的关系.方法:应用聚合酶链反应(PCR),对63例POF患者(POF组)和62例正常对照者(对照组)的BMP-15编码区特异扩增,PCR产物进行DNA测序,检测基因突变.结果:发现3个突变位点,位于第1外显子的rs3810682(-9C ＞G)和rs79377927 (788_789insTCT),第2外显子的rs17003221(852C＞ T).其中rs3810682(-9C＞G)和rs17003221 (852C＞ T)在POF组和对照组中均有发现,且该两个位点的基因型及等位基因频率在POF组和对照组分布比较,差异均无统计学意义(P＞0.05).仅在POF组中发现2例rs79377927( 788_789insTCT),该位点基因型在POF组中为3.17％.结论:BMP-15的rs79377927 (788_789insTCT)位点基因突变与POF发病可能相关.     

抗苗勒氏管激素及其受体Ⅱ的基因多态性与卵巢过度刺激综合征关系初探

目的:初步探讨抗苗勒氏管激素(AMH)及其受体Ⅱ(AMHRⅡ)的基因多态性与卵巢过度刺激综合征(OHSS)的关系。方法:采用聚合酶链反应(PCR)和DNA测序法,分别检测27例OHSS患者和22例促排卵后非OHSS患者(对照组)的AMH及其受体AMHRⅡ的基因外显子DNA序列,行单核苷酸多态性(SNP)分析。结果:OHSS组AMH的第1外显子146位G>T、第2外显子134位G>A基因型分布与对照组比较,有统计学差异(P<0.05)。OHSS组AMH基因的第1外显子303位G>A基因型与对照组比较,无统计学差异(P>0.05)。OHSS组和对照组AMHRⅡ基因的1~11号外显子均未检测出SNP突变。结论:AMH基因多态性可能是导致卵巢对外源性激素敏感性增强、OHSS发病的因素之一。     

58例苗勒管发育异常患者HOXA13基因分析

目的:探讨中国汉族苗勒管发育异常患者中HOXA13基因是否存在突变。方法:对58例中国汉族苗勒管发育异常患者(包括51例苗勒管融合不全患者、7例先天性无阴道无子宫患者)和54例正常对照者进行HOXA13基因检测。PCR扩增目的片断后,毛细管电泳分析HOXA13基因1号外显子多聚丙氨酸束和自动化测序分析基因2号外显子同源结构域。结果:HOXA13基因1号外显子毛细管电泳分析结果显示,患者和对照者毛细管电泳图均显示单一的PCR产物峰,提示没有发现多聚丙氨酸束片断长度改变或多态现象。2号外显子直接自动化测序分析结果显示,在患者和对照者中均没有突变发生。结论:中国汉族妇女苗勒管发育异常的发生可能与HOXA13基因的多聚丙氨酸束长度扩增或同源结构域突变无关。     

妊娠期肝内胆汁淤积症中MDR3基因表达的研究

目的:探讨MDR3基因mRNA的表达与妊娠期肝内胆汁淤积症(ICP)发病的关系.方法:从36例ICP患者和38例正常孕妇外周血标本中抽提总RNA,反转录.聚合酶链反应(RTPCR)技术扩增MDR3基因和β-Actin基因,采用凝胶分析软件对RT-PCR产物进行半定量分析,比较两组间差异.结果:MDR3基因mRNA的表达在ICP患者和正常孕妇组间差异无统计学意义(P＞0.05).结论:MDR3基因mRNA的表达异常可能与中国皖南地区ICP发生无关或关联很小,中国人ICP的发生可能存在其他的发病机制.     

X-连锁脊柱骨骺发育不良家系孕早期产前诊断一例

2007年本实验室对1例X-连锁脊柱骨骺发育不良家系(X-linked spondyloepiphyseal dysplasia tarda,X-SEDL)进行基因诊断[1],确诊该家系的先证者(图1:I:1)为位于染色体Xp22.2的SEDL基因的4号外显子c.209G＞A突变致病,其女儿(图1:Ⅱ:1)为该致病基因突变携带者(c.209G/A杂合突变).2008年,该女性携带者于妊娠19周时曾在本院对胎儿(图1:Ⅲ:1)采集的羊水细胞进行基因诊断,证实为男性胎儿伴遗传突变基因而引产.此次妊娠11周时要求在孕早期对胎儿(图1:Ⅲ:2)进行产前诊断.签署手术知情同意书后,行绒毛膜穿刺术取绒毛组织.部分绒毛组织标本经常规细胞培养、收获制片后G显带染色体核型分析.部分绒毛组织提取基因组DNA,进行Y染色体性别决定基因(sex determining region of Y chromosome,SRY)检测,判断性别,聚合酶链反应(polymerase chain reaction,PCR)条件参照文献[2].参照相关研究[1]直接PCR扩增SEDL基因4号外显子,PCR产物经琼脂糖凝胶电泳、回收纯化后进行DNA序列分析.同时选择Xp22区域内位于SEDL基因附近的4个短串联重复(shorttandemrepeat,STR)位点(包括DXYS10085、DXYS10092、DXYS10093和DXYS233)进行PCR扩增检测,PCR扩增引物及反应条件见参考文献[3],进行单体型分析.     

CYP1B1基因多态性与卵巢癌易感性的研究

目的:研究CYP1B1基因外显子2密码子119(G-T)、外显子3密码子432(C-G)多态性与卵巢癌遗传易感性的关系。方法:应用等位基因特异性聚合酶链反应(AS-PCR)法对53例卵巢癌患者和30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,用免疫组化SP法进一步研究雌激素受体(ER)、孕激素受体(PR)的表达,分析其是否受CYP1B1基因多态性的影响。结果:CYP1B1基因密码子432中等位基因C、G在卵巢癌组和对照组分布的差异有统计学意义(P<0.05),其中等位基因G使卵巢癌发病风险增加2.71倍。CYP1B1基因密码子432C/G各基因型分布两组间差异有统计学意义(P<0.01),纯合突变(G/G)基因型、杂合突变(C/G)基因型与野生(C/C)基因型相比,患卵巢癌的危险度分别提高了4.53倍和4.43倍。此外,432G/G、C/G基因型者ER阳性表达率高于432(C/C)基因型,三者间有显著差异(P<0.05)。结论:CYP1B1基因密码子432突变等位基因与卵巢癌的发生有一定关系,突变基因型增加了卵巢癌的发病风险,且与ER的表达相关。     

苗勒管发育异常对生育结局的影响及HOXA13基因的突变研究

目的:分析苗勒管发育异常患者不同表型对生育结局的影响,探讨这些患者HOXA13基因的第二号外显子编码区是否存在突变。方法:回顾分析348例本中心苗勒管发育异常患者的不同表型对生育结局的影响;应用聚合酶链反应(PCR)扩增目的片段后,自动化测序分析HOXA13基因的第二号外显子编码区,与NCBI提供的正常序列比较,分析HOXA13基因的第二个外显子是否存在突变。结果:(1)苗勒管发育异常患者中不同表型对是否妊娠的影响有统计学差异(χ2=32.656,P<0.0001);(2)苗勒管发育异常患者的不同表型对妊娠结局的影响有统计学差异(χ2=12.817,P=0.005);(3)本研究中,所有患者第二号外显子均无突变发生。结论:(1)苗勒管发育异常患者的不同表型对生育结局可能有不同的影响;(2)苗勒管发育异常可能与HOXA13基因的第二个外显子编码区突变无明显关系。     

卵泡刺激素受体基因与多囊卵巢综合征关系的初步研究

目的:探讨卵泡刺激素受体基因第7、10(A)外显子基因突变与多囊卵巢综合征(PCOS)发病的关系。 方法:提取56例PCOS患者及38例对照者的外周血基因组DNA,采用PCR及RFLP(限制性片段长度多态性)方法研究FSHR第7、10(A)外显子基因多态性。 结果:PCOS组FSHR第7外显子49/49型96.43%,86/49型3.57%,第10(A)外显子128/128型98.21%,128/216型1.79%,与对照组相比较,其基因型分布差异无显著性(P>0.05)。PCOS组第7外显子49型等位基因频率为98.21%,86型等位基因频率为1.79%,第10(A)外显子128型等位基因频率为99.11%,216型等位基因频率为0.89%。结论:中国人群存在以上两位点突变,但突变率在PCOS组及对照组不存在差异。PCOS组LH /FSH比值、高雄激素血症与等位基因86型、216型存在一定的相关趋势。     

Androgen receptor gene mutation identified by PCR-SSCP and sequencing in 4 patients with complete androgen insensitivity syndrome

To study the genetic defect of the human androgen receptor (hAR) gene in the complete androgen insensitivity syndrome (CAIS), we amplified each of the eight exons by PCR in genomic DNA extracted from the paraffin blocks of the resected gonads. We analyzed using SSCP, and directly sequenced the abnormally shifted bands. Mutations were found in 4 cases of CAIS. Patient 1 carried a point mutation; a G to A transition in exon 7 resulted in a change from arginine to glutamine at codon 831. Patient 2 carried a point mutation; a C to T transition in exon 7 resulted in a change from arginine to stop at codon 831. Patient 3 carried a point mutation and deletion in exon 7. A point mutation was an A to G transition that caused a glutamine to be substituted for the asparagine present at codon 819. A deletion of a G at codon 820 resulted in a frameshift and consequently in the introduction of a premature stop at codon 821. Patient 4 carried a mutation in 5’ splice donor site of intron 7; a G to T transition might have caused an abnormal splicing of the exon 7. All of the mutations were found in exon 7. These mutations of hAR gene might be related to the pathogenesis of CAIS. Received: May 1999 / Accepted: 17 August 1999     

Genetic derangements in the tumor suppressor gene PTEN in endometrial precancers as prognostic markers for cancer development: a population-based study from northern Norway with long-term follow-up

OBJECTIVES: The purpose of the current study was to characterize the role of PTEN in malignant transformation and to evaluate the significance of mutated PTEN exons as prognostic markers in the carcinogenesis of endometrial hyperplasia. A comparison of PTEN mutations as prognostic markers with former investigated prognosticators was also intended. METHODS: Histological material from 68 patients with endometrial hyperplasia and 10-20 years of follow-up of whom 18 later developed cancer was examined. PCR amplification and DNA sequencing were performed, screening the most frequently mutated exons 5a-8b of the PTEN gene. RESULTS: Mutations were demonstrated in 13.2% of the patients. Of the patients with cancer development, five showed to have PTEN mutations corresponding to 28%. Of the patients remaining without carcinoma, only 8% had PTEN mutations (P = 0.04). In total, there were three missense, three nonsense, and four frameshift mutations, and twice as many mutations leading to a truncated protein (six) than mutations altering one amino acid in the entire protein (three). Mutations were distributed in the following manner: three in exon 5a, two in exon 5b, two in exon 6, two in exon 7, and one in exon 8b. Only mutations in exons 6, 7, and 8a were connected with cancer development or coexisting cancer and six out of seven mutations within these exons were frameshift or nonsense mutations. CONCLUSIONS: Our results showed that mutations in the PTEN gene were statistically more frequent in cases with cancer development or coexisting cancer. Although the specificity was acceptable, the sensitivity of PTEN mutations was too low to make it suitable as a tumor marker (sensitivity of 27% and specificity of 91%) in clinical practice.     

Follicle-stimulating hormone receptor gene mutations are rare in Japanese women with premature ovarian failure and polycystic ovary syndrome

OBJECTIVE: To determine whether the known inactivating FSH receptor gene mutations are present in Japanese women with secondary amenorrhea because of premature ovarian failure (POF) and polycystic ovary syndrome (PCOS). DESIGN: Clinical and molecular studies. SETTING: An outpatient clinic in a university hospital. PATIENT(S): Fifteen women with idiopathic POF, 38 women with PCOS, and three normal controls. INTERVENTION(S): Extraction of DNA from blood samples for subsequent polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S): PCR fragments digested with MunI, BsmI, and HhaI were compared in patients and controls. PCR fragments were also analyzed by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. RESULT(S): No inactivating mutations reported thus far in exons 6, 7, 9, and 10 of the FSH receptor gene were identified in Japanese women with POF and PCOS. DGGE analysis of PCR fragments of exon 10 also revealed no FSH receptor gene mutations in this region. CONCLUSION(S): Although we cannot exclude the presence of point mutations in other regions of the FSH receptor gene, the described FSH receptor mutations may be uncommon in Japanese patients with POF and PCOS.     

STUDY ON THE HOMOZYGOUS DElETIONS AND MUTATIONS OF P16 GENE IN OVARIAN EPITHELIAL CARCINOMAS AND THEIR CLINICAL SIGNIFICANCE

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不同表型肌营养不良症患者抗肌营养不良蛋白基因缺失类型的研究

目的探讨我国东北地区杜氏型肌营养不良症（DMD）及贝克型肌营养不良症（BMD）患者抗肌营养不良蛋白基因缺失类型分布与表型的关系,并用于产前基因诊断。方法采用多重PCR法检测124例来自东北地区的DMD（106例）及BMD（18例）男性患者的抗肌营养不良蛋白基因缺失情况,并对30例高危胎儿行产前抗肌营养不良蛋白基因缺失检测。结果124例患者中,抗肌营养不良蛋白基因缺失检出率为49％（61／124）,其中41例（41／61,67％）缺失分布于外显子45—53,13例（13／61,21％）缺失分布于外显子8—19,5例（5／61,8％）在上述两个外显子缺失区内均有缺失,2例（2／61,3％）缺失分布于外显子34和43;缺失型患者中有9例发生整码缺失（为BMD患者）,49例发生移码突变（为DMD患者）。30例高危胎儿中,17例为男性胎儿,其中10例为抗肌营养不良蛋白基因缺失型,缺失位点与先证者相同;13例为女性胎儿,无一例抗肌营养不良蛋白基因缺失。结论DMD及BMD患者抗肌营养不良蛋白基因缺失主要分布于两个区域,外显子8附近区域可能是东北地区该基因缺失高发区;缺失类型与临床表型有一定的关系,当基因发生整码缺失时,临床表型为BMD,而发生移码突变时,临床表型为DMD。     

自然流产与绒毛中维甲酸X受体α基因突变关系的研究

目的 :探讨人类自然流产与绒毛中维甲酸X受体α(RXRα)基因突变的关系。方法 :用PCR SSCP 银染法和DNA测序法检测了自然流产 5 0例和正常早孕要求人工流产4 0例的胚胎绒毛组织中RXRα基因的外显子 2～ 10的突变。结果 :4 9例患者和正常对照组绒毛组织中RXRα基因的各个外显子在PCR SSCP 银染检测中均未显示异常。 1例患者SSCP 银染中显示异常的第 9外显子序列经DNA测序后未发现突变。结论 :目前尚不能认为人类自然流产与绒毛中RXRα基因突变有关     

卵巢肿瘤中c-kit基因突变的临床研究

目的探讨c-kit基因在卵巢恶性上皮性肿瘤中的突变情况及其在卵巢肿瘤发生发展中的作用。方法2004年1月至2005年12月在哈尔滨医科大学附属第一临床医学院、第三临床医学院应用PCR扩增和基因测序的方法检测卵巢恶性上皮性肿瘤、卵巢交界性肿瘤、卵巢良性肿瘤及正常卵巢组织中c-kit基因第11号外显子序列。结果c-kit基因在卵巢恶性上皮性肿瘤、卵巢交界性肿瘤、卵巢良性肿瘤及正常卵巢组织中的突变率分别为72.1％、44．4％、11．5％、0，卵巢恶性上皮性肿瘤及交界性肿瘤中c-kit基因突变率均高于卵巢良性肿瘤及正常卵巢组织，差异有显著性意义（P〈0．05）。卵巢恶性上皮性肿瘤与卵巢交界性肿瘤中c-kit基因突变率差异无显著性意义（P〉0.05）。在卵巢恶性上皮性肿瘤中，低分化组的c—kit基因突变率为88．9％，明显高于中、高分化组（P〈0．05），c—kit基因的突变率随FIGO分期的进展及淋巴结的转移而升高（P〈0．05），c—kit基因的突变与卵巢肿瘤的病理类型无关（P〉0．05）。结论c-kit基因突变在卵巢恶性上皮性肿瘤的发生发展中可能发挥重要作用，可作为诊断卵巢良恶性肿瘤的参考指标。c-kit基因突变与卵巢恶性上皮性肿瘤的预后有关，可作为判断卵巢恶性上皮性肿瘤患者预后的指标之一。     

Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma

OBJECTIVE: In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene. METHODS: We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery. RESULTS: Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhD-negative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D-C-E-c+e+ C(w)- and the infant D+C-E-c+e+ C(w)+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DC(w)- paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (C(T)) for RHD exon 10 or 7 amplicons reached nearly the same value like C(T) for control beta-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined. CONCLUSION: Prediction offoetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products.     

The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study

The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.