β地中海贫血[-28(A→G)·CD17(A→T)/N]双重突变杂合子的基因鉴定及家系分析

目的：报道β地中海贫血（β地贫）［－２８（Ａ→Ｇ）·ＣＤ１７（Ａ→Ｔ）／Ｎ］双重突变杂合子的基因鉴定及家系分析结果。方法：采用聚合酶链反应结合等位基因特异的寡核苷酸探针杂交（ＰＣＲ／ＡＳＯ）技术。结果：对４例先证者及其家系成员计３５人进行基因鉴定，共检出２２人同时携带有β地贫－２８（Ａ→Ｇ）和ＣＤ１７（Ａ→Ｔ）的突变基因，结合临床体征、血液及生化指标、家系调查资料综合分析，确定他们是β地贫［－２８（Ａ→Ｇ）·ＣＤ１７（Ａ→Ｔ）／Ｎ］双重突变杂合子，为同一条染色体上基因的双重点突变。结论：［－２８（Ａ→Ｇ）·ＣＤ１７（Ａ→Ｔ）／Ｎ］双重突变杂合子是一种新的β地贫类型，这种β地贫的发现，为认识我国地贫的高度异质性积累了资料。     

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。     

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     

羟基脲治疗一例-28/654-2型β地中海贫血疗效观察

β地中海贫血（β地贫）危害最大的是重型β地贫。在基因替代治疗还未取得实质性进展的情况下,应用某些药物,如羟基脲（ｈｙｄｒｏｘｙｕｒｅａ,ＨＵ）等调节珠蛋白基因表达的开关,激活非α珠蛋白基因的合成,从而恢复α与非α珠蛋白肽链的平衡,即基因调控治疗,被认为是目前治疗β地贫的一种有效途径［１］。我们与我校附属第二医院协作,将ＨＵ试用于几例具有４１４２（－４ｂｐ）、６５４２（Ｃ→Ｔ）、１７（Ａ→Ｔ）、－２８（Ａ→Ｇ）以及７１７２（＋Ａ）的双重杂合子或纯合子的治疗,发现只有对具有－２８突变的双重杂合…     

丝绸之路沿线陕甘新地区β地中海贫血CD17（A→T）的分布特点

目的：探讨β地中海贫血（β地贫）ＣＤ１７（Ａ→Ｔ）在中国丝绸之路地区的分布特点。方法：应用ＤＮＡ扩增结合等位基因特异的寡核苷酸探针杂交（ＰＣＲ／ＡＳＯ）技术。结果：对该地区的８５例β地贫先证者进行了基因分析,检出２４例ＣＤ１７突变型,其中陕西３例、甘肃２０例、新疆仅１例,以甘肃地区检出率为最高。结论：根据ＣＤ１７在丝绸之路地区的分布特点,结合国内外文献综合分析,推测这种β地贫的突变基因可能起源于甘肃陇西。     

应用SSCP及测序方法检出三种罕见的β地中海贫血突变型

为明确广西３例不能被多聚酶链反应－寡核苷酸探针斑点杂交法检出的β地中海贫血（β地贫）基因携带者的突变型，用银染法单链构象多态性（ＳＳＣＰ）分析和化学发光直接测序法对该３例β地贫基因携带者进行检测。结果发现３种中国人罕见的β地贫突变型，即ＣＤ３０（ＡＧＧ→ＧＧＧ），ＣＤ９５（＋Ａ）和ＣＤＳ２７－２８（＋Ｃ）。其中ＣＤ３０（ＡＧＧ→ＧＧＧ）为国内外首次报道，ＣＤ９５（＋Ａ）首次在中国人中发现     

一个HPFH复合β地中海贫血家系的^Aγ珠蛋白基因分析

一个ＨＰＦＨ复合β地中海贫血家系的Ａγ珠蛋白基因分析孙琼陈美珏任兆瑞曾溢滔我们已报道过一个β地中海贫血（β地贫）复合遗传性持续性胎儿血红蛋白症（ＨＰＦＨ）的家系［１］。研究结果表明，该家系的β地贫基因为ＩＶＳ－Ⅱ－６５４Ｃ→Ｔ剪接缺陷型突变，来自母系...     

广西壮族人HLA-DQB1等位基因与β地中海贫血的相关性

目的：探讨ＨＬＡ抗原与β地中海贫血（地贫）的相关性。方法：应用聚合酶链反应序列特异引物（ＰＣＲＳＳＰ）方法对６４名随机抽样的，无血缘关系，三代以上均为壮族并三代以上居住在广西，籍贯分布广西各地，均在本实验室经基因诊断证实为β地贫的患者进行ＨＬＡＤＱＢ１基因分型。结果：发现ＨＬＡＤＱＢ１０６０４等位基因在该群体中频率为１２．５％，对照组为１．４％；其相对危险度为９．９，与对照组比较，有非常显著差异（显著性检验χ２＝２０．７８，Ｐ＜０．００５）。结论：提示ＨＬＡＤＱＢ１０６０４等位基因与广西壮族居民β地贫的易感性有关联     

应用ARMS法对β地中海贫血进行基因型分析

β地中海贫血 (β地贫 )是我国南方和西南各省常见的遗传病 ,从婚前或产前检查中检出 β地贫携带者 (轻型 )并鉴定他们的基因突变类型是降低重型 β地贫患儿出生的有效预防方法。已经报告的中国人中 β珠蛋白基因突变型已达 2 9种。对于常见突变使用的反向点杂交法 (ＲＤＢ)已经得到广泛应用。毛跃华等[1] 报告了多重ＰＣＲ检测我国常见 4种 β地贫的突变特异性扩增系统 (ＡＲＭＳ)法 ,杜传书等[2 ] 补充了占第 2位的ＩＶＳ Ⅱ 6 5 4(Ｃ→Ｔ)突变的ＡＲＭＳ检测法。我们用此法对 95例β地贫进行基因型分析 ,评价其作为常规分子生物学诊断方…     

β地中海贫血多突变位点多聚酶链反应与反向斑点杂交基因分析

目的：研究３８例β地中海贫血（β地贫）基因突变以及与临床表型的相关性。方法：采用ＰＣＲ／反向斑点杂交（ＰＣＲ／ＲＤＢ）技术同时检测１１种β地贫基因突变位上 对所有病例作血液学分析和临床资料收集对比。结果：本组病例共栓出７种基因空谈类型涉及４６个位点突变,包括４种常见类型（３５／４６,７６．１％）,三种罕见类型（１１／４６,２３．９％）。３２例轻型β地贫中,３０例为基因突变杂合子,２例是双重基因突变     

Gene identification and pedigree analysis of the double mutation heterozygote of beta-thalassemia[-28(A-->G).CD17(A-->T)/N]]

OBJECTIVE: To report the result of gene identification and pedigree analysis of the double mutation heterozygote of beta-thalassemia[-28(A-->G).CD17(A-->T)/N]. METHODS: Polymerase chain reaction in combination with dot-blot hybridization of allele-specific oligonucleotide(PCR/ASO) probes was used. RESULTS: Gene identification was carried out in four probands and 35 other family members. There were 22 patients with beta-thalassemia -28(A-->G) and CD17(A-->T) mutant gene. Based on the clinical features, the results of hematological and biochemical examinations and pedigree analysis, they proved to be the double mutation heterozygote of beta-thalassemia[-28(A-->G).CD17(A-->T)/N], i.e., double point mutations on the same chromosome. CONCLUSION: Double mutation heterozygote[-28(A-->G).CD17(A-->T)/N] is a new type of beta-thalassemia.     

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.     

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。     

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.     

应用液态基因芯片技术对132例β-地中海贫血基因检测分析

目的用液态基因芯片技术检测深圳东部地区人群中β-地中海贫血分析常见类型。方法采用液态基因芯片技术，使用Luminex100多功能悬浮点阵检测仪及Lumi2 nex Data Collectorvisionl．7数据收集软件检测并通过突变标本与正常标本的莹光强度比值（Mutan／Normal）分析深圳东部地区人群5637例标本。结果发现132例携带β-地中海贫血突变基因，携带率为2．34%。其中常见点突变模式中CD41／42（TCTT）突变61例（占46．2％），IVS2—654（C→T）29例（21．9％），-25（A→G）突变14例（10．6％），-13（A→T）突变11例（8．3％），CD71／72（＋A）5例（3．8％），-29（A→G）突变杂合3例（2．27％），28（A→G）复合CD41／42-TCTT突变7例，其他少见杂合点突变2例，常见单点突变β-地中海贫血患者共123例为93．18％，少见模式和多点杂合突变共9例，为6．82％。     

A family with multiple mutations and sequence variations in the alpha- and beta-globin gene clusters.

BACKGROUND: Usually, laboratory diagnostics of hereditary hemoglobin disorders is fairly straightforward. Sometimes, however, correct diagnosis can be difficult. In this study, we describe a family with multiple mutations and sequence variations in the alpha- and beta-globin gene clusters. METHODS: Hemocytometry results were obtained using an automated cell counter. Hemoglobin variant analysis was performed by cation-exchange HPLC. PCR and DNA sequence analyses were used to identify mutations in the globin genes. RESULTS: The proposita was referred to our laboratory for hematological evaluation [hemoglobin 145 g/L (119-155 g/L) mean corpuscular volume 72 fl (80-97 fl), mean corpuscular hemoglobin 26 pg (28-36 pg), erythrocytes 5.6 x 10(12)/L (3.7-5.0 10(12)/L)]. Characterization and quantification of hemoglobin variants showed 11.3% HbA1, 4.4% HbA2, 58.9% HbC and 23.0% HbF. Subsequent analysis revealed, in addition to a heterozygous HbC mutation, the presence of a beta-thalassemia causing mutation (-90C>T), a heterozygous alpha-thalassemia (-alpha(-3.7)/alpha alpha) and three different gamma-globin sequence variations. Additional molecular analysis was performed in all family members. CONCLUSIONS: In the family presented in this study, 10 different mutations were found in the globin genes. Molecular analysis was necessary to clarify hemoglobin variant analysis, in particular the low amount of HbA1 in the proposita. Knowledge of the molecular background facilitates in the understanding of the hematological parameters and proper counseling of the patient.     

缺失型α—地中海贫血中的基因诊断新技术研究

目的 建立诊断我国最常见的三种缺失型α-地中海贫血（α-地贫）,即东南亚型缺失（--^SEA)、右侧缺失（-α^3.7)和左侧缺失（－α^4.2)的PCR技术。方法 设计三组PCR引物,优化PCR反应条件,运用PCR、琼脂糖凝胶电泳、UVP凝胶成像技术,根据电泳图谱检测并诊断三种缺失型α-地贫。结果 成功地检测这三种缺失型的纯合子、杂合子或双重杂合子,结果与Southern blotting分析一致。完成42例α-地贫样本的基因诊断。结论 本研究所建立的检测α-地贫缺失型的技术,准确、简便、重复性好,便于推广应用。