100例β-地中海贫血患者产前基因诊断分析

目的应用基因诊断技术对携带β-地中海贫血基因的夫妇所孕胎儿进行β-地中海贫血产前诊断,避免重型地中海贫血患儿的出生。方法应用聚合酶链技术（PCR）结合反向点杂交技术（RDB）,对羊水样本进行中国人常见的17种β-地中海贫血突变基因的检测。结果在受检的100例羊水样本中,共检出正常基因33例、杂合子49例、纯合子或双重杂合子18例。结论运用RDB-PCR技术对β-地中海贫血进行产前基因诊断,是降低出生缺陷的好办法。     

地中海贫血产前基因诊断中的护理配合

目的通过对地中海贫血(地贫)高危孕妇的产前基因诊断,早期发现重症地贫胎儿,以达到优生目的。方法通过Gap-PCR和RDB技术及配合临床医护人员介入,对高危孕妇113例进行α地贫和β-地贫基因诊断。产前诊断术前、术中、术后给予护理配合,解释和心理安慰。结果113例地贫家系中,完全正常胎儿32例,α地贫杂合子14例,β地贫杂合子43例,血红蛋白H病3例,重症胎儿21例。重症胎儿中α地贫5例,β地贫16例。重症总检出率18.6%。结论地贫高危孕妇在与护理良好配合中进行产前基因诊断,可取得更好临床疗效。     

反向点杂交法用于80例β地中海贫血的快速产前诊断

为探讨反向点杂交法（ＲＤＢ）在β地中海贫血（β地贫）基因诊断中的优越性，应用该方法对７６个家庭的８０例β地贫高风险胎儿进行产前诊断。在８０例被检胎儿中，完全诊断的有７７例：包括正常个体２２例；β地贫杂合子４９例；双重杂合子４例及纯合子２例。另有３例为不完全诊断，其中２例被排除了重症的可能；余１例有１／２的机会为β地贫双重杂合子。ＲＤＢ法可同时筛查１８种中国人β地贫突变，且操作简便快速，在异质性大的β地贫的基因诊断中有明显的技术优势     

孕早期绒毛膜细胞原位培养法用于地中海贫血的产前诊断

目的探讨孕早期绒毛膜细胞原位培养及在地中海贫血(简称地贫)产前诊断中的应用价值,评价其在产前诊断中的可行性。方法用COOK公司的绒毛活检吸管,经宫颈吸取绒毛膜细胞(chorion ic villous samp lings,CVS)标本,分两组:1组直接行地贫基因诊断,另1组进行绒毛膜细胞原位培养,收获细胞后行地贫基因诊断。结果取材手术69例,64例取材成功,63例绒毛膜细胞原位培养成功,1例培养失败,培养成功率为98.44%。64例中有6例绒毛取材量极少,无法直接提取DNA行地贫基因诊断,经培养后完成基因诊断。产前诊断结果:63例中29例正常儿,异常34例,其中:Bart’s胎儿2例,HbH胎儿1例,α地贫基因携带者12例,HbCS胎儿2例,β地贫纯合子3例,β重型地贫6例,β地贫基因携带者5例,α β复合地贫纯合子1例,α β复合地贫杂合子2例。结论孕早期绒毛膜细胞原位培养方法简单易行,技术稳定,需标本量少,安全,结果可靠。可应用于地贫的产前诊断。     

应用多重等位基因特异性PCR技术对β地中海贫血进行产前诊断

为研究β地中海贫血（β地贫）产前诊断的新途径，应用多重等位基因特异性多聚酶链反应（ＰＣＲ）技术对１０例有重型β地贫风险的胎儿进行了产前诊断。将相应于－２８Ａ→Ｇ，ＣＤ１７→０，ＣＤ４１－４２（－４ｂｐ），ＣＤ７１－７２（＋Ａ）和ＩＶＳ－Ⅱ－６５４Ｃ→Ｔ等中国人最常见的五种β地贫突变类型的五组扩增引物组合成一个ＰＣＲ体系，以羊水细胞以及父母的ＤＮＡ为模板进行多重等位基因特异性扩增，结果显示：４例胎儿为２种不同突变类型复合杂合子，２例胎儿为一种突变类型的纯合子，３例胎儿为一种突变类型的杂合子，还有１例为正常胎儿。所有产前诊断的结果均与ＰＣＲ／等位基因特异性寡核苷酸探针点杂交或ＤＮＡ测序结果一致。该方法为β地贫的产前诊断提供了一种简便、快速和可靠的新途径。     

郴州地区103例胎儿地中海贫血产前基因诊断分析

[目的]通过对孕妇进行产前地中海贫血(简称地贫)基因分析,以避免中、重型地贫患儿出生,降低出生缺陷.[方法]收集2013年1月至2015年4月在本院确诊的同型地贫高风险夫妇103对,根据孕妇妊娠月份抽取羊水或脐带血进行胎儿地贫基因分析.采用Gap-PCR技术检测3种常见缺失型α-地贫,PCR-RDB技术检测17种常见β-地贫及3种非缺失型α-地贫.[结果]同型地贫高危夫妇103对,90例接受产前诊断,产前基因诊断率87.38％.检出重型α地贫胎儿12例(13.33％),中间型α地贫胎儿8例(8.89％),中重型β地贫胎儿14例(15.56％).经产前诊断检出的中重型地贫胎儿中,除3例血红蛋白H病(HbH)病胎儿家属选择继续妊娠,其余均在知情同意下终止妊娠.引产标本结果与产前诊断一致.[结论]对同型地贫高危夫妇行产前诊断,可有效阻断Bart's胎儿水肿及中、重型β地贫患儿的出生.     

α地中海贫血的基因诊断及产前诊断研究

目的建立同时检测中国人中最常见的3种α地中海贫血(地贫)基因缺失的单管多重PCR(mPCR)技术，并运用于河北省一个α地贫家系的基因诊断与产前诊断。方法mPCR包含7条引物．运用mPCR、凝胶电泳及DNA测序技术进行α地贫的基因诊断和胎儿羊水细胞DNA的产前基因诊断：结果运用mPCR技术检测了42份α地贫患者DNA标本，证明其能准确、灵敏、快速、简便地诊断出完整的α珠蛋白基因型。查明河北省1例α地贫患儿为--^SEA和Hb^CS、双重杂合子，该家系第2胎胎儿羊水细胞DNA检查证明为完全正常胎儿，产后检测与产前诊断结果相符。结论所建立的α地贫基因诊断mPCR技术可用于3种缺失型α地贫的快速诊断和产前诊断。我国北方也有少量散在α地贫患者及家系，应引起重视，以免贻误诊断和治疗     

某市孕中期产前筛查诊断结果分析

目的：通过对孕中期孕妇进行产前筛查及产前诊断,探讨其临床应用价值。方法对于35岁以下的孕妇,采用化学发光分析系统,测定孕中期（15～20＋6周）血清中甲胎蛋白（A FP ）、游离β人绒毛膜促性腺激素（Freeβ-HCG）及游离雌三醇（uE3）的浓度。采用配套风险评估软件计算出唐氏综合征（21-三体）、爱德华综合征（18-三体综合征）和神经管缺陷（ONTD）的风险值。筛查高风险者进行羊水穿刺确证。适应羊水穿刺及年龄大于或等于35岁的孕妇在知情同意的情况下直接进行产前诊断并行羊水穿刺。结果年龄小于35岁的孕中期孕妇2117例中筛查出高风险145例,筛查阳性率6．8％。其中有124例经过羊水培养对胎儿染色体进行分析,检查出1例唐氏综合征、1例18-三体综合征以及3例其他染色体异常。69例年龄大于或等于35岁的及51例适应羊水穿刺的孕妇中发现8例染色体异常。结论遂宁市孕中期产前筛查异常率高,为有效预防和减少出生缺陷,在孕中期进行产前筛查诊断意义重大。     

一个HPFH复合β地中海贫血家系的基因分析及其高危胎儿的产前基因诊断

目的调查一种缺失型遗传性持续性胎儿血红蛋白综合征（ＨＰＦＨ）的基因型和表型的关系;探索进行β地中海贫血（地贫）复合这种缺失型ＨＰＦＨ的高风险胎儿的快速产前诊断方法。方法用跨越断裂点的三引物聚合酶链反应直接分析法检测ＨＰＦＨ缺失突变;用反向点杂交技术筛查β地贫基因突变。结果发现先证者（中间型地贫患儿）为一种缺失型ＨＰＦＨ和β地贫双重杂合子,其ＨＰＦＨ基因自母方遗传,在该家系三代６名成员中共检测到４例携带了这一基因;β地贫基因［密码子１４１５（＋Ｇ）移码突变］自父方遗传。在此基础上,我们对该家庭的中间型地贫高风险胎儿进行了产前基因诊断,结果显示胎儿的基因型与先证者完全相同,为ＨＰＦＨ和β地贫双重杂合子,故建议终止妊娠。人工流产后取样分析进一步证实了产前诊断的结果。结论这是国内首次完成并报告的对由ＨＰＦＨ基因与β地贫点突变双重杂合导致的中间型地贫高风险胎儿的产前诊断。所采用的技术简便、快速,可用作解决同类事件的参考。     

284例珠蛋白生成障碍性贫血产前基因诊断结果分析

目的 对284例珠蛋白生成障碍性贫血(简称地贫)产前基因诊断结果进行回顾性分析.方法 缺失型α地贫的产前诊断采用跨越断裂点PCR技术,非缺失型α地贫和β地贫的产前诊断采用反向斑点杂交技术进行检测.产前基因诊断完成6～8个月后,实验室检测的284例样本均由产前诊断科医生进行随访.结果 284例产前诊断样本均来自清远地区,α地贫基因共检测215例,其中产前诊断严重类型地贫47例(21.86％),包括重型α地贫36例(16.74％)和中间型α地贫11例(5.12％),轻型α地贫111例(51.63％),正常基因型αα/αα57例(26.51％);β地贫基因共检测52例,其中严重类型β地贫17例(32.08％),轻型β地贫20例(35.09％),正常基因型αα/αα15例(28.30％).复合型地贫17例,检出重型α地贫1例(6.67％),重型β地贫1例(6.67％),中间型α地贫复合轻型β地贫1例(6.67％),轻型α地贫复合轻型β地贫14例(82.35％).3例样本结果为轻型α地贫同时合并18三体综合征.37例重型α地贫、9例中间型α地贫、18例严重类型β地贫和3例轻型α地贫伴随18三体综合征,均在知情同意和自愿选择的原则下接受了终止妊娠的处理.结论 地贫高风险夫妇进行产前基因诊断,可有效检出严重类型地贫患儿,并能有效地防止重症地贫患儿的出生.     

α与β地中海贫血双重杂合子基因诊断

目的：探讨α与β地中海贫血双重杂合子的基因诊断。方法：采用聚合酶链反应（ＰＣＲ）和β地中海贫血等位特异寡核苷酸探针／反向点杂交（ＡＳＯ／ＲＤＢ）技术，对在遗传咨询和地中海贫血产前诊断病例中发现的６例疑为α与β地中海贫血双重杂合子个体，分别进行了α地中海贫血基因和β地中海贫血基因分析。结果：该６个病例均属东南亚缺失型α地中海贫血１和β地中海贫血双重杂合子（－－ＳＥＡ／αα，βＴ／βＡ），其中３例为α地中海贫血１和β４１４２（－ＴＣＴＴ），２例为α地中海贫血１和β－２８（Ａ→Ｔ）和１例α地中海贫血１与βＩＶＳⅡ６５４（Ｃ→Ｔ）双重杂合子。结论：α与β地中海贫血双重杂合子的检出对临床准确开展产前诊断有重要意义     

β-地中海贫血复合缺失型α-地中海贫血双重杂合子的分子检测及血液学分析

本研究对β-地中海贫血复合缺失型α-地中海贫血双重杂合子进行分子检测及血液学表型分析，以了解其检出情况及基因分布状况。采用单管多重gap—PCR技术检测3种常见的缺失型α-地中海贫血基因；采用PCR结合反向点杂交法检测β-珠蛋白基因17个位点的18种突变；进行血细胞常规分析。结果表明：81例β-地中海贫血杂合子中有15例复合缺失型α-地中海贫血，占18．52％。共有9种基因型，包括6例（7．41％）β-地中海贫血杂合子携带α-地中海贫血-1基因（--^SEA／αα-）；8例（9．88％）携带α-地中海贫血-2基因，其中6例（7．41％）为右侧缺失型（-α^3.7／αα），2例（2．47％）为左侧缺失型（-α^4.2／αα）；1例（1．23％）携带缺失型HbH基因（--^SEA／-α^3.7）。β-地中海贫血复合缺失型α-地中海贫血双重杂合子的各项红细胞参数与单纯β-地中海贫血杂合子比较差异无显著性意义（P〉0．05）。结论：梧州市β-地中海贫血复合缺失型α-地中海贫血双重杂合子的发生率很高，而血液学指标缺乏特异性。采用gap—PCR作为临床上地中海贫血筛查的一线方法，可以有效减少β-地中海贫血复合α-地中海贫血双重杂合子漏检的可能，对遗传咨询和准确进行产前诊断具有重要意义。     

Rapid screening of multiple beta-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes

BACKGROUND: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. METHODS: As a model, we designed a protocol based on the LightCycler technology to screen for a spectrum of beta-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of beta-globin gene mutations in the Greek population, including IVSII-745C-->G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G-->A, IVSI-5G-->A, IVSI-6T-->C, IVSI-110G-->A, and Cd39 C-->T. RESULTS: The protocol, standardized by analysis of 100 beta-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek beta-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations HbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five beta-globin gene mutations from other population groups (IVSI-1G-->T, IVSI-5G-->C, IVSI-116T-->G, Cd37 TGG-->TGA, and Cd41/42 -TCTT). CONCLUSIONS: The described LightCycler system protocol can rapidly screen for many beta-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.     

一个HPFH复合β地中海贫血家系的基因分析及其高危胎儿的产 …

目的 调查一种缺失型遗传性持续性肿儿血红蛋白综合征的基因型和表型的关系;探索进行β地中海贫血复合这种缺失型ＨＰＦＨ的高风险胎儿的快速产前诊断方法。方法 用跨越断裂点的三引物聚合酶链反应直接分析法检测ＨＰＦＨ缺失突变;用反向点杂交技术筛查β地贫基因突变。     

对142例β地中海贫血基因携带者进行缺失型α地中海贫血1基因分析

目的了解广东地区常见的β地中海贫血（地贫）与α地贫１复合存在的发生率。方法应用聚合酶链反应（ＰＣＲ）技术对１４２例经筛查确定为轻型β地贫的成人血样ＤＮＡ进行α地贫１基因检测,阳性者又应用突变引物ＰＣＲ特异扩增或用等位基因特异寡核苷酸探针反向点杂交（ＡＳＯ／ＲＤＢ）技术,确定其β地贫基因突变类型。结果１４２例中有１３例轻型β地贫样本同时合并有α地贫１基因,占总数的９１５％,其β地贫基因的突变类型分别是：ＣＤ４１４２（－ＴＣＴＴ）突变５例,ＩＶＳ２６５４（Ｃ→Ｔ）突变３例,ＣＤ１７（Ａ→Ｔ）突变２例,ＣＤ７１７２（＋Ａ）、ＣＤ４３（Ｇ→Ｔ）和－２８（Ａ→Ｇ）突变各１例。结论β地贫复合α地贫１的双重杂合子在轻型β地贫个体中的发生率较高,是该地区在地贫的遗传咨询和产前诊断工作中值得重视的一个问题。     

Molecular detection of Spanish deltabeta-thalassemia associated with beta-thalassemia identified during prenatal diagnosis

BACKGROUND: beta-Thalassemia is one of the most common single gene disorders and is widely distributed in the Mediterranean region. The deltabeta-thalassemias are a rare group of disorders, however in Spain the Spanish deltabeta0 thalassemia that removes 114 kb of beta-globin cluster is quite frequent. METHODS: To establish a molecular diagnosis of beta-thalassemia in a second-trimester fetus from a couple who are beta-thalassemia carriers, a rapid real-time PCR method was performed to detect major mutations prevalent in people of the Mediterranean basin (IVS I-1, IVS I-6, IVS I-110, CD-6, CD-37, and CD-39). The Spanish deltabeta0 deletion was detected using PCR with deletion-specific primers. RESULTS: The father was diagnosed as a carrier for the IVSI-6 (T>C) mutation and the mother as carrier for the Spanish deltabeta0 deletion. The fetus was a compound heterozygote for both those mutations. The phenotype of compound heterozygosity for deltabeta/beta-thalassemia can range from mild thalassemia intermedia to thalassemia major. After confirming these results in the DNA from amniocentesis obtained at the 20th week of gestation, the couple decided to terminate the pregnancy. CONCLUSIONS: This paper reports the first molecular characterization of Spanishdeltabeta0-thalassemia associated with beta-thalassemia IVSI-6, and confirms the importance of prenatal genetic testing for single gene disorder such as beta-thalassemia.     

βIVS—Ⅱ—1 G→A复合三联α基因所致的韩国人中间型β地中？…

目的 研究中间型β地中海贫血的分子机制,为中间型β地中海贫血的基因诊断提供科学依据。方法 应用聚合酶链反应（ＰＣＲ）,Ｓｏｕｔｈｅｒｎ印迹杂交和双链ＤＮＡ循环测序等技术,对在韩国首次发现的中间型β地中海贫血家系的α和β珠蛋白基因进行分析。结果 基因分析表明,该家系属典型的中间型β地中海贫血,它的分子病因是β珠蛋白基因的第２内含子５‘端的第１个碱基Ｇ→Ａ（ＩＶＳ－Ⅱ－１Ｇ→Ａ）突变和三联α珠蛋白基因     

Simultaneous PCR detection of beta - thalassemia and alpha - thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome

OBJECTIVE: To establish a rapid PCR method for simultaneous detection of beta-thalassemia and alpha-thalassemia 1 genes for diagnosis of complex alphabeta-thalassemia syndrome. DESIGN AND METHODS: Using multiplex allele specific PCR approach, we evaluated a simultaneous detection of the SEA type alpha-thalassemia 1 and the common Southeast Asian beta-thalassemia and hemoglobin E genes. The system was tested on known cases of double heterozygote for alpha- and beta-thalassemias and in a prenatal diagnosis of complex alphabeta-thalassemia syndrome. RESULTS: Co-inheritance of alpha-thalassemia 1 (SEA type) with each of the common beta-thalassemia genes in Southeast Asian and with hemoglobin E could be identified in a single PCR reaction. A successful application of this simultaneous detection system in prenatal diagnosis of a complex thalassemia syndrome caused by an EFBart's disease was demonstrated in a Thai family. CONCLUSION: We have shown that correct diagnosis of double heterozygosity for alpha-thalassemia 1 and beta-thalassemia or hemoglobin E could be obtained using a simultaneous multiplex PCR. These rapid PCR assays would facilitate characterization and prenatal diagnosis of complex thalassemia syndromes in the regions where both alpha- and beta-thalassemias and hemoglobin E are common.     

The studies of hemoglobinopathies and thalassemia in China--the experiences in Shanghai Institute of Medical Genetics.

BACKGROUND: In the past two decades, a large-scale survey of hemoglobinopathies and thalassemia was carried out in mainland China, involving nearly one million people in 28 provinces. The incidences of hemoglobin (Hb) variants, alpha-thalassemia and beta-thalassemia were 0.33%, 2.64% and 0.66%, respectively. The chemical structural analysis identified 67 Hb variants. Among them, 20 are new variants. The analysis of the alpha-globin gene organization in 111 HbH patients showed 76 cases (68.5%) were of the deletion type, 8 had Hb Constant Spring and the other cases were of non-deletion type. The results of the molecular characterization of more than 200 beta-thalassemia alleles showed that the most common types of beta-thalassemia mutations in China are CD 41/42 (-4 bp), IVS-II-nt.654 C-->T, CD 17 A-->T, CD 71/72 (+A) and -28 A-->G. METHODS: To explore the simple method for molecular diagnosis of beta-thalassemia, multiplex allele-specific amplification (MAS-PCR) was used that could simultaneously detect the above five common types of beta-thalassemia mutations. RESULTS: Based on the molecular analysis of beta-thalassemia intermedia, beta-thalassemia homozygotes or compound heterozygotes combined with alpha-thalassemia, as well as the conjunctive abnormalities of beta-thalassemia heterozygote with triplicated haplotype of alpha-globin genes, were the most common cause of thalassemia intermedia in China. We also used the RT-PCR quantitation method to show that the most common beta-thalassemia allele, IVS-II-nt.654 C-->T, still produced a small amount (about 15%) of normally spliced beta-globin mRNA, therefore, causing beta+-thalassemia. In clinical trials of hydroxyurea (HU) treatment for beta-thalassemia patients, we found that HU may enhance the expression of the beta-globin gene in some patients, leading to an alleviation of clinical symptoms. In the studies of the reversal of aberrant splicing of IVS-II-nt.654 C-->T allele by the antisense approach, we constructed a mammalian expression vector that can produce an antisense RNA targeting against the aberrant splice sites of IVS-II-nt.654 C-->T pre-mRNA. CONCLUSIONS: The results indicated that the antisense RNA produced from the vector could efficiently suppress the aberrant splicing pattern and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemia cells.     

母血中胎儿有核红细胞的分离及无创性产前基因诊断研究

目的 从母血循环中分离、富集、鉴定和提取胎儿有核红细胞，建立遗传病无创性产前基因诊断方法。方法 取２５名妊娠８～３６周的孕妇静脉血，加载在细胞分离液Ｈｉｓｔｏｐａｑｕｅ１．０７７上分离单个核细胞（ＭＮＣ）；用包被ＣＤ７１抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ２１免疫磁珠或包被ＣＤ４５抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ４５免疫磁珠富集ＭＮＣ中的胎儿有核红细胞：ａｎｔｉ－ξ－ｂｉｏｔｉｎ或ａｎｔｉ