地中海贫血的分子基础及产前诊断-徐湘民

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,和,地中海贫血的分子基础及产前诊断,徐湘民,南方医科大学，医学遗传学教研室,南方医院产前诊断和遗传病诊断技术中心,Zhongshan,2007, November 30,遗传性血红蛋白病,Genetic disorders of human hemoglobin,异常血红蛋白,Abnormal hemoglobin, Structural variants,地中海贫血,(,地贫,),Thalassemias,-,thalassemia, -,thalassemia,遗传性持续性胎儿血红蛋白,Hereditary persistence of fetal hemoglobin(HPFH),Thalassemia,地中海贫血是全球最大的单基因遗传病之一,中国南方不同高发地,区人群携带率,1,23%,研究背景,全球血红蛋白病人群负荷,WHO,地区,人口,(,百万,),出生,(,百万,),纯合子出生,(,千,),非洲,650,30.0,230,美洲,730,17.5,5.0,亚洲,3150,84.0,120.0,欧洲,780,11.0,1.6,大洋洲,30,0.5,0.2,合计,5340,143.0,356.8,中国南方地中海贫血人群携带率,地区,携带率,(%),地贫,地贫,广东,8.53,2.54,广西,14.95,6.78,四川,1.92,2.18,台湾,4.20,1.10,香港,5.02,3.41,Xu, et al.,J,Clin,Pathol,2004, 57:517-22.,Cai, et al.,Chi J,Epidemiol,2002,23:281-5.,排序,出生缺陷分析,例数,发生频率,(/,万,),1,先天性心脏病,260,19.66,2,重型,地贫,208,15.73,3,多指,200,15.12,4,肢体短缩,111,8.39,5,唇裂,/,腭裂,109,8.24,广州市主要出生缺陷的发生率,(1998-2001),Pedigree for,-,thalassemia,in a typical family.,地中海贫血,:,常染色体隐性遗传病,地贫是严重致死,、,致残性遗传性血液病,常染色体隐性遗传病,受累基因：,地贫,-,珠蛋白基因,（,16p13.3,）,地贫,-,珠蛋白基因,（,11p15.3,）,突变纯合子：,重型地贫,-,致死,中间型地贫,-,致残,病理学基础：,/,链比例失衡,地贫纯合子：致死性出生缺陷,重型,地贫,:,胎儿水肿综合征,早产死胎或出生前后死亡,可导致严重产科并发症,无理想治疗方法,重型,地贫,出生后,1,年内发病,多于未成年前因严重贫血而夭折,输血和去铁治疗可维持,代价昂贵,家系样品采集现场,中间型地贫是,严重致残性遗传病,研究地中海贫血的意义,重症地贫患儿的出生是世界公认的公共卫生问题,通过产前诊断阻止重症患儿出生是首选预防措施,WHO,优先推荐在发展中国家实施预防的遗传性疾病,预防地贫是中国南方高发区减少出生缺陷的战略需求,研究背景,中国需要解决的研究主题,发展用于临床和预防的分子诊断技术,高发地区人群中的遗传流行病学调查,开展人群水平的地中海贫血预防,指导制定计划,大人群监控和预防,用于基因分型,用于产前诊断,诊断,筛查,预防,地中海贫血的研究策略,研究背景,遗传流行病学调查,建立一系列诊断标准和诊断技术,地中海贫血的临床特征,小细胞低色素性溶血型贫血,(,中、重度,),黄疸,肝脾肿大,(,脾大明显,),骨髓扩增,发育迟缓,合并感染,Bart,s,水肿胎,(,地贫,宫内或出生后半小时内死亡,),地中海贫血的血液学表型特征,RBC,参数,MCV,MCH,细胞形态变化,Hb,电泳分析,Hb,A,2,(,),或,(,),Hb,Barts,Hb,H (,),Hb,E (,),地贫血象：红细胞着色不足、异形红细胞和骨髓成红血细胞增多症,血红蛋白电泳图谱,1 2 3 4 5,1.,正常人,HB,2.,轻型,地贫,3.,异常,HB E,杂合子,4.,新生儿,HB Barts (,地贫标准型,）,5. HB H,Normal AA,2,AE Barts AF HA,+,-,500V, 25min,地贫,发生的,分子病理学经典机制,地贫,:,基因缺失使基因功能丢失,地贫,:,结构基因变异使,转录异常,-,50kb -10kb 0kb 10kb 20kb 30kb,(-SEA) Deletion,(-,4.2) Deletion,(-,3.7) Deletion,2 ,1,导致,地贫的不同类型的基因缺失,Globin,genes affected by,thalassemia,deletion,Silent carrier,Thalassemia,trait,Hb,H disease,Hydrops,fetalis,Normal,地贫,疾病严重程度的单倍型顺序,2,1,T,2, ,1,2,T,1, ,1,T, ,Hematological data of the,proposituse,and her family members.,1,2,1,2,3,4,5,1,2,Sex/,age(Y,),F/49,M/54,M/33,F/27,F/25,M/22,F/35,F/1,M/5,Hb(g,/L),118,150,173,126,135,13 5,118,112,141,RBC(1012),5.63,4.96,5.70,6.23,4.51,4.57,5.87,6.09,5.02,MCV(fL,),64.5,85.7,88. 1,62.1,87.8,82.3,63.0,56.2,78.5,MCH(pg,),21.0,30.2,30.4,20.2,29.9,29.5,20.1,18.4,28.1,HbA,(%),97.8,97.5,93.1,97.0,96.6,95.7,96.9,90.0,97.4,HbA2(%),2.20,2.50,3.27,2.98,3.37,3. 28,3.12,2.18,2. 65,HbF,(%),0,0,3.62,0,0,0,0,7.79,0,Genotype,-11.1,/,/,/,-11.1,/,/,/,-11.1,/,- 11.1,/,/,Jia,SQ, et al. J,Clin,Pathol, 2004; 57:164-7.,Different point mutations,reducing,globin,gene expression,EXON 1,IVS 1,EXON 2,EXON 3,IVS 2,Promoter mutation,RNA splicing,Initiation mutation,Frameshift,Nonsense,Poly(A,) signal,Rund,. &,Rachmilewitz,.,N. Engl. J. Med.,2005, 353:1135-46.,世界上不同种族的常见,地贫突变,类型,种 族 突变类型,地中海人,IVS-1, position 110 (G,A),CD39, nonsense (CAG,TAG),IVS-1, position 1 (G,A),IVS-2, position 745 (C,G),IVS-1, position 6 (T,C),IVS-2, position 1 (G,A),黑人,29,(A,G);,88,(C,T),Poly(A), (AATAAA,AACAAA),东南亚人,中国人,CD 41-42,Frameshift,(,CTTT),IVS-2, position 654 (C,T),28,(A,G);,Hb,E (G,A),CD17, nonsense (AAG,TAG),IVS-1, position 5 (G,C),亚洲印地安人,IVS-1, position 5 (G,C),619-bp deletion,CD 8-9,Frameshift,(,+G,),IVS-1, position 1 (G,T),CD 41-42,Frameshift,(,CTTT),Sex,Proband,female,Mother,female,Father,male,Brother,male,Husband,male,RBC(x1012),5.7,4.8,4.7,5.8,6.4,Hb,(g/L),139,105,154,144,145,MCV (fl),73.6,68.2,88.2,73.4,67.1,MCH (pg),23.6,21.8,33.0,24.7,22.8,MCHC (g/L),357,320,374,336,340,RDW (%),0.149,0.152,0.132,0.144,0.159,HbA,(%),94.3,93.6,97.2,94.0,97.1,HbA,2,(%),5.7,6.4,2.8,6.0,2.9,Genotype,-90,/,N,-90,/,N,N,/,N,-90,/,N,N,/,N,/ ratio,2.130,2.219,3.716,2.238,5.341,Results of blood analysis, DNA diagnosis and mRNA detection in the family members.,Sequencing of the,-,globin,gene amplified from the,proband,Heterozygosity,for the C,T substitution,at position-90 of,the-globin,gene,近年来发现的部分中国南方新突变,基 因,突 变,珠蛋白基因,11.1,大片段缺失,珠蛋白基因,2,CD118(+TCA),珠蛋白基因,90(CT,),珠蛋白基因,73(AT),珠蛋白基因,28(AC),珠蛋白基因,CDs15/16(+G),珠蛋白基因,CD38(,A),地中海贫血的产前诊断,胎儿取样技术,DNA,诊断技术,-,地贫的诊断,-,地贫的诊断,羊膜穿刺,(amniocentesis),抽取胎儿样品示意图,植入前基因诊断,Preimplantation,genetic diagnosis(PGD),超声产前诊断,Prenatal diagnosis: Ultrasound measurement,母体外周血分离胎儿细胞进行产前诊断,Prenatal diagnosis: Fetal cells in the maternal circulation,母体血浆或血清中胎儿,DNA,分析,Analysis of fetal DNA in maternal plasma and serum,目前及今后的胎儿产前诊断新技术,Ding C, Chiu RW, Lau TK, Leung TN, Chan LC, Chan AY,Charoenkwan,P, Ng IS, Law HY, Ma ES,Xu,X,Wanapirak,C,Sanguansermsri,T, Liao C, Ai MA, Chui DH, Cantor CR, Lo YM.,MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis.,Proc,Natl,Acad,Sci,U S A. 2004 20;101(29):10762-7.,单碱基延伸产物的质谱分析,M F,F,父亲的突变类型,SABER:,single allele base extension reaction,分子量,F,M,Standard protocol,SABER protocol,在母血浆,DNA,中,排除,父亲的,地贫突变,+,+,双亲基因型不同：,Case,CD 41/42,- CTTT,IVS2 654,C,T,nt,28,A,G,CD 17,A,T,Standard protocol,SABER,Fetal genotype,Weeks gestation,1,F,M,N,/ N,11,2,F,M,N,/ N,18,3,F,M,N,/ N,21,4,M,F,F,/ N,18,5,M,F,F,/ M,17,6,F,M,F,/ N,11,7,F,M,F,/ N,14,8,F,N,/ N,7,9,F,F,/ N,12,10,M,F,N,/ N,17,11,F,F,/ N,20,12,M & F,N.A.,N.A.,N / N,18,鉴定母血浆,DNA,中的父亲,地贫突变,单倍型分析用于鉴定母血浆,DNA,中的父亲等位基因,Case,Mother,Father,HBB mutant,allele,HBB WT,allele,SNP,G,allele,Paternal HBB mutation,Fetal HBB genotype,3,CC,G,C,G,C,N / N,11,CC,G,C,G,C,F / N,12,CC,G,C,G,C,N.A.,N / N,地中海贫血的产前诊断,胎儿取样技术,DNA,诊断技术,-,地贫的诊断,-,地贫的诊断,DNA,诊断技术,多重,PCR,（,multiplex PCR,）,等位基因特异性性寡核苷酸杂交,（,ASO,）,反向点杂交 （,RDB,）,变性高效液相色谱（,DHPLC,）,基于,微孔板,(,microtiter,well-based ),高通量检测,实时,PCR (real-time PCR),-,50kb -10kb 0kb 10kb 20kb 30kb,(-SEA) Deletion,(-,4.2) Deletion,(-,3.7) Deletion,2 ,1,导致,地贫的不同类型的基因缺失,Location of primers and detection of -,SEA,deletion by gap-PCR,Genotyping of the-,SEA,alleles by Gap-PCR,Chong,SS, et al. Blood. 2000; 95(1):360-2.,寡核苷酸探针杂交技术,ASO,探针检测,-,地贫基因诊断结果,PCR,B,B,B,B,B,B,gDNA,Hybridization,Detection,Streptavidin,-POD,Color Signal,M,Membrane,ASO,N M N M N M,Substrate(TMB),反,向,点,杂,交,技,术,RDB analysis: application to rapid prenatal diagnosis,for eight,thalassaemia,mutations.,Genotype,Father,CDs41-42 (,CTTT) / N,Mother,IVS-2-654 (C,T) / N,Fetus,CDs41-42 / IVS-2-654,Normal control,N / N,Human,-globin gene and the PCR amplicons,5UTR,EXON 1,IVS 1,EXON 2,IVS 2 IVS2,EXON 3,3UTR,FA(453bp),FB(364bp),FC(423bp),-90,-73,-29,-28,cd2,cd13-14,cd14-15,cd15-16,cd17,cd26,cd27-28,IVS1-1,IVS1-5,cd37,cd38,cd41-42,cd43,cd71-72,IVS2-5,IVS2-16,IVS2-654,IVS2-666,Fragment,Mutation patterns,Normal patterns,Total,Mutation,Mutation + SNP,Heterozygous,Homozygous,FA,12,22,1,2,37,FB,5,15,2,2,24,FC,0,3,2,2,7,Total,17,40,5,6,68,68 uniquely profiled DHPLC patterns,observed from 795 DNA samples,实验结果,实验结果,实验结果,实验结果,实验结果,中国南方,和,地中海贫血的预防计划,人群筛查预防计划的策略,医院水平,(Hospital-based),的预防计划,社区水平,(community-based),的预防计划,家系扩展筛查,(screening extended families),Carrier screening for,and,thalassemia,in pregnancy: the results of an 11-year prospective program in Guangzhou Maternal and Neonatal Hospital,Guangzhou Maternal and Neonatal Hospital,Liao C,Li J Huang YN Li QM,Feng,Q,Zhong,HZ Cui YY,Southern Medical University,Mo QH,Li LY,Hua,L Zhang JZ,Zeng,R,Jia,SQ,Xu,XM,Prenat,Diagn,2004, 25:163-71,地贫携带者筛查及产前诊断流程,被检对象,（婚前或孕中期）,血液学分析,（,MCV,，,Hb,A2,变化）,排除地贫,地贫携带者,遗传咨询，配偶检查,血液学分析,（,MCV,Hb,A2,变化）,地贫携带者,排除地贫,遗传咨询,双亲基因分析,胎儿产前诊断,年份 筛查 广州总 筛查,孕妇数 孕妇数 丈夫数,筛查发现的 检 测 出 的 完 成 的 产 前,阳性孕妇数 阳 性 夫 妇 数 诊 断 数,地贫,(%),地贫,(%),地贫,(%),地贫,(%),/,地贫,(%),地贫,(%),地贫,(%),1993,3989,68393,350,232 (5.8),129 (3.2),13,(3.7),8,(2.3),10,(2.9),12 (92.3),7,(87.5),1994,4564,71617,421,265 (5.8),164 (3.6),18,(4.3),8,(1.9),9,(2.1),16 (88.9),8,(100),1995,3902,71751,361,227 (5.8),142 (3.6),17,(4.7),7,(1.9),12,(3.3),15 (88.2),7,(100),1996,4994,74460,464,285 (5.7),181 (3.6),19,(4.1),8,(1.7),12,(2.6),17 (89.5),8,(100),1997,4392,71989,401,250 (5.7),158 (3.6),21,(5.2),7,(1.7),10,(2.5),18 (85.7),7,(100),1998,4532,75378,425,263 (5.8),172 (3.8),17,(4.0),8,(1.9),12,(2.8),17,(100),8,(100),1999,5016,80141,465,285 (5.7),186 (3.7),24,(5.2),8,(1.7),14,(3.0),23,(95.8),8,(100),Outcomes of screening program in GZMNH 1993-2003,年份 筛查 广州总 筛查,孕妇数 孕妇数 丈夫数,筛查发现的 检 测 出 的 完 成 的 产 前,阳性孕妇数 阳 性 夫 妇 数 诊 断 数,地贫,(%),地贫,(%),地贫,(%),地贫,(%),/,地贫,(%),地贫,(%),地贫,(%),2000,4492,81817,418,256 (5.7),172 (3.8),21 (5.0),7 (1.7),16 (3.8),21 (100),7,(100),2001,4521,80152,411,254 (5.6),164 (3.6),17 (4.1),8 (1.9),12 (2.9),17 (100),8,(100),2002,4695,80667,415,260 (5.6),163 (3.4),16 (3.9),8 (1.9),11 (2.7),16 (100),8,(100),2003,4124,86393,372,227 (5.5),152 (3.6),15 (4.0),6 (1.6),13 (3.5),15 (100),6,(100),Total,49221,860758,4503,2804 (5.7),1783 (3.6),198 (4.4),83 (1.8),131 (2.9),187 (95.8),82 (98.8),( contd ),诊断结果,基因型,例数,正常,30,杂合子,(total),35,CDs 41-42 (,TCTT) / N,14,IVS-2-654 (CT) / N,8,-28 (AG) / N,5,CD17 (AT) / N,3,CD26 (GA) / N,2,-29 (AG) / N,1,CDs27-28 (+C) / N,1,Unknown / N or Normal,1,Prenatal diagnosis in 83 fetuses at-risk for,thalassemia,诊断结果,基因型,例数,纯合子或双重杂合子,(,总数,),18,CDs 41-42 (,TCTT) / IVS-2-654 (CT),6,CDs 41-42 (,TCTT) / -28 (AG),4,CDs 41-42 (,TCTT) / CD17 (AT),2,IVS-2-654 (CT) / CD17 (AT),2,CDs 41-42 (,TCTT) / CDs 41-42 (,TCTT),1,CDs 41-42 (,TCTT) / CDs 27-28 (+C),1,IVS-2-654 (CT) / CDs 71-72 (+A),1,IVS-2-654(CT) / CD43 (GT),1,总数,83,( contd ),胎儿诊断,产前诊断例数,地贫,地贫,总数,(%),正常,41,30,69 (25.6),携带者,92,35,125 (46.3),受累胎儿,*,54,18,72 (26.7),胎儿流产,2,1,3 (1.11),误诊,0,0,0.0,总数,(%),187 (69.3),83 (30.7),270 (100),Results in 266 pregnancies where a precise,diagnosis was achieved,*,Three,Hb,H disease involved,Detection of the -,SEA,/ deletion by 1%,agarose,gel,electrophoresis of amplified fragments using gap-PCR.,M N P B,Fa,Mo Fe,Fa,Mo Fe,Fa,Mo Fe,Normal Heterozygote Homozygote,Family 1 Family 2 Family3,Normal,Mutant,筛查发现的阳性孕妇或阳性夫妇,地贫,1,地贫,地贫,地贫,5.7,%,3.6,%,4.4,%,1.8,%,Total of 4503 couples screened,Outcomes of screening program in GZMNH 1993-2003,54,例,Barts,水肿儿和,Hb,H,病,18,例,重型,地贫,2804 1783 198,对,83,对,产前诊断：,187,例,地贫,82,例,地贫,49221,例,被筛查的孕妇,4587(9.3%),地贫特征,/,病例,2804 (5.7%),地贫,1783 (3.6%) ,地贫,139,例其他异常,Hb,个体,44495,例正常表型个体,4503(98.2%),阳性孕妇的配偶被筛查,281(6.2%),例高风险妊娠,198,重型,地贫,83,重型,地贫,84(1.8%),例配偶未接受筛查,281(100%),例高风险妊娠孕妇,进入跟踪随访,4222,例非风险妊娠,269 (95.7%),孕妇实施产前诊断,187,地贫,83 ,地贫,(1,双胞胎,),72(26.7%),孕妇要求终止妊娠,54,重型,地贫,18,重型,地贫,(1,双胞胎,),69,重型地贫被预防,51,水肿胎,18,重型,地贫,(1,双胞胎,),12,孕妇不接受产前诊断,197,例孕妇继续妊娠,3,例,HbH,病出生,2,例,水肿胎出生,自行研制的东南亚型,地贫,链免疫学检测试剂盒,A,B,产前遗传学的临床和公共卫生问题,利用有效的公共卫生教育资源，首先使医生,和公众了解更多的地贫知识和知晓选择遗传,服务及其对待产前诊断的态度是我们面临的,一个重要课题；,2.,为满足大规模预防工作的需要，我们须大力,加强专科医生,遗传咨询师，及其他相关专业,人员的培训工作,;,3.,预防监测系统建立过程中，要特别注重转诊、,病例登记、实验室质量控制的把关,;,4.,政府在组织、基础建设、制定政策法规和行,政管理上的主导作用是推动预防计划的关键,因素。,( contd ),A,cknowIedg,ments,第一军医大学 南方医科大学,广州市妇婴医院,珠海市妇幼保健医院,韶关市,妇幼保健医院,广东医学院附属一医院,汕头市第二人民医院,柳州市妇幼保健医院,美国,Transgenomic,公司,珠海市海泰生物制药有限公司,NSFC, 973 Program,广东省科技厅,广东省卫生厅,广州市卫生局,四会市卫生局,