【作者】 李泽松；

【导师】 蔡绍皙；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【摘要】 α-地中海贫血是世界上最常见的单基因遗传病,也是我国南方各省最常见、危害最大的遗传病。本病是由于第16号染色体短臂末端α-珠蛋白基因缺陷所致,有缺失型和非缺失型两种,缺失型是主要类型。在广东人中,超过96%的α-地中海贫血是由东南亚缺失(--SEA)、左侧缺失(-α4.2)和右侧缺失(-α3.7)引起。本病尚无有效治疗方法,携带者筛查及产前基因诊断是唯一防止新的患儿出生、提高人口素质的有效措施。实施该措施的前提是对人群中α-地中海贫血的流行病学的了解和相应的诊断技术的建立。目前,多种分子诊断技术已用于α-地中海贫血的诊断检测。其中多重Gap-PCR已广泛用于α-地中海贫血的分子筛查和临床诊断,但其在检测陈旧DNA时尚有一定局限性。基因芯片技术已用于多种遗传性疾病的诊断和研究。该技术可为α-地中海贫血的分子诊断提供进一步发展完善的可能。因此,本课题将研制一种可用于临床的、具有稳定性好、重复性高的快速检测中国人中常见的三种缺失型α-地中海贫血的基因芯片。在参照国内外文献的基础上,我们首先设计了特异性的PCR引物,建立并优化了PCR反应体系和条件。我们也设计了一系列长短不同的特异性的探针。在对醛基片的质量、探针的合成、点样液成分、探针浓度、杂交和洗涤等条件进行了研究比较后,我们获得了最适合的制备工艺和条件,并制备了含有70个寡核苷酸的探针的芯片。通过对临床标本的检测,设定了检测信号的判定标准:仅信噪比大于10,并且信号强度大于1000的探针才被认为是阳性的;如果-α3.7探针检测为阳性,那么α2的信号值必须大于-α3.7的一半时才能被判定为阳性。在上世纪八十年代使用血液学方法对全国范围的地中海贫血的流行病学研究结果对地中海贫血的预防起了巨大的作用。但是伴随我国经济高速发展的大规模的人口移动以及几十年来对地中海贫血的有效防治,我国人群中地中海贫血的流行病学可能发生变化。同时,血液学研究的结果与实际情况可能有偏差,需要从基因水平进一步验证和核实。因此,新一轮的基于分子分析的全国范围的地中海贫血流行病学的调查研究势在必行。这将是一个浩大的工程。一个小规模具有代表性的人群中地中海贫血流行病学的调查研究,将具有积极意义。深圳作为一座移民城市,其人口来源于全国各地,深圳市人群中地中海贫血的流行病学对于全中国人群有一定的代表性。因此,我们也进行了深圳市人群中地中海贫血流行病学的研究。使用自制的芯片我们对广东省深圳市人群地中海贫血的分子流行病学进行了调查。在检测过程中,我们检测到了一例特殊的病例,病人--SEA、?α3.7和α2均为阳性。血液血表型表现为典型的α-地中海贫血特点。通过分子分析鉴定其为HKαα和--SEA的复合杂合子。病人母亲基因型也为HKαα/--SEA,其父亲基因型为αα/--SEA。据我们所知,这是首次报道HKαα和--SEA的复合杂合子。由于HKαα/--SEA有典型的α-地中海贫血特点,与αα/--SEA的临床表型一致,因此在HKαα和αα之间并无明显的血液学和临床差异。这也反映了HKαα与αα基因序列的一致性。通过对到深圳几家大的医院就诊和寻求产前咨询的3713个人进行了表型筛查和基因分析,我们获得了深圳市人群中地中海贫血的流行病学和突变谱带。深圳人口地中海贫血基因携带率为6.49%,其中α-地中海贫血为4.34%,β-地中海贫血为1.99%;α-地中海贫血和β-地中海贫血双重杂合子0.16%。较广东省其他地区的低。这可能与深圳市人口多为外来移民有关。我们共检测到3种缺失型α-地中海贫血突变、1种非缺失型α-地中海贫血点突变和9种β-地中海贫血。与广东省其他地区相比,深圳人口中地中海贫血基因的突变谱带并无明显差异。综上所述,我们通过设计特异性的探针,与使用单管多重PCR扩增产物直接杂交,从而建立了一种新的检测缺失型α-地中海贫血的方法。该方法不仅方便快速,而且灵敏度高、特异性好,有利于在临床中推广应用。通过对深圳市人口地中海贫血流行病学的研究显示,该方法可应用于大规模人口地中海贫血流行病学调查,为地中海贫血的预防、诊断和基因治疗提供科学的理论根据。更多还原

【Abstract】 α-thalassemia is the most common human monogenic hereditary diseases in the world. Generally,α-thalassemia is mainly resulted fromα-globin gene defects which located in 16p13.3.α-thalassemia is classified as deletional or non-deletional according to the mutational mechanism involved. The deletional types comprise the majority of cases ofα-thalassemia. The Southeast Asian deletion (--SEA), rightward deletion (-α3.7), and leftward deletion (-α4.2) are the most common causes of this disorder in the Guangdong population, accounting for more than 96% of allα-thalassemia. Populations in southern China have such high prevalence rates ofα-thalassemia that they present a public health concern. The screen ofα-thalassemia carrier, antenatal gene diagnosis and selective abortion is the only choice to controlα-thalassemia due to the lack of ideal treatment forα-thalassemia in clinic. The prerequisites for the purpose are the detailed genetic epidemiology of a defined population and the correspondent optimized gene analysis strategy.At present, various molecular diagnosis technologies have been applied to theα-thalassemia assay. Gap-PCR is widely applied to the clinical diagnosis and molecular screening of deletionalα-thalassemia carriers. But the method has limits on assay for the old genomic DNA. The DNA microarray technology which has been applied to diagnosis and research of various genetic diseases could provide the further improvement for the diagnosis of deletionalα-thalassemia. Thus, a simple, fast, and highly reproducible protocol for detection of deletionalα-thalassemia using an oligonucleotide microarray was developed.Specific PCR primers for the three deletionalα-thalassemia and normalα2 gene were designed through consulting and contrasting the domestic and foreign literatures. A single tube quadruple PCR reaction system was set up and the conditions were optimized. A series of probes specified to different PCR products were designed. The microarray with 70mer oligonucleotide probes was prepared using the optimized technological conditions. PCR products were directly hybridized to the microarrays with different specific probes. Genotypes were determined by quantitative analysis of the fluorescent signals detected by fluorescence scanning. The detection criteria are as follows: (a) spots, with signal-to-background ratios greater than 10.0 and intensities subtracted local background signals more than 1000, were considered positive; (b) spots forα2 were considered positive only when signal of spots forα2 was more than half of that for -α3.7 when the spots for-α3.7 are positive. The preparation protocols and the detection criteria were further optimized based on the analysis of 32 DNA samples.As far as the epidemiology of thalassemia is concerned, the epidemiology study of hemoglobinpathies in China mainland in the middle of 80’s of last century played critical poles in controlling thalassemia. But a new round epidemiology investigation all over the country is imperative because previous study emphasized on abnormal hemoglobin diseases rather than thalassemia, and the epidemiology of thalassemia may be changed due to massive and long standing migration across the country with the fast social and economic development of China. This will be a tremendous task. A small scale investigation on thalassemia in representative population will be of certain guiding significance for the new round epidemiology. The prevalence and spectrum of thalassemia in Shenzhen is representative for the whole China because its population comes from all of the country. Thus, the molecular epidemiology of thalassemia in Shenzhen populations was carried out by using the home-made microarray.In the molecular epidemiological study of thalassemia from Shenzhen population by using the home-made microarray, we found a special case that was positive for the ?α3.7 junction fragment, --SEA junction fragment andα2. Phenotypic analysis revealed that the proband presented a typicalα-thalassemic trait. The molecular analysis identified that the genotype of proband was compound heterozygosity for HKααand --SEA (HKαα/--SEA). To our knowledge, this is the first report on heterozygosity for HKααand --SEA. Thus, the current case provided a chance to investigate the hematological and clinical impact of the HKαα. Asαα/--SEA, HKαα/--SEA presented a typicalα-thalassemic trait and there is no evident haematological and clinical difference between the HKαα/--SEA andαα/--SEAWe have obtained the prevalence and spectrum ofα- andβ-thalassemia mutations in Shenzhn by screening clinical blood samples. Of total 3721 samples, 241 (6.49%) were carriers of thalassemia, of which 161 (4.34%) hadα-thalassemia, 74 (1.99%) hadβ-thalassemia, and 6 (0.16%) had bothα- andβ-thalassemia. Thus, the prevalence of thalassemia mutations in the Shenzhen population is 6.49%. We identified three deletionalα-thalassemia mutations but only one nondeletional point mutation (–αCS). Of theseα-thalassemia mutations, the Southeast Asian deletion accounted for about 80% ofα-thalassemia chromosomes. More than 90% of theβ-mutations were accounted for by codon 41/42 (–CTTT), IVS-II-654 (C→T), codon 17 (A→T), and–28 (A→G).Compared with other areas in Guangdong Province, the prevalence of thalassemia in Shenzhen was lower, while there was no evident difference for the spectrum of mutations.To sum up, an oligonucleotide microarray for detection of the three most frequently observed deletionalα-thalassemia (--SEA, -α3.7, -α4.2) was developed. Purification, fragmentation, and nested PCR are not needed, which makes it possible to complete the entire protocol in a work day. A novel genotype was discovered while the prevalence and spectrum of thalassemia mutations in Shenzhen was investigated by using the home-made microarray. These results showed that the simple, fast, and highly reproducible protocol may be suitable for routine clinical use and population screening for deletionalα-thalassemia.更多还原