全基因组测序对一例无痛无汗症伴白化病患儿的基因变异分析

目的:对1例无痛无汗症合并白化病患儿及其父母进行基因变异分析。方法:对患儿及其父母进行全外显子组测序(whole exome sequencing,WES),在未完全找到变异位点的情况下对先证者进行了全基因组测序(whole genome sequencing,WGS),用Sanger测序对患儿及其父母的变异位点进行了...     

MYO15A基因突变致聋家系的遗传学分析

目的对一常染色体隐性遗传性耳聋家系行遗传学分析及产前诊断。方法收集该家系成员的临床资料及外周血血样,运用十五项遗传性耳聋微阵列芯片及二代测序技术对先证者进行基因组全外显子序列分析,对检出的致病突变进行Sanger测序验证,结合应用STR检测技术对该家系行产前分子诊断。结果该家系先证者为极重度感音神经性耳聋患者,存在MYO15A基因c.9400CT(p.R3134X)纯合突变,父母亲均为c.9400CT(p.R3134X)杂合突变携带者,孕母亲产前诊断结果提示胎儿该位点基因型与先证者相同。结论 MYO15A基因c.9400CT(p.R3134X)突变为该家系耳聋患者的致聋遗传因素;二代测序结合Sanger测序法有助于明确耳聋患者热点基因突变以外的罕见致聋基因及其位点。     

COL1A1/COL1A2基因突变分析与成骨不全的产前基因诊断

目的对有成骨不全(Osteogenesis Imperfecta,OI)孕史的患者,进行系统B超及COL1A1/COL1A2基因检测,希望建立OI患儿产前诊断方案,为OI患儿进行产前诊断提供技术保障。方法对于有OI孕史的孕妇,进行系统B超监测;根据胎儿股骨、长骨的超声影像学表现,初诊为成骨不全。抽取羊水,采用直接测序法对羊水DNA的COL1A1和COL1A2基因全编码外显子及启动子区域进行突变位点检测。检出的新突变,对孕妇夫妇及家系其他成员直接测序证实。产前诊断标本均需做母血污染鉴定。结果胎儿超声影像学表现为股骨短小,胫腓骨弯曲成角,颅骨变薄且发现多处骨折,考虑OI。STR法鉴定,羊水无母血污染。DNA序列分析结果显示COL1A1基因鉴定出19个SNP位点,没有鉴定出突变位点;COL1A2基因鉴定出13个SNP位点及第36外显子的第2180位置碱基发生错义突变位点(c.2180G>A,p.Gly727Asp)。孕妇在COL1A2基因的第36外显子亦存在错义突变位点(c.2180G>A,p.Gly727Asp),但其临床特征不一致。其他成员均未检测到Gly727Asp突变。结论有OI孕史的孕妇,采取B超和COL1A1/COL1A2基因诊断技术,可以快速、有效对高危胎儿做出确诊,为预防患病胎儿出生提供技术保障。     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

目的 评价多重连接依赖式探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)在染色体非整倍体诊断中的应用价值,为我国羊水染色体诊断提供一种快速、特异、高通量的分子诊断手段.方法 应用MLPA技术检测了500份羊水标本,所有标本均进行荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测和常规染色体核型分析,应用RH-MLPA-v511数据分析软件获得MLPA结果,比较MLPA技术与FISH和染色体核型分析结果的准确性,总结MLPA技术临床应用过程中的关键要点.结果 在500份羊水标本中,MLPA检测成功率97%.3个工作日完成结果的为92%,需重复检测的为5%,失败为3%.对染色体非整倍体异常检测敏感性和准确性100%.证实38例非整倍体病例探针信号比值＞正常二倍体4s,2例疑似三体结果＞2s.分析了21号染色体8条探针的杂交效率,21三体患者8条探针中平均4条探针比值＞1.3.结论 MLPA技术具有快速、特异、敏感、高通量、成本低等特点,可用于产前染色体非整倍体数目的快速检测,是传统染色体培养方法的补充,临床应用价值较高.

Abstract：

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

两个Coffin-Siris综合征家系的 ARID1B基因变异分析

目的:通过对两个Coffin-Siris综合征家系先证者的临床表征及基因变异分析,揭示其可能的遗传发病机制,为家系的遗传咨询提供依据。方法:应用全外显子组测序技术对两例先证者进行致病原因筛查,结合临床表型确定可能致病的候选基因,用Sanger测序法对先证者及其家系成员进行变异验证。结果:WES检测结果显示家系1先证者 ...     

应用多重连接探针扩增筛查高风险胎儿染色体亚端粒区变异北大核心CSCD

目的探讨多重连接探针扩增（multiplex ligation—dependent probe amplification,MLPA）技术在产前大规模筛查染色体亚端粒区变异中的应用。方法选取1050例高风险胎儿,抽取羊水或脐带血,进行核型分析和MLPA检测。染色体亚端粒区变异结果用染色体微阵列验证。结果核型分析发现染色体非整倍体23例,末端异常8例。MLPA检出了所有核型分析发现的染色体非整倍体和末端异常,并对4例染色体末端异常提供了更清晰的描述。此外,MLPA发现5例核型分析为正常的样本存在染色体亚端粒区变异,经微阵列验证2例为假阳性结果,假阳性率为0．19％。MLPA实际检出染色体亚端粒区变异11例,检出率为1．05％。结论将MLPA技术应用于大规模筛查高风险胎儿的染色体异常,可以缩短检测周期,发现亚微观染色体变异,为遗传咨询和产前诊断提供依据。     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.     

焦磷酸测序技术检测温州地区1573例孕妇MTHFR基因C677T多态性分析

目的运用焦磷酸测序技术对温州地区孕早期妇女行亚甲基四氢叶酸还原酶基因(MTHFR)分型检测,以指导孕妇的叶酸补充,同时也为浙江地区人群MTHFR基因分型遗传构成提供基础数据。方法 EDTA抗凝管收集1573例孕早期妇女外周血2ml,抽提DNA并结合焦磷酸测序法测定MTHFR基因677位点,统计本地区孕妇群体该位点碱基C/T的分布频率,并与文献所报道的我国南北方群体遗传数据行统计学分析。结果温州地区孕妇群体中携带C/C、C/T、T/T基因型分别占总群体数的41.7%、45.8%和12.5%;等位基因型C与T群体分布频率分别为64.6%和35.4%;统计学χ2检验结果显示温州孕妇群体该位点基因型分布频率显著区别于我国北方(山东,河南)群体(P0.01),与我国南方(四川、海南)群体也存在统计学差异(P=0.028)。结论本研究说明,焦磷酸测序技术作为一种短片段测序技术可用于准确、快速地检测MTHFR基因多态性位点;温州地区女性群体MTHFR基因多态性分布具有地区特异性。     

百会穴长留针法对慢性应激抑郁大鼠中枢5-HT和DA水平的影响

目的:观察百会穴长留针法对慢性应激抑郁模型大鼠的效应,并从神经生化角度探讨针灸抗抑郁的作用机理。方法:选用36只SD雄性大鼠随机分为空白对照组、模型对照组、百会穴长留针组和百会穴普通留针组。除空白组外,其余3组大鼠采用孤养结合慢性轻度不可预见性应激进行造模,共接受21 d不可预见的刺激,造模同时百会穴长留针组和普通留针组分别给予百会穴长留针和普通留针治疗。用高效液相电化学法(HPLCECD)测定实验后大鼠海马和下丘脑组织匀浆的单胺类神经递质5-羟色胺(5-HT)和多巴胺(DA)的水平。结果:实验后与空白组相比,模型对照组大鼠下丘脑DA含量明显下降(P<0.05)。与模型对照组相比,百会穴长留针组下丘脑5-HT和DA及海马5-HT水平显著性升高(P<0.05),百会穴普通留针组下丘脑DA水平显著升高(P<0.05);同期组间比较显示,百会穴长留针组较普通留针组各指标(P>0.05),但有升高趋势。结论:百会穴长留针法可提高海马及下丘脑5-HT和DA水平,其机制可能与调节脑内单胺类递质有关。