【作者】 陈万群；

【导师】 徐湘民；

【作者基本信息】 南方医科大学 ， 细胞生物学， 2009， 博士

【摘要】 背景与目的β地中海贫血（简称β地贫,β-thalassemia）是世界上最常见的单基因遗传病之一,尤其是在热带和亚热带地区（包括华南地区）,该病的发生率特别高。广东和广西是中国发生率最高的两个省区,分别高达2.54%和6.78%。β地贫是由于β珠蛋白肽链合成减少或缺失而导致的一种疾病,主要由β珠蛋白基因的点突变或小片段的插入或缺失引起。从功能上看,β突变可以分为β⁰（完全没有β珠蛋白产生）,β⁺（有少量β珠蛋白产生）,β⁺⁺和β^silent（对β珠蛋白的合成没有或影响很小）。根据临床表型,β地贫可以分为三种主要的类型:重型地贫（thalassemia majior,TM）,轻型地贫（thalassemia trait,TT）和中间型地贫（thalassemia intermediam,TI）。重型地贫是病情最为严重的一类地贫,患者从幼儿期就要定期输血才能存活。轻型地贫的患者一般表型正常,没有地贫表征。而中间型地贫是介于重型地贫和轻型地贫之间的一类地贫。中间型地贫的表型轻重不一,跨越了很大的表现谱:轻者只有轻微的地贫表征,可无明显的临床症状;重者需要偶尔不定时输血,出现肝脾肿大等明显的地贫特征。中间型β地贫的表型多样,它的分子基础也相当复杂,具有很大的遗传异质性。Thein SL总结了β地贫的主要遗传修饰因子有:β珠蛋白基因和α珠蛋白基因,以及影响γ珠蛋白基因型表达的一些遗传因素。研究表明与γ珠蛋白基因表达有关的遗传修饰因素主要有:p珠蛋白基因簇的3’HS1（+179C→T）多态位点,位于β珠蛋白基因簇5’HS2的模序（AT）_XN_Y（AT）_Z,β珠蛋白基因上游-540区的模序（AT）_XT_Y,BCL11A基因内的SNP位点rs11886868（T→C）等。此外,有些遗传修饰因子可以调节或导致β地贫,如GATA-1,α血红蛋白稳定蛋白（Alpha HaemoglobinStabilizing Protein,AHSP）和血红素调节抑制因子（Heme-regulated eIF2αkinase,HRI）等。目前在意大利、印度、伊朗等多个国家,中间型β地贫的分子基础都已经有了比较系统的研究。然而目前我国针对中间型β地贫的研究仅有少数个案报道,缺乏系统的研究,导致人们对于我国的中间型β地贫的遗传基础了解很少,妨碍了我国南方这一重要遗传病临床诊治和遗传咨询等临床工作的深入开展。本研究立足于地贫的高发区收集中间型β地中海贫血样本,系统分析来自华南地区的117例中间型β地贫患者的分子基础,研究靶点包括β珠蛋白基因型,α珠蛋白基因型和其他影响α/β珠蛋白基因比例的一些遗传修饰因素。本研究拟通过对中间型β地贫患者的临床资料,血液学资料和分子基础进行综合分析,阐明华南地区的中间型β地贫的分子基础,为临床实践提供必要的支持。病例样本与方法1.病例样本及表型分析:纳入本研究的样本来自地中海贫血高发区（主要是广西省和广东省）的109个家系,共117例中间型β地贫患者（纳入标准:Hb含量在60-105 g/L;MCV<80 fL;MCH<27 pg;在幼儿期间可以不依赖输血而能够存活;发病年龄在2岁以后）。统计样本的输血情况,首次发病时的年龄,肝脾是否肿大,有无进行脾切除手术,有无地贫面容等。采集117例样本的EDTA抗凝外周静脉血。2.实验方法:（1）对117例样本进行如下分析:①进行血常规和血红蛋白含量分析,提取所有样本的DNA,-20℃低温保存备用。②对α、β珠蛋白基因的突变进行分析:利用反向点杂交技术（reverse dot blot,RDB）对中国人群中常见的α、β珠蛋白基因点突变或小的缺失或插入突变进行分析;利用Gap-PCR技术分析中国常见的珠蛋白基因缺失突变(3种常见的α珠蛋白基因缺失突变,包括-α^3.7、-α^4.2和--^SEA。2种β珠蛋白基因簇缺失,包括东南亚型遗传性持续性胎儿血红蛋白综合征（SEA-HPHF）缺失和中国型^Gγ⁺（Aγδβ）⁰缺失);利用PCR技术对ααα^anti3.7和ααα（anti4.2）这两种α珠蛋白基因的三联体进行分析。③利用基于PCR基础上的Xmn1酶切技术,分析^Gγ珠蛋白基因的-158位的Xmn1酶切位点。经过上述分析,117例样本可以分为两类,一类是基因型可以解释表型的样本;一类是基因型尚不能解释表型的样本（β⁰/β⁰,β⁺/β^N或β⁰/β^N）（2）对基因型为β⁰/β⁰,β⁺/β^N或β⁰/β^N的样本进一步分析。针对前者,PCR扩增α₂和α₁珠蛋白基因全长,对PCR产物直接测序。采用基于PCR基础上的直接测序技术进一步对多个可使HbF水平升高的遗传修饰因素进行分析,这些因素包括:3’HS1,5’HS2核心区,^Gγ和^Aγ珠蛋白基因的启动子区,β珠蛋白基因-540区的（AT）x（T）y序列,BCL11A基因中rs11886868的SNP位点信息。针对后者,对以下多个位点进行了分析:β珠蛋白基因全长,5’HS2和5’HS3的核心区,AHSP基因全长,以及GATA-1和HRI的cDNA序列。对部分样本5’HS2核心区的PCR产物通过克隆测序进一步验证。（3）对本课题中可能发现的新突变进行分析:①评价新突变对β珠蛋白基因表达水平的影响②利用基于PCR的限制性片段长度多态性（restriction fragmentlength polymorphism,RFLP）技术对β珠蛋白基因簇进行单倍型分析。（4）利用半变性高效液相色谱（DHPLC）技术分析新突变和BCL11A基因的SNP位点rs11886868在对照样本中的情况。（5）统计分析:采用统计软件SPSS13.0。主要是利用两独立样本的t检验方法分析突变对β珠蛋白基因mRNA水平的影响。结果与讨论本研究我们共收集到117例中间型β地贫样本。年龄介于2岁和60岁,发病年龄则处于1.5岁和27岁。75例（64.1%）有地贫面容,29例（24.8%）尚未出现地贫面容。62例（52.5%）没有输过血,15例（12.7%）输血1次,31例（26.3%）需要偶尔输血。69例（59.0%）患者存在肝脾肿大,其中实行脾切除手术的患者有23例（19.5%）,36例（30.8%）尚未出现肝脾肿大。可见,这些中间型β地贫患者的表型差别很大,即具有很大的表型异质性。本课题在117例中间型β地贫患者中共检测到18种β珠蛋白基因突变,这些突变的出现频率明显不同,它们的出现频率从大到小的顺序排列如下:-28（A→G）,CD 41-42（-CTTT）,CD 17（A→T）,βE（CD 26 G→A）,IVS-2-654（C→T）,IVS-2-5（G→C）,CD 71-72（+A）,SEA-HPFH,IVS-1-1（G→T）和-29（A→G）,中国型^Gγ⁺（^Aγδβ）⁰,CD 43（G→T）,-73（A→T）、CD15-16（+G）、CD27-28（+C）、CD 53（-T）、Cap+39（C→T）和Term CD+32（A→C）等6种突变各1例,最后两种突变是在本课题中首次发现的,相关信息已经提交到GenBank数据库,序列号分别为FJ876835和FJ876836。除了检测到上述的β珠蛋白基因突变外,有22例样本同时合并α珠蛋白基因突变,共涉及5种α珠蛋白基因突变,其中CD142（T→C）(α^CS)2例,-α^3.7例,--^SEA 11例(其中1例合并-α^4.2),-α^4.21例(同时合并--^SEA),6例存在ααα^anti3.7,未发现ααα^anti4.2三联体。18种β珠蛋白基因突变和5种α-珠蛋白基因突变组成了51种β和α珠蛋白基因型组合。根据β珠蛋白基因型,117例中间型β地贫样本可以分为两类:（1）β珠蛋白基因突变纯合子或者双重杂合子,有97例（82.9%）。（2）β珠蛋白基因突变杂合子,有20例（17.1%）。分析表明这两类患者的主要血液学指标都存在显著差异（Mann-Whitney test,P<0.05）。在第一类97例中间型β地贫样本中:（1）α珠蛋白基因正常,β珠蛋白基因突变纯合子或双重杂合子有44例,占总样本的37.6%（44/117）。（2）α珠蛋白基因正常,HbE合并β⁰珠蛋白基因突变有27例,占总样本的23.1%（27/117）。（3）β珠蛋白基因突变纯合子或双重杂合子合并α珠蛋白基因突变(包括-α^3.7,-α^4.2,--^SEA,α^CS和ααα^anti3.7)15例。我们检测到2例特殊病例:1例为β珠蛋白基因突变双重杂合子合并HbH病,基因型具体为β⁽CD17（A→T）/β^{IVS-2-654（C→T）}合并--^SEA/-α^4.2;另一例是β-^{28（A→G）}/β-^{28（A→G）}合并ααα^anti3.7/αα。（4）7例为β珠蛋白基因突变杂合子合并SEA-HPFH,4例为β⁺/中国型^Gγ⁺（^Aγδβ）⁰或β⁰/中国型^Gγ⁺（^Aγδβ）⁰。其中,有两例样本的基因型分别是β^{IVS-2-654（C→T）}/中国型^Gγ⁺（^Aγδβ）⁰和β-^{28（A→G）}/中国型^Gγ⁺（^Aγδβ）⁰,此外,这两例样本的α珠蛋白基因型均为--^SEA/αα。在第二类20例中间型β地贫样本中:（1）α珠蛋白基因正常,β珠蛋白基因突变杂合子样本14例,占12.0%（14/117）。其中,β⁺/β^N样本1例,具体基因型为β-^{28（A→G）}/β^N;其余13例样本为β⁰/β^N,具体为7例β^{IVS-2-654（C→T）}/β^N,3例β^{CD17（A→T）}/β^N,2例β^{CD71-72（+A）}/β^N,1例为β^{41-42（-CTTT）}/β^N。（2）α珠蛋白基因正常,基因型为β^CD53（-T）/β^N的β珠蛋白基因显性突变杂合子样本1例。（3）β珠蛋白基因突变杂合子合并ααα^anti3.7/αα有5例。在117例样本中未发现1例ααα^anti4.2阳性样本。在117例中间型β地贫样本中,有3例β⁰/β⁰样本,基因型分别为β^{CD17（A→T）}/β^{IVS-2-654（C→T）},β^{IVS-1-1（G→T）}/β^{CD41-42（-CTTT）}和β^{CD17（A→T）}/β^{CD17（A→T）}。对这三例样本的α珠蛋白基因进行多种缺失突变检测,PCR扩增它们的α2和α1的珠蛋白基因全长、β和γ珠蛋白基因的的启动子区,然后对PCR产物测序,结果未发现突变。三例样本中,有两例样本的Xmn1分析结果为+/-,一例为-/-。通过对包括3’HS1的（+179）位点、β和γ珠蛋白基因的启动子区、HS2和HS3核心序列等在内的一些与HbF表达增加相关的遗传修饰因素的测序分析,没有发现明确的相关因素。这说明还有未知的遗传修饰因素,需要进一步的研究。值得注意的是,在117例中间型β地贫样本中,有14例为p珠蛋白基因突变杂合子,其中1例为β-^{28（A→G）}/β^N,7例β^{IVS-2-654（C→T）}/β^N,3例β^{CD17（A→T）}/β^N,2例β^{CD71-72（+A）}/β^N,1例为β^{41-42（-CTTT）}/β^N。对这批样本进行α珠蛋白基因的ααα^anti3.7和ααα^anti4.2三联体分析,没有发现α珠蛋白基因三联体。分析其他的一些相关靶点,包括β珠蛋白基因5’HS2和5’HS3的核心序列、AHSP基因全长和GATA-1的cDNA序列,仍然没有发现相关信息。在HRI中发现两个多态位点rs2639和rs2640,其他各靶点未发现突变。Rs2639（A→G）是一个同义突变,而rs2640（A→G）属于错义突变,导致K558R的氨基酸改变。然而,由于赖氨酸（K）和精氨酸（R）都属于碱性氨基酸,它们都属于性质相似的氨基酸,这种改变对HRI功能的影响难以判定。总之,我们认为在这些样本中,应该还有一些未知遗传修饰因素的参与。针对Term CD+32（A→C）和Cap+39（C→T）这两种新的β珠蛋白基因突变,通过反转录实时定量PCR技术分析突变对β珠蛋白基因mRNA水平的影响。前者位于β珠蛋白基因的3’UTR,在终止密码子TAA后第32位核苷酸,因此被称作Term CD+32（A→C）突变。对先证者进行^Gγ珠蛋白基因的-158位点的Xmn1酶切分析,结果为+/+,这是在本研究中发现的唯一1例阳性纯合子样本。通过对7个限制性性内切酶位点的单倍型分析,发现与Term CD+32（A→C）突变连锁的单倍型为"--+++-+"。另一种新的突变Cap+39（C→T）,突变位点位于β珠蛋白基因的5’加帽位点后的第39位核苷酸,与β珠蛋白基因的起始密码子AUG间隔11个碱基对。应用反转录实时定量PCR评价两例新突变对β珠蛋白基因mRNA水平的影响。采用内参和靶基因的双标准曲线相对定量的方法,内参采用β肌动蛋白基因。靶基因β珠蛋白基因mRNA的标准曲线为:y=-3.672x+19.787（R²=0.999）;内参β肌动蛋白mRNA的标准曲线为:y=-3.466x+24.000（R²=0.999）。Term CD+32（A→C）突变组β珠蛋白mRNA相对含量为0.835±0.048（n=3）,Cap+39（C→T）突变组β珠蛋白mRNA相对含量为1.093±0.118（n=5）,正常对照组β珠蛋白mRNA相对含量为1.016±0.098（n=6）。应用两独立样本的t检验方法进行统计学分析表明,Term CD+32（A→C）突变组与正常对照组的β珠蛋白mRNA相对含量有显著性差异（P=0.021）,突变可以使β珠蛋白基因的mRNA水平下降17.8%,属于β⁺突变。Cap+32（C→T）突变组与正常对照组的β珠蛋白mRNA相对水平没有显著性差异（P=0.270）,突变不影响β珠蛋白基因的mRNA水平。此外,该突变的5个携带者的血液学指标均正常。因此,该突变应该属于沉默突变(silent mutation,β⁺⁺)。序列分析表明,发现的两例新突变所在的基因序列在进化过程中是很保守的。结论1.研究表明华南地区的中间型β地贫患者具有很大的遗传异质性。117例中间型β地贫样本的分子基础可归结为两大类,即20例（占17.1%）β珠蛋白基因突变杂合子和97例（占82.9%）β珠蛋白基因突变纯合子或者双重杂合子。前者又可分为:α珠蛋白基因正常,β珠蛋白基因突变杂合子样本14例;α珠蛋白基因正常,基因型为β^CD53（-T）/β^N的β珠蛋白基因显性突变杂合子样本1例;β珠蛋白基因突变杂合子合并ααα^anti3.7/αα5例。后者又包括:α珠蛋白基因正常,β珠蛋白基因突变纯合子或双重杂合子有44例;α珠蛋白基因正常,HbE合并β⁰珠蛋白基因突变有27例;β珠蛋白基因突变纯合子或双重杂合子合并α珠蛋白基因突变15例;7例为β珠蛋白基因突变杂合子合并SEA-HPFH,4例为β⁺/中国型^Gγ⁺（^Aγδβ）⁰或β⁰/中国型^Gγ⁺（^Aγδβ）⁰(其中2例样本合并--^SEA/αα)。总之,我们已经基本阐明了华南地区中间型β地贫的分子基础。2.在117例中间型β地贫样本中,有3例β⁰/β⁰样本和14例β珠蛋白基因突变杂合子样本。针对这些样本,我们对可导致中间型β地贫的主要及次要遗传修饰因子进行了系统的分析,但是未能阐明他们的分子基础,需要进一步的研究。3.发现了2例新的β珠蛋白基因突变类型,Cap+39（C→T）和Term CD+32（A→C）,丰富了人类β珠蛋白基因突变数据库,对临床具有指导意义。评价了突变对mRNA水平的影响,Term CD+32（A→C）突变可以使β珠蛋白基因的mRNA水平下降17.8%,属于β⁺突变。Cap+32（C→T）突变不影响β珠蛋白基因的mRNA水平,属于沉默突变(silent mutation,β⁺⁺)。研究表明与新突变TermCD+32（A→C）连锁的单倍型为“--+++-+”。更多还原

【Abstract】 Background and Objectiveβ-thalassemia is one of the most common monogenic disorders in the world. The incidence for this disease is high in areas of the tropics and subtropics including southern China. In southern China, the carrier rate ofβ-thalassemia is 2.54% in Guangdong and 6.78% in Guangxi where are two provinces of the most frequently thalassemia occoured. According to the clinical phenotypes,β-thalassemia can be divided into three main types: thalassemia major （TM）, thalassemia trait （TT） and thalassemia intermedia （TI）. TM is a severe form that requires transfusions from infancy for survival, whereas TT is usually asymptomatic. TI is used to indicate a clinical condition of intermediate gravity between TT and TM, which encompasses a wide phenotypic spectrum spanning from mild anemia to more severe anemia with required occasional transfusions.Corresponding to the phenotypic diversity, the molecular basis of TI is also variable. Thein has reviewed the major genetic modifiers ofβ-thalassemia: genotypes ofβ- andα-globin and expression ofγ-globin. Some genotypic factors have been reported to affect synthesis of theγ-globin chain, such as the 3’HS1 （+179 C→T） polymorphism, the （AT）xNy（AT）z motif in the 5 ’HS2 site, and the （AT）x（T）y motif in the -540 region of theβ-globin gene. Variation of rs11886868 （T→C） in the BCL11A gene has also been shown to correlate with increased HbF in European TI patients. In addition, those factors that can moderate globin imbalances indirectly or cause theβ-thalassemia-like phenotype, such as GATA-1,AHSP, and heme-regulated initiation factor 2 alpha kinase （ HRI）,are also thought to contribute to the phenotypic diversity of TI.Researchers have described molecular characterization of TI in Iranian, Indian, Italian, and other populations. However, to date, the genetic basis of TI in Chinese patients is poorly understood. In this study, we performed a comprehensive analysis of the molecular basis underlying TI in southern China. Genotypes ofβ-globin and other known modifiers linked toα/βimbalance were investigated in 117 patients withβ-thalassemia intermedia phenotypes. Their clinical, hematological, and molecular data were analyzed systematically with the aim of creating a genotype-phenotype correlation.Samples and MethodsSamplesWe recruited the TI patients in this study according to previously described criteria, in whom the classical clinical diagnosis of TI patients, such as the steady state Hb level of 60-105g/L, age at onset over two years old, and transfusion independence were emphasized for all our patients. A total of 117 patients from 109families with TI phenotypes were recruited for this study.MethodsClinical events analysis: Complete blood counts and red cell indices were determined by automated cell counting （Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan）; the levels of HbA, HbA2 and HbF were analyzed on the Bio-Rad VariantⅡHPLC system （HPLC, VARIANTM, Bio-Rad, Hercules, CA, USA）. The information about blood transfusions, thalassemia appearance, age at onset, hepatosplenomegaly and splenectomy was obtained by retrospective clinical data.DNA analysis: We collected the blood with EDTA anticoagulation of 117 Chinese TI patients. Genomic DNA was extracted from peripheral blood by standard phenol/chloroform methods. The 11 knownβ-thalassemia mutations, the two common deletions including Chinese ^Gγ⁺（^Aγδβ）⁰ thalassemia and Southeast Asian hereditary persistence of fetal hemoglobin （SEA-HPFH） , the three commonα-thalassemia deletions (--^SEA,-α^3.7 and -α^4.2),the six non-deletional mutations (α^CD30,α^CD31,α^CD59,α^QS,α^CS andα^WS), theααα^anti3.7 orααα^anti4.2 triplication and Xmn1 site -158 of the ^Gγ-globin gene were analyzed. Further sequence analysis was applied on bothβ⁰/β⁰ andβ⁺/β^N orβ⁰/β^N samples, analyzed targets for the former （β⁰/β⁰） include both 3’HS1 and 5’HS2 core region, the promoters of the ^Gγ- and ^Aγ-globin genes, the （AT）x（T）y sequence variations at the position -540 of theβ-globin gene, SNP polymorphism of rs11886868 in the BCL11A gene, as well as the wholeα2- andα1-globin genes; and those for the latter （β⁺/β^N orβ⁰/β^N） include the core regions of both 5’HS2 and 5’HS3,the wholeβ-globin gene and AHSP gene, and the cDNA of GATA-1 generated by RT-PCR from mRNA.Since HRI has been shown to modify the phenotypic severity ofβ-thalassemia in murine models, we also sequenced the HRI cDNA inβ⁺/β^N orβ⁰/β^N samples by RT-PCR.RNA analysis: Total cellular RNA was isolated from fresh peripheral blood using Gentra Purescript RNA Kit （Gentra, Americia）. The cDNA synthesis was performed using the ExScript RT reagent Kit （TaKaRa Biotechnology, China）. The expression levels ofβ-globin （target gene） andβ-actin （control） were measured by SYBR Green-based relative quantitative RT-PCR assays. Five heterozygous subjects carrying the Cap+39（C→T） mutation and three heterozygous subjects carrying the Term CD+32（A→C） mutation were selected respectively, as two patient groups, while six normal subjects served as the control group.β-globin gene haplotype analysis: Theβ-globin haplotypes associated with the two novel mutations were analyzed with PCR amplification followed by restrictionenzyme digestion. Seven classical polymorphic restriction enzyme sites selected for haplotype analysis were HincⅡ-5’ε,HindⅢ-^Gγ,HindⅢ-^Aγ,HincⅡ-ψβ,HincⅡ-3‘ψβ, AvaⅡ-βand Bam HI-3’β. Each individual was scored for the presence （+） or absence （-） of each of the seven RFLP sites.Statistical methods: Statistical analysis was performed using SPSS software （Version 13.0, SPSS inc, USA）. The difference of the relative mean mRNA concentration between mutation carriers and normal individuals were analyzed by the independent samples t-test. Ap-value < 0.05 was considered statistically significant.Results and DiscussionIn this study,117 Chinese individuals between 2 and 61 years old were enrolled to characterize the molecular basis ofβ-thalassemia intermedia in southern China. The level of Hb is between 60 and 105g/L. The age at onset is between 1.5 and 27 years old. Seventy-five TI patients have thalassemia-like appearance. Sixty-two ones have no transfusion, fifteen ones have single time transfusion, and thirty-one TI patients need occasional transfusion. Sixty-nine ones are of hepatosplenomegaly, twenty-three ones are of splenectomy, and thirty-six ones have normal liver and spleen.According to their genotype ofβ-globin, we divided 117 TI patients into two types: TypeⅠβ-thalassemia homozygotes （n=97） who inherited two deficientβ-globin alleles and TypeⅡβ-thalassemia heterozygotes （n=20） who had only a singleβ-thalassemia allele. In total, we detected 18β-thalassemia alterations including two novel ones which were termed as Term CD+32（A→C） and Cap+39（C→T） respectively,β-globin variant HbE, the two deletional mutations which can lead to SEA-HPFH or （δβ）⁰-thalassemia Chinese ^Gγ⁺（^Aγδβ）⁰.TypeⅠβ-thalassemia homozygosity was detected in 97 TI patients （82.9%） as follows: （i） Forty-four （37.6%） had homozygous or compound heterozygousβ-thalassemia alleles and normalα-globin genes,（ii） Twenty-seven （23.1%） were HbE/β⁰ compound heterozygotes. No HbE/β⁺ heterozygote or co-incidentα-globin mutations were found in this group,（iii） Fifteen （12.8%） had twoβ-thalassemiamutations, and in addition carried theα-thalassemia alterations including the -α^3.7, -α^4.2,--^SEA alleles orα^CS.We observed two particular patient cases. One was caused by co-existence of Hb H disease (--^SEA/-α^4.2) andβ-thalassemia compound heterozygosityβ^{CD17（A→T）}/β^{IVS-2-654（C→T）},while the other was caused byαα/ααα^anti3.7andβ-thalassemia homozygosity of the -28 （A→G） mutation, （iv） Eleven （9.4%） carried SEA-HPFH （7 cases） or Chinese ^Gγ⁺（^Aγδβ）⁰ （4 cases） deletions in addition to having oneβ-thalassemia mutation. Among them, two were IVS-2-654 （C→T） / Chinese ^Gγ⁺（^Aγδβ）⁰ and -28 （A→G） / Chinese ^Gγ⁺（^Aγδβ）⁰,respectively, both plus one --^SEA deletion.TypeⅡβ-thalassemia heterozygosity was detected in 20 TI patients （17.1%） as follows: （i） Fourteen （12.0%） carried a singleβ-thalassemia allele and normalα-globin genes.（ii） Five （4.2%） had a singleβ-thalassemia allele and co-inheritedααα^anti3.7 triplication. Noααα^anti4.2 triplication was detected.（iii） One patient （0.9%）, in a Miao family, was a heterozygote for a frameshiftβ-thalassemia mutation at CD 53 （-T）. There were threeβ⁰/β⁰ TI cases whose genotypes wereβ^{CD17（A→T）}/β^{IVS- 2-654（C→T）},β^{IVS-1-1（G→T）}/β^{CD41-42（-CTTT）} andβ^{CD17（A→T）}/β^{CD17（A→T）}.We detectedα-globingene and other modifiers which could increase the production of HbF including the promoter region of bothβ- andγ-globin gene, the core regions of both HS2 and HS3, 3’HS1,and the SNP rs11886868 in BCL11A gene. The analysis of XmnI site -158 of the ^Gγ-globin gene showed that two patients were +/- and one case was -/-.The three patients all had C/C genotypes of both at +179 of 3’HS1 and rs11886868. However, we could not consider that C/C genotypes of rs11886868 contributed to the high HbF level based on two facts. One is that the frequency of C/C genotypes was 0.867 in Chinese people from the HapMap data （http://www.hapmap.org/）.The second is that an additional 115 samples were analyzed, including 30 TI patients （ 25β⁰/β⁺,5β⁺/β⁺） and 85 normal individuals, in which all were found to be the C/C genotype. As to other modifiers, the results of analysis showed that they were normal.It was notable that we found 14 patients with heterozygous forβ-thalassemia (1 case with the genotype ofβ-^{28（A→G）}/β^N,7 cases with the genotype ofβ^{IVS-2-654（C→T）} /β^N,3 cases with the genotype ofβ^{CD17（A→T）}/Β^N,2 cases with the genotype ofβ^{CD71-72（+A）}/Β^N and 1 case with the genotype ofβ^{CD41-42（-CTTT）}/Β^N) among our 117 TI patient cohort, in whom theβ-globin gene was found to be structurally intact by sequence analysis and excluded theα-globin gene triplication. As for the underlying defective targets, we focused on primary genetic modifiers that could modulate the imbalance ofα/βfor investigation in ten TI patients. We evaluated the effects of some potential modifiers involving one linked to theβ-globin gene cluster, the LCR resides in the 5’HS2 and 5’HS3 regions, and three ones not to be linked to theβ-globin cluster, AHSP, GATA-1 and HRI.Sequence analysis of the core regions of 5’HS2 and 5’HS3 showed wild type sequences, thus excluding mutations in these control regions in reducing expression ofβ-globin gene. The sequence of AHSP gene was normal. No mutations in the cDNA sequences of GATA-1 have been found. In the case of HRI, the polymorphism of rs2639 and rs2640 were detected. The rs2639 （A→G） is a synonymous mutation and the rs2640 （A→G） is a missense mutation resulting in a K558R substitution. However, the affect on the function of this substitution is unknown since Lys （K） and Arg（R） are similar in amino acid properties. These results indicate the existence of causative genetic determinants not yet molecularly defined.Two novel mutations ofβglobin were analyzed. The proband of Term CD +32（A→C） mutation was a compound heterozygote ofβ^{Term CD+32（A→C）}/β^{CD 27-28（+C）} with XmnI （+/+） homozygosity. By using PCR-based RFLP method and family linkage study, the haplotypes linked to the TermCD+32（A→C） mutation and CD27-28（+C） were identified as "- -+++-+" and "--++++-", respectively. The proband of Cap+39 （C→T） mutation was a compound heterozygote ofβ^{Cap+39（C→T）} /β^{CD41-42（-CTTT）} with Xmn I（-/-） homozygosity. Interestingly, his father and four other family members were all heterozygotes of Cap+39（C→T） mutations. Since they do not have any evident hematologic phenotype, we regard this mutation asβ⁺⁺ （silentβthalassemia）.We used real-time PCR to analyze the mRNA level. Two standard curves were generated: y =-3.672x +19.787 （R²=0.999） forβ-globin mRNA and y= -3.466x+24.000 （R²=0.999） for p-actin mRNA. The mean relative mRNA concentrations were 0.835±0.048 （n=3） for the Term CD+32（A→C） group, 1.093±0.118 （n=5） for the Cap+39（C→T） group, and 1.016±0.098 （n=6） for the normal control group after a normalization procedure using linear regression equations. Statistical analysis showed that there was a significant difference （P=0.021） of mean relativeβ-globin mRNA concentration between the Term CD+32（A→C） group and the control group. The decreased level ofβ-globin mRNA in the patient group compared with that of the normal control group was calculated to be 17.8%. In contrast, no significant difference （P=0.270） was found between the Cap+39（C→T） group and the control group. Sequence alignment showed that Term CD+32（A） and Cap+39（C） were conserved sites during evolution. These two novel mutations weren’t detected in 156 random samples from southern Chinese individuals.ConclusionsChinese TI patients showed a high degree of heterogeneity in both phenotypic and genotypic aspects. According to their molecular basis,117 TI patients can divide into two types which are 97β-thalassemia homozygotes who inherited two deficientβ-globin alleles and 20β-thalassemia heterozygotes who had only a singleβ-thalassemia allele. The former includes five sub-types: Forty-four had homozygous or compound heterozygousβ-thalassemia alleles and normalα-globin genes; Twenty-seven were HbE/β⁰ compound heterozygotes; Fifteen had twoβ-thalassemia mutations, and in addition carried theα-thalassemia alterations including the -α^3.7,-α^4.2,--^SEA alleles orα^CS;Eleven carried SEA-HPFH （7 cases） or Chinese ^Gγ⁺（^Aγδβ）⁰ （4 cases） deletions in addition to having oneβ-thalassemia mutation. Among them, two wereβ^{IVS-2-654（C→T）}/ Chinese ^Gγ⁺（^Aγδβ）⁰ andβ-^{28（A→G）}/Chinese ^Gγ⁺（^Aγδβ）⁰,respectively, both plus one --^SEA deletion.The latter includes three sub-types: Fourteen carried a singleβ-thalassemia allele and normalα-globin genes; Five had a singleβ-thalassemia allele and co-inheritedααα^anti3.7 triplication; One patient was a heterozygote for a frameshiftβ-thalassemia mutation at CD 53 （-T）.In a word, we have basically clarified the molecular basis of the 117 TI patients.The molecular basis of 14 TI patients （13β⁰/β^N and 1β⁺/β^N cases） with known heterozygous mutations ofβ-thalassemia and three ones with homozygousβ-thalassemia （β⁰/β⁰） were uncertain, the existence of other causative genetic determinants are remaining to be molecularly defined.We have detected two novel mutations which are Term CD+32（A→C） and Cap+39（C→T）.After assessing them, we think that the former mutation can decrease the level ofβ-globin mRNA and the latter has no influence upon the level ofβ-globin mRNA. In addition to the pedigrees data of the two novel mutations, we regard Cap+39（C→T） mutation asβ⁺⁺ （silentβ-thalassemia） and Term CD +32（A→C） mutation asβ⁺.The haplotypes associated with Term CD+32（A→C） mutation is"--+++-+".更多还原