【作者】 余文芳；

【导师】 徐湘民；

【作者基本信息】 南方医科大学 ， 医学遗传学， 2010， 硕士

【摘要】 背景与目的遗传性血红蛋白病是世界上最常见的人类单基因遗传病之一,其中α-地中海贫血（a-thalassemia,α-地贫）和β-地中海贫血（β-thalassemia,β-地贫）为遗传性血红蛋白病的主要类型,地理分布主要在全球的热带与亚热带地区。α-地贫以α-珠蛋白基因大片段缺失为主,非缺失型α-地贫较少见；β-地贫主要是由于β-珠蛋白基因（HBB）发生突变所致,β-地贫的大片段缺失也较少见；另外,在α-地贫与β-地贫的一些不常见的病例中可有罕见的自发改变,包括少数α-珠蛋白基因的自发缺失,特别是导致的伴α-地中海贫血智力低下综合征（ATR-16综合征）,以及一些发生在不同人群β-珠蛋白基因上的自发突变。α地贫是一组以珠蛋白合成减少,α-链／非α-链比例失衡为特征的遗传性溶血性血红蛋白病,是发生于包括中国南方在内的世界上最常见的一类对人类健康影响最大的单基因遗传病之一,全国90万人的流行病学调查得出的总发病率为2.64%,其中广东省的发病率为4.11%。Hb H病是一种中间型α-地贫的类型,流行于东南亚地区及中国南部；患者由于三个α-珠蛋白基因缺失或缺陷,导致α-珠蛋白肽链的合成速率的明显降低,过剩的β珠蛋白肽链形成β4四聚体-Hb H,并在红细胞内形成Hb H包涵体,引起慢性溶血性贫血。东南亚地区Hb H病主要由于缺失型α-地贫如（--SEA）缺失型α-地贫与缺失型α+-地贫如-α3.7缺失型或-α4.2缺失型,或与非缺失型α+-地贫如HbConstant Spring组合而成。只有Waye JS等首次报道过整个ζ-α-珠蛋白基因簇的自发缺失复合-α3.7缺失所导致的Hb H病。在本研究中,我们鉴定了一种α2-珠蛋白基因的自发移码突变复合（--SEA）缺失型α-地中海贫血所导致Hb H病。我们探讨了基因型／血液学表型与α-地贫包括引起Hb H病的自发突变之间关系,在家系分析的基础上,通过实时荧光定量反转录PCR技术,我们在mRNA水平上分析了这一新突变对基因转录效应的影响。我们在PubMed上进行文献检索后,发现这是首次报道人α-珠蛋白基因的单个基因自发缺失突变复合（--SEA）缺失型α-地中海贫血所导致的Hb H病。材料与方法研究对象先证者,为一名2岁的男孩,来自于中国南方广西省桂林市,其父母无亲缘关系；顺产,出生时体重为3650g；出生时无黄疸,无明显的生长发育迟缓；6月大时,家人注意到其面色苍白；1岁时,先证者表现出如下临床和血液学表型而首次被诊断为Hb H病：轻度肝肿大；实验室检查示小细胞低色素贫血,血红蛋白（Hb）8.0 g／dL,平均红细胞体积（MCV）56.3fL,平均红细胞血红蛋白量（MCH）17.8 pg；血红蛋白电泳显示Hb H带,铁缺乏被排除。接下来的1年里,先证者的贫血未恶化,且未接受输血治疗,当先证者2岁的时候,对先证者进行基因检测以确诊。在签署知情同意书后,我们抽取先证者及其4位家庭成员外周血标本进行此次研究,外周血以EDTA抗凝。血液学方法红细胞参数分析（血常规）采用全自动血细胞计数仪（Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan）进行检测,血红蛋白定量检测采用毛细管电泳法（Capillarys, Sebia, Montpellier, France）。DNA分析基因组DNA的提取采用酚／氯仿方法。Gap-PCR技术和反向点杂交技术分别用于检测中国人常见α-地中海贫血缺失和6种α-地中海贫血点突变（αcd30α／,αcd31α／,αcd59α／,αQSα／,αCSα／和αWSα／）的诊断,这9种突变占到已知中国人α地贫突变的98%。11种中国人常见β-地中海贫血突变的分析采用反向点杂交方法。我们进行了直接α1和α2珠蛋白基因的测序（α1 andα2, GenBank Accession NG₀00006）来鉴定未知的突变。为了排除α2-珠蛋白基因自发点突变体细胞嵌合的可能性,我们将先证者淋巴细胞、毛发及口腔粘膜剥脱细胞来源的DNA分别进行α2-珠蛋白基因的测序。我们还进行了亲子鉴定以排除非自发突变的可能。RNA分析为了评价α-地贫自发突变对α-珠蛋白mRNA表达水平的影响,我们设计了实时荧光定量反转录PCR实验,采用基于双标准曲线法的定量分析,我们对该家系的先证者（Hb H病）,先证者母亲（--SEA缺失型α-地贫携带者）,正常人的α-珠蛋白mRNA进行了基因表达水平检测,每种样品均进行4次独立的重复测定。结果血液学分析结果先证者（Ⅲ：1）表现为Hb H病的表型,具有小细胞低色素贫血（Hb 8.4 g/dL, MCV 60.7 fL and MCH 15.9 pg）, Hb H和HbBart’s升高分别为3.2%和5.4%。先证者母亲（Ⅱ：1）表现为小细胞低色素贫血,其它3个家庭成员（Ⅰ：1,Ⅱ：2,Ⅱ：3）均为正常血液学表型。DNA分析结果通过PCR扩增先证者及其家系成员α-珠蛋白基因全长片断,并对PCR产物直接进行DNA序列测定分析及Gap-PCR技术分析表明先证者是α2-珠蛋白基因CD44（-C）缺失和（--SEA）缺失型α-地贫的复合杂合子,在PubMed上进行文献检索,以及查询珠蛋白基因服务网站（http://globin.cse.psu.edu/）,均未发现CD44（-C）突变类型,证实此突变是世界首报的α-地贫突变类型；（--SEA）缺失型遗传自母亲。该家庭成员中,先证者母亲是（--SEA）缺失型α-地贫携带者,其余三个成员（先证者爷爷、父亲、姑姑）均为正常基因型。先证者淋巴细胞来源的DNA与毛发及口腔粘膜剥脱细胞来源的DNA其α2-珠蛋白基因的测序结果完全一致,因此排除了α2-珠蛋白基因自发点突变体细胞嵌合的可能性。为了排除非自发突变,我们进行了亲子鉴定,亲子鉴定结果强力支持先证者与先证者父亲的亲缘关系,因此证实为自发突变。RNA分析结果实时定量反转录PCR分析显示当正常人α-珠蛋白mRNA相对浓度作为标准定义为1.0时,先证者（Hb H病）平均α-珠蛋白mRNA相对浓度为0.0856±0.01,先证者母亲（--SEA缺失型α-地贫携带者）平均α-珠蛋白mRNA相对浓度为0.598±0.069。由此可见先证者的α-珠蛋白基因mRNA表达水平显著低于先证者母亲。讨论尽管遗传性α-珠蛋白基因的改变占大多数,少数自发的异常α-珠蛋白基因的改变也能引起α-地贫表型,特别是α-珠蛋白基因的自发缺失导致的伴α-地中海贫血智力低下综合征（ATR-16综合征）或非常罕见的Hb H病；另外,仅有很少发生在α-珠蛋白基因上的自发突变导致血红蛋白结构异常的病例被报道。在本研究中,我们首次报道了在中国人非缺失型Hb H病患者中发现了一种新的α-珠蛋白基因单个基因的自发缺失突变。通过对该家系的研究,我们发现先证者的（--SEA）缺失型α-地贫基因型遗传自其母亲,而先证者父亲的血液学表型及α-珠蛋白基因型均为正常,亲子鉴定排除了非亲缘关系,表明先证者的α2-珠蛋白基因CD44（-C）缺失为自发突变。在排除了α2-珠蛋白基因自发点突变体细胞嵌合的可能后,我们推测该自发突变在精子的发生过程或早期胚胎的过程产生；因此,该自发突变的表达效应该与遗传性突变相对应,由该α2-珠蛋白基因的自发移码突变CD44（-C）复合（--SEA）缺失型α-地中海贫血即可导致Hb H病表型；本研究中的先证者,2岁的中国男孩,根据其Hb H病的典型临床表现,将其归类为中度贫血,非缺失型Hb H病。我们进行的实时定量反转录PCR分析显示：当把正常人a-珠蛋白mRNA相对浓度作为标准定义为1.0时,先证者（Hb H病）平均α-珠蛋白mRNA相对浓度为0.0856±0.01,先证者母亲（--SEA缺失型α-地贫携带者）平均α-珠蛋白mRNA相对浓度为0.598±0.069。由此可见α2-珠蛋白基因自发的移码突变能显著的降低α2-珠蛋白基因mRNA转录水平,主要是因为α2-珠蛋白基因CD44缺失碱基C,导致在其下游CD48处出现了一个提前终止密码子（premature termination codons, PTC）。大多数的无义突变和移码突变可以触发无义介导的mRNA降解（nonsense-mediated mRNA decay, NMD）,来清除含有PTC的mRNA,本研究中先证者α2-珠蛋白基因CD44缺失碱基C触发了的无义介导的mRNA降解,尽管我们尚不清楚影响α2-珠蛋白基因表达的终止密码子作用效应。更多还原

【Abstract】 Background and ObjectiveThe inherited hemoglobin disorders are one of the most common human monogenic diseases worldwide. Among them,α-andβ-thalassemia are two most common forms mainly occurred in the tropical and sub-tropical regions of the world, which mostly result from the gross deletions ofα-globin gene cluster and the point mutations in theβ-globin gene (HBB), respectively. Both nondeletional a-thalassemia mutations and gross deletions of P-thalassemia are relatively uncommon. In addition to this, rare de novo alterations leaded to these two disorders have been characterized in some unusual cases withα-orβ-thalassemia phenotypes, which included a few de novo deletions located on theα-globin locus, particularly those abnormality causing ATR-16 syndrome and a group of spontaneous mutations occurred within the P-globin gene identified in various populations.α-thalassemia is one of the most common hereditary disorder in which a-globin chain synthesis is either decreased or absent. The resulting imbalance of globin chain synthesis leads to the accumulation of excess y-globin or P-globin chains which eventually causes the destruction of affected red blood cells. According to the epidemiologic data, the incidence of this disease screened by electrophoresis in seven provinces of China was calculated to be 2.64%. The data show that the incidence of a-thalassemia in Guangdong province is 4.11%.Hemoglobin (Hb) H disease is a moderate form of a-thalassaemia resulting from various genetic defects and it is particularly prevalent in Southeast Asia and in southern China. There is the heterogeneity of Hb H disease genotypes in the different endemic populations. Interaction of common types ofα0-thalassemia deletions (such as--SEA/allele) with the silentα+-thalassemia deletions (such as-α3.7/or-α4.2/allele) or nondeletionalα-thalassemia mutations in theα2-globin gene (HBA2) (such asαConstant Springα/allele)have been extensively demonstrated to be causes of giving rise to a Hb H disease phenotype. Only one case with Hb H disease attributed to the phenotype of (--de novo/-α3.7) involving a de novo deletion of the entireξ-αglobin gene cluster was firstly reported by Waye.In this report, we identified a novel de novo frameshift mutation (-C) occurred at the HBA2 gene in a Chinese boy with Hemoglobin H disease. We have performed establishment of a genotype-phenotype correlation in a Chinese family with a-thalassemia involving this de novo mutation defined as a cause of Hb H disease in our study. To our knowledge, this is the first time the de novo single-base lesion causing a-thalassemia has been reported to occur in human a-globin gene.Materialss and MethodsSubjectsThe proband, a 2-year-old boy who was the offspring of unrelated parents that originated from Guilin city, Guangxi Province of southern China. The pregnancy was uneventful, and he was delivered vaginally with a birth weight of 3650g. There was no history of jaundice at birth and no obvious retardation of growth. He looked pale when he was 6 months old. He was first diagnosed as Hb H disease at age one year according to the clinical phenotype of moderate anemia and mild hepatomegaly, as well as a typical hematological features of hypochromic microcytosis with Hb level of 8.0g/dL, a mean cell volume (MCV) of 56.3fL, and a mean cell hemoglobin (MCH) of 17.8 pg. Analysis of his hemoglobin showed Hb H band. Iron deficiency was excluded.During the following one year, his anemia had not worsened and he never received a blood transfusion up till now. He was referred for molecular test for confirmatory diagnosis at age of 2 years. He and his family member’s peripheral blood samples were collected using EDTA as anticoagulant after informed consent was obtained.Hematologic MethodsHematological parameters were measured by an automated cell counting (Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan) and quantification of hemoglobin were conducted on the capillary electrophoresis (CE) device (Capillarys, Sebia, Montpellier, France).DNA analysis ofα-andβ-globin geneGenomic DNA was extracted from peripheral blood leucocytes by standard phenol/chloroform method. The genotype of known types of defects in Chinese population both in a-globin and in P-globin genes was performed using our protocols as described previously. Direct DNA sequencing of the entire both a-globin genes (α1 andα2, GenBank Accession NG.000006) was performed for identifying the unknown mutation as previously described. To exclude the possibility of a somatic mosaicism of a point mutation in the HBA2 gene, that is a phenomenon previously defined as a cause of thalassemia intermedia (TI), we performed a sequencing of a2-globin gene of the patient’s genomic DNAs derived from lymphocytes, hair root and oral mucosal exfoliated cells, respectively. Their paternity tests were conducted by using a set of fifteen short tandem repeat (STR) markers and amelogenin gene.RNA analysisWe design a real-time quantitative reverse-transcript PCR asaay to evaluate the expression level ofα-globin gene mRNA. On the basis of two standard curves method, the gene expression at the mRNA level of both the mutant and wild-type a-globin alleles were measured by using SYBR Green-based relative quantitative RT-PCR method and theβ-globin gene served as a control for assessment of equivalent RNA loading as our previous protocol. Four independent tests for each of samples with three different a-globin genotypes (the proband, his mother and a normal people) were conducted in order to calculate the mean mRNA concentration.ResultsHematological data analysisThe proband (Ⅲ:1) was found to have a Hb H disease phenotype with a hypochromic microcytic anemia (Hb 8.4g/dL, MCV 60.7 fL and MCH 15.9 pg), 3.2%Hb H,5.4%HbBart’s. His mother (Ⅱ:1) also have a hypochromic microcytic anemia. In addition, his three other family members (Ⅰ:1,Ⅱ:2,Ⅱ:3) showed normal red-cell parameters and hemoglobin electrophoretic profile. DNA analysisDirect DNA sequencing of the entire human a-globin gene was performed and mutation cd44 (-C) was identified in the proband. After retrieving PubMed and online database of human hemoglobin variants and thalassamias on the Globin Gene Server website (http://globin.cse.psu.edu/), it was confirmed to be a novelα-thalassemia mutation, which had never been reported. Results of molecular studies showed that the proband’s mother carries the Southeast Asianα-thalassemia deletion, which the proband inherited, whereas his father does not have any commonα-thalassaemia deletions or point mutations. The sequence results of the proband’s DNA derived from lymphocytes were completely consistent with that of testing his hair root and oral mucosal exfoliated cells.thereby excluding the possibility of a somatic mosaicism of a point mutation in the HBA2 gene. The results of the DNA paternity testing proved that the alleged father is the biological father of the proband and the probability of paternity is 99.99%. RNA analysisWe determined that the mean relative a-globin mRNA levels of 0.0856±0.01 in the proband versus of 0.598±0.069 in his mother (a SEA deletion carrier) when the normal standard was defined as 1.0.DiscussionAlthough parentally inherited alterations involved in a-globin genes are responsible for a large majority of cases, a few de novo abnormalities can result in a-thalassemia phenotypes, particularly de novo gross deletions defined as the cause of ATR-syndrome or a very rare form of Hb disease. In addition to this, only a few cases of spontaneous mutations identified at both a-globin genes (α1 andα2), that give rise to the structural Hb variants were previously reported in very small individuals. We report here for the first time, in a Chinese patient, a de novo single mutation event at the a-globin gene detected in a nondeletional Hb disease.According to the data observed from our family study, we report a proband with Hb H disease who is constitutionally heterozygous for the SEA type ofα0-thalassemia deletion inherited from his mother. The proband’s father shows a normal both in the hematological phenotype and a-globin genotype, thereby indicating that the abnormality arose as de novo events. Based on the results of excluding the possibility of a somatic mosaicism of a point mutation in the HBA2 gene, it is suggested that the de novo single-base deletion should have arisen during the spermatogenic process or earlier embryonic stage. Thus, the effects on expression of this de novo mutant allele should correspond to that of an inherited mutation. Therefore, interaction of this de novo frameshift mutation (-C) at theα2-globin gene with the SEA type ofα0-thalassemia deletion in the proband could give rise to a Hb H disease phenotype). Our observation that the clinical manifestation of classic Hb H disease presented in the boy aged 2 may be classified into anemia of moderate severity known as the nondeletional form of Hb H disease.We determined that the mean relative a-globin mRNA levels of 0.0856±0.01 in the proband versus of 0.598±0.069 in his mother (a SEA deletion carrier) when the normal standard was defined as 1.0. Thus indicating that this de novo frameshift mutation in the HBA2 gives rise to a significantly reduced the mRNA levels ofα2-globin gene expression. As the frameshift mutation generates a premature translation termination codon at position 48, it most likely that the mechanism of a nonsense-mediated mRNA decay (NMD), that had been demonstrated previously by the very similar case with the inherited microdeletion (-C) at the a2-globin gene, should be responsible for the reduced mRNA levels although we could not directly investigate the effect of the nonsense codon on theα2-globin gene expression.更多还原