中国南方人群β-地中海贫血CD41-42（-CTTT）突变的起源与扩散的群体遗传学研究
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摘要
背景与目的
β-地中海贫血(β-thalassemia,简称β-地海)是全球最常见、对人类健康危害最大的单基因遗传病之一,其分子遗传学基础是由于β-珠蛋白基因缺陷,导致血红蛋白分子中β-珠蛋白肽链的合成减少或缺如,从而引起血红蛋白的α/β珠蛋白肽链比例失衡,进而导致血红蛋白不稳定、红细胞破坏而产生以溶血性贫血为主的症状群,在临床上表现为轻重不等的慢性进行性溶血性贫血。β-地贫是我国长江以南各省区人群中发生率最高、危害最为严重的遗传病之一,其中尤以广西、广东、海南等地发生率为甚,广西和广东人群携带率分别为4.8%～6.78%和2.54%～3.36%,海南人群的携带率估计约为2.7%,而海南黎族人群高达8%～11.6%。
流行病学、遗传学和体外实验研究结果表明,人类β珠蛋白基因是受疟疾选择作用最明显的基因之一。流行病学研究结果显示,β珠蛋白基因常见的变异HbS(β6Glu→Val)、HbC(βGlu→Lys)和HbE(β26Glu→Lys)以及β-地贫主要分布在非洲、地中海地区、中东、东南亚和中国南方等热带、亚热带地区,与疟疾的流行区域基本一致。细胞学和流行病学研究发现,血红蛋白病HbS、HbC、HbE以及β-地贫的个体和红细胞均具有抵抗疟原虫感染和降低恶性疟严重程度的作用。最近对β-珠蛋白基因簇单核苷酸多态性的连锁不平衡及单体型的分析发现,HbS、HbC和HbE的发生可能是人类进化史上近5000年内独立发生的事件,提示人类β珠蛋白基因变异与疟疾选择作用密切相关。由于疟疾流行区人群中杂合子优势具有保护人类免患疟疾或减缓恶性疟严重程度的作用。因此,这些具有选择优势的基因在疟疾发生地人群中的高频率有利于人类抵抗疟疾感染而得以生存和繁衍。
β-地贫的基因突变具有高度的异质性,不同种族人群拥有自己一组独特的β-地贫基因突变谱,表现为明显的地域性或群体特异性。到目前为止,在我国南方人群中至少已发现36种β地贫突变类型。其中,常见的突变基因主要有8种,包括CD41-42(-CTTT)、IVS-2-654C→T、CD71-72(+A)、CD17A→T、-28A→G,CD26 G→A(HbE)、-29 A→G和CD43G→T突变,分布在我国东南和西南等南方地区的人群中。值得注意的是,我国人群的β-地贫基因突变类型中,CD41-42(-CTTT)缺失是一种最常见和分布最广泛的突变,在我国南方各省人群中,该突变约占地贫突变的40%,而黎族人中β地贫基因突变几乎全为CD41-42(-CTTT)突变。我们最近的研究说明基因转换(gene conversion)可能是人群中CD41-42(-CTTT)突变扩散的主要分子机制。对于特定人群来说,常见和广泛分布的突变往往与其历史和种群变迁相关,因此,可以通过遗传病突变来追朔其建立者或祖先,进而阐述人群起源和进化史,这也是目前国际上分子进化研究的前沿课题。
壮族是我国人口最多的少数民族,主要聚居在广西壮族自治区的南部和中西部地区,源于古代百越的一支—骆越和瓯越,是我国历史上民族的主体很少迁徙的民族之一。黎族被认为是海南的土著民族,其先民也源于骆越,在海南岛至少有3000年以上的历史,与我国南方属汉藏语系的操壮侗语族诸语言的壮族、侗族、水族、傣族、布依族等民族有密切的渊源关系。尽管β珠蛋白基因CD41-42(-CTTT)突变是包括壮族和黎族人群在内的我国南方地区人群中最常见的β-地贫突变,但是,该突变何时出现?在黎族人群迁徙至海南前出现还是迁徙至海南后出现?在起源上,黎族、壮族和汉族人群的CD41-42(-CTTT)之间有何关系?是否与疟疾选择作用存在相关性?黎族人群为什么有如此高的携带率?到目前为止尚未有确切的分子遗传学依据。本研究根据壮族和黎族密切的历史渊源关系以及黎族在海南有史可证的时代背景,选择在广西和海南当地人群中CD41-42(-CTTT)这一最常见的β-地贫突变为对象,通过群体遗传学分析来阐述中国南方人群中这一具有选择优势的致病突变的起源与扩散,并企图解释人群自然选择的分子进化机制。
材料与方法
1.实验设计和样品:在海南岛采集2291例黎族人随机样品,在广西地区采集1627例壮族人随机样品,用于当地人群的β-地贫基因频率调查。以随机数方式分别从上述海南2291例黎族人和广西1627例壮族人中选出201例黎族人和354例壮族人样品,并分别采集海南岛黎族人居住地的200例汉族人随机样品和广西地区壮族人居住地的262例汉族人样品,用于中国南方人群β-地贫CD41-42(-CTTT)突变的分子进化研究。血液学参数和血红蛋白A_2定量用于筛查样品中的β-地贫携带者,DNA诊断技术用于进行β-地贫携带者突变的分子诊断。经突变分析确诊,并在上述二地的四组样品中排除了其他类型的β-地贫突变样品后,核准用于群体遗传学分析的样品合计907例,分别为:海南黎族人201例,海南汉族人200例,广西壮族人280例和广西汉族人226例。此外,我们还采集了8例β-地贫CD41-42(CTTT)纯合子患者样品用于本研究,分别为海南黎族人1例,海南汉族人3例、广西汉族人2例和广东汉族人2例。
2.方法:用盐析法或磁珠法提取外周血白细胞DNA。用全血细胞计数(FBC)和HbA_2含量测定筛查β-地贫携带者;用等位基因特异PCR和反向点杂交诊断41-42(-CTTT)突变。用PCR扩增β-珠蛋白全长基因及其周围序列(3.5 kb片段),用双向核苷酸序列测定分析β-珠蛋白基因的单核苷酸多态性(SNPs)。用基因记数方法计算出各个位点的基因型频率和等位基因频率,用x~2检验进行基因型Hardy-Weinberg平衡吻合度和各人群的SNP等位基因频率比较,用Fisher's确切概率法检测各个人群之间的各种CD41-42(-CTTT)单倍型的频率有无统计学差异。计算各个人群的单核苷酸多态性杂合度,用方差分析比较各个人群单核苷酸多态性杂合度的差异,并计算人群的分化指数,分析各个人群之间β-珠蛋白基因遗传结构的分化情况。用Phase2.1.1软件进行单倍型分析,用x~2检验进行各个人群单倍型频率的比较。用DnaSP4.10进行Four Gamete分析,进行β-珠蛋白基因中重组事件以及发生位置的评估。用Alequin3.01软件计算各个位点之间的两两相对连锁不平衡系数D'值、两两相对连锁不平衡相关系数r~2值、各个人群β-珠蛋白基因的核苷酸分离位点数θ值和任意两序列之间核苷酸差异的平均数π值,并计算Tajima's D值进行分析对象的中性检验,评估SNP之间的两两不平衡关系及与选择作用的关系。用Net-Work4.1.1.2软件构建样品的单倍型系统树,观察各种单倍型之间的演变关系。参考相关史料来解释和讨论基因突变及其人群起源和进化历史。
结果与讨论
在海南岛黎族人群随机大样品调查得出的β-地贫CD41-42(-CTTT)突变携带率为8.16%,未发现其它突变,随机抽取的201例黎族人CD41-42(-CTTT)突变的携带率为10.45%,抽样样本和人群的CD41-42(-CTTT)突变携带率结果之间没有统计学差异(P＞0.05)。根据血液学参数和HbA_2含量,在1624份广西壮族随机大样品中调查得出β-地贫突变携带率为6.64%。在随机抽取的354份壮族样品和262例汉族样品中,CD41-42(-CTTT)突变携带率的结果分别为3.39%和3.05%,在剔除其它β-地贫突变后,最终入选的280例壮族样品和226例汉族样品中CD41-42(-CTTT)突变携带率的结果分别为3.92%和3.54%,两组壮族样本或两组汉族样本之间CD41-42(-CTTT)突变携带率没有统计学差异(P＞0.05)。200例海南汉族人检出CD41-42(-CTTT)突变携带者2例,未发现其它β-地贫突变,其CD41-42(-CTTT)突变携带率为1.00%。在上述二地的四组人群样品的β-珠蛋白基因中,除了CD41-42(-CTTT)缺失突变外,还存在13个SNP位点,四组人群中的14个多态性位点的基因型均达到Hardy-Weinberg平衡;四组人群中,除了CD41-42(-CTTT)突变的等位基因存在统计学差异外(P＜0.05),其它位点的等位基因频率没有统计学差异。本研究使用这些多态性位点进行单倍型分析。
Four Gamete分析结果显示,四个人群β-珠蛋白基因中,除了少数位点外,大部分位点均有重组事件发生。单倍型分析显示,四组人群中共发现47种单倍型,其中,海南黎族和广西壮族人群分别存在26种和20种单倍型,海南汉族人群和广西汉族人群中分别存在20种和27种单倍型,频率较高的单倍型为HP3、HP11、HP30和HP43,这四种单倍型所占的比例为89.14%。统计学分析表明,频率较高的单倍型HP3、HP11、HP30和HP43在四组人群中的分布没有显著性差异,而CD41-42(-CTTT)突变出现的主要单倍型HP20和HP47在四组人群中的分布存在显著性差异(P＜0.05),他们分别由HP3和HP43发生一次突变衍生而成。在CD41-42(-CTTT)突变的单倍型中,黎族人群中带有CD41-42(-CTTT)突变的单倍型HP20的频率(95.6%)明显高于其他三组人群的单倍型HP20频率(广西壮族27.2%,广西汉族33.3%,海南汉族62.5%)(P＜0.05)。由于黎族人群与壮族人群均起源于骆越,他们的分化时间约在3000～5000年以前,根据黎族人群CD41-42(-CTTT)突变单倍型遗传结构单一的特点,我们推测我国人群中的CD41-42(-CTTT)突变为单一起源,该突变发生在黎族和壮族人群分化之前,至少已有3000年的历史,HP20可能是最早出现的CD41-42(-CTTT)突变单倍型,然后在进化过程中通过基因转换的方式产生HP47。
连锁不平衡分析显示,14个SNP位点中,除了CD41-42(-CTTT)突变及个别位点与其它位点的连锁程度比较弱外,其它位点之间呈较强连锁不平衡。结合Four Gamete分析的结果,我们在大样本研究中进一步证实了我们新近发现的结果,即基因转换是进化过程中β-珠蛋白基因变异的形成以及CD41-42(-CTTT)在人群中播散的主要机制。中性检验显示,四个人群β-珠蛋白基因的π和θ值相差较大,Tajima'D值均为正值(P＜0.05),表明四个人群β-珠蛋白基因的常见SNP位点增多。同时,我们还观察到47种单倍型中存在三种主要单倍型,而且这三种单倍型之间的核苷酸差异比较大,表明这些人群的β-珠蛋白基因存在两个以上频率较高的等位基因,提示β-珠蛋白基因受到平衡选择的作用。根据史料,其结果可能与疟疾选择作用相关。我们推测β-地贫基因CD41-42(-CTTT)突变可能首先发生于长期居住在疟疾流行的广西境内、广东西部等地区的骆越群体中,然后由于受到疟疾的选择作用,在人群中达到多态性水平。
除了疟疾选择作用外,漂变和人群迁徙也可能是造成目前CD41-42(-CTTT)突变分布格局的原因。我们在Net-Work系统树中观察到黎族人群中的绝大部分CD41-42(-CTTT)突变单倍型HP20均由HP3发生4-bp缺失突变衍生而成,其单倍型的起源和结构上的单一性表明,黎族人群的CD41-42(-CTTT)突变的发生和分布符合建立者效应。与汉族和壮族比较,黎族人群体较小,而且是一个相对隔离的人群,建立者效应和人口的快速增长可以解释这一致病基因的频率增长,即使该群体中少数始祖所具有的CD41-42(-CTTT)突变基因逐渐达到较高水平。而在海南汉族、广西汉族和广西壮族人群的CD41-42(-CTTT)突变的单倍型中,我们没有观察到建立者效应。由于汉族和壮族的群体较大,遗传漂变可能导致CD41-42(-CTTT)突变的等位基因频率逐渐降低或变动不大。历史上,我国人群的迁徙主要是自北往南迁,而南方的群体很少往北迁;广西汉族是在不同时期从北方逐渐迁徙到广西的,而壮族基本上没有往北迁徙,汉族与壮族曾发生比较频繁的基因交流。这种基因流的走向可能是形成目前广西汉族和壮族人群中CD41-42(-CTTT)突变分布格局的重要因素。
历史上海南汉族人迁徙到海南晚于黎族人的迁徙,其β-地贫的突变谱、CD41-42(-CTTT)突变的携带率及该突变的单倍型分布与黎族人不同,但与广西、广东地区人群相似,提示海南汉族人群的CD41-42(-CTTT)突变可能是随着汉族迁入海南过程中带入海南汉族群体,该突变在汉族人群迁徙至海南之前已经发生,汉族人群与黎族人群之间的基因交流对海南汉族人群的CD41-42(-CTTT)突变的贡献可能较小。遗传漂变可能也是影响海南汉族人群CD41-42(-CTTT)突变频率的重要因素。
此外,我们还分析了四个人群β-珠蛋白基因单核苷酸多态性的杂合度及分化指数,结果表明β-珠蛋白基因的遗传结构没有分化。
结论
1.根据人群单倍型的特点、LD类型、Four Gamete结果以及重组和突变或缺失发生的机率,结合黎族人群与壮族人群的起源和分化的时间以及黎族迁徙史,我们推测中国人群的CD41-42(-CTTT)突变可能是单一起源,发生在黎族和壮族人群分化之前,至少已有3000的历史,CD41-42(-CTTT)缺失突变单倍型HP20可能是最早出现的单倍型,然后在人群传播过程中,通过基因转换的方式产生HP47。
2.连锁不平衡和遗传多样性分析结果表明海南汉族、海南黎族、广西汉族和广西壮族人群的β-珠蛋白基因在进化过程中受到平衡选择的作用,可能与疟疾选择作用相关。疟疾选择作用可能是造成我国南方地区人群β-地贫携带率高于北方人群的重要原因之一。
3.建立者效应可能是黎族人群CD41-42(-CTTT)突变携带率增高的重要原因。遗传漂变可能是导致海南汉族、广西汉族和广西壮族人群CD41-42(-CTTT)突变携带率低于黎族人群的原因之一。
4.人群迁徙也是影响海南汉族、广西汉族和广西壮族人群CD41-42(-CTTT)突变分布的因素之一。广西汉族人群和广西壮族人群之间的CD41-42(-CTTT)突变可能是由于两个民族之间基因交流的结果,而海南汉族人群的CD41-42(-CTTT)突变是随着汉族迁入海南带入汉族群体,汉族人群与黎族人群之间基因交流的贡献可能较小。
5.四个人群的β-珠蛋白基因的遗传结构非常相似,没有发生分化。
Background and Abjective
β-thalassaemia is one of the most common monogenic diseases of humans, constituting a major global health problem.It results from defective production ofβ-globin chain which arrises from mutations inβ-globin gene,causing imbalanced production ofαandβglobin chains,unstable hemoglobin and chronic hemolytic anemia.The disorder is very common in the south of China,especially in Guangxi, Guangdong and Hainan.The carrier frequencies are 4.8%～6.78%and 2.54%～3.36% respectively in Guangxi and Guangdong.The carrier frequency is estimated to be about 2.7%in Hainan Han population.The carrier frequency is 8%～11.6%in the Li population in Hainan.
Epidemiological,population-based and cellular studies showed strong evidences that humanβ-globin is one of the genes subject to malaria selection.Previous studies showed that the common hemoglobin variants HbS(β6Glu→Val),HbC(βGlu→Lys), HbE(β26Glu→Lys) andβ-thalassaemia are widespread in the tropical and subtropical regions such as Africa,Mediterranean,Middle East,Southeast Asia and the south of China,which is well correlated with the endemic of malaria in the history.Cellular and population-based studies indicated that the some individuals and erythrocytes of HbS,HbC,HbE andβ-thalassaemia confered resistance to the invasion of malaria parasites and increase in protection from life-threatening forms of malaria.Recently, analysis of haplotype structure associated with HbS、HbC,and HbE suggested that these variants are relatively recent and may arise within the past 5000 years, indicating that the variants are strongly associated with malaria selection in the human evolution.Under malaria selection,the variants had risen rapidly in allellic frequency due to heterozygote advantage which protected human from invasion of malaria and reduced the severity of life-threatening forms of malaria.
Mutations causingβ-thalassemia are highly herterozygous and each high-frequency population has different sets of mutations.At least 36 mutations ofβ-thalassemia have been identified in Chinese population,8 of which,CD41/42(-CTTT), IVS-2-654C→T,-28 A→G,CD17 A→T,CD71/72 +A,CD26 G→A(HbE),-29 A→G and CD43G→T,are the most common mutations.It is noteworthy that CD41/42(-CTTT) mutationis is the most frequent and widespread in the population of southern China and accounts for about 40%ofβ-thalassemic mutations,while theβ-thalassemia mutations in Li population is almost CD41/42(-CTTT) mutation. Zhuang is China's largest minority population,with most of its population living in the Guangxi autonomous region,who migrates less in the history.It is thought to be originated from the 'Luo-Yue' and 'Xi Ou' groups.The Li population is considered to be the native population in Hainan Island,which also originated from the 'Luo-Yue' group and lived in Hainan for at least 3000 years.The Li population has close origin to the populations of Zhuang,Dong,Shui,Dai and Buyi whose language belongs to the Zhuang-Dong linguistic subfamily of Han-Tibetan family.Although the CD41/42(-CTTT) mutation is the most common mutation in the populations of southern China,including the Zhuang and Li populations,it is unclear that when the mutation occurred and lack of evidence for the origin and spread of the mutation.Did it happen before the migration of Li population to Hainan or after its migration to Hainan? What is the relationship of the mutation in origin among the Li,Zhuang and Han populations? Is the high requencies of mutation in southern China relative to malaria selection? Why the carrier frequency is higher in the Li population than in other population? Based on the origin of the Li and Zhuang populations from common ancestors and the traceable history of the Li population in Hainan,we analyzed the single nucleotide polymorphisms and haplotypes inβ-globin gene to reveal the origin and spread of the CD41-42(-CTTT) mutation in south of China.
Materials and methods
1.Study design and population sampling:Blood samples from 2291 individuals of Li population in Hainan and 1627 Guangxi Zhuang individuals in Guangxi were randomly collected for screeningβ-thalassemia.201 Li samples and 354 Guangxi Zhuang samples were ramdomly selected from 2291 individuals and 1627 Guangxi Zhuang individuals.At the same time,blood samples of 200 Hainan Han individuals and 262 Guangxi Han individuals were collected for molecular evolution analysis of CD41-42(-CTTT) mutation.β-thalassemia carriers were screened by using haematological parameters and HbA_2 concentration.DNA analysis techniques were used to diagnose thalassemic mutations.After excluding the samples of other mutations and the unknown mutations,a total of 907 samples were finally chosen for further analysis,including samples of 201 Hainan Li,200 Haina Han,280 Guangxi Zhuang and 226 Guangxi Han.In addition,8β-thalassemia homozygous patients of CD 41-42(-CTTT) mutation were recruited for the study,including 1 Li patient,3 Hainan-Han patients,2 Guangxi-Han patients and 2 Guangdong-Han patients.
2.Methods:The DNA was prepared from blood leukocytes by salt-out method or magnetic bead method.β-thalassemia carriers were screened by analysis of peripheral blood cell counts,hemoglobin concentration,mean cell volume of red cell(MCV) and HbA_2 concentration.The thalassemic mutations of all samples were determined by allelic-specific polymerase chain reaction(PCR) or reverse dot blot(RDB).A 3.5-kb fragment ofβ-globin gene and the flanking region was amplified by PCR for all samples.The single nucleotide polymorphisms ofβ-globin gene were detected by direct sequencing bi-directionally for all samples.Nucleotide changes were detected by visual inspection of chromatograms.The allelic frequencies and genotype frequencies were calculated by counting.x~2 test was used to test Hardy-Weinberg equilibrium and to compare the allelic frequencies among the populations.Fisher's exact test was used to test the difference between the frequencies of CD41-42(-CTTT) haplotypes in the populations.The heterozygosity of the SNPs was estimated and the differences between the heterozygosities among the populations were tested by using ANOVA test.The differenciation measures among the populations were estimated to analyze the genetic structrure ofβ-globin gene in the populations. Haplotypes were estimated using program Phase 2.1.1 and the frequencies of haplotypes among the populations were compared by x~2 test.By using DnaSP4.10 program,the four-gamete test was applied to reveal the recombination events and its position in theβ-globin gene.Pairwise |D'| and r~2,the mean number of nucleotide differences(π) and the proportion of segregating sites(θ),for the whole sample and for subpopulations,were calculated by Arlequin3.01 based on the haplotype inference results.Tajima's D was calculated to assess deviations from a neutral equilibrium frequency distribution for all loci.To root the network showing the relationship between each of haplotypes,the network of the haplotyes was constructed by the program NETWORK4.1.1.2.Combined the results with the history data of the ethnics,the origin and spread of the mutation will be discussed.
Results and discussion
The carrier frequency of CD41-42(-CTTT) mutation is 8.16%in 2291 Li individuals and no other mutations were found.In the randomly chosen 201 samples,the carrier frequency of CD41-42(-CTTT) mutation is 10.45%.There is no significant difference between the carrier frequencies in the Li population and in the randomly selected Li samples(P＞0.05).Based on haematological parameters and HbA_2 concentration,the carrier frequencies ofβ-thalassemia is 6.64%in 1624 Guangxi Zhuang individuals.In the randomly selected samples of 354 Guangxi Zhuang individuals and 262 Guangxi Han individuals,the carrier frequencies of CD41-42 (-CTTT) mutation are 3.39%and 3.05%.After excluding the samples of the other mutations and unknown mutations,the the carrier frequencies of CD41-42(-CTTT) mutation 3.92%and 3.54%respectively in the populations of Guangxi Zhuang and Guangxi Han.There are no significant differences between the carrier frequencies of CD41-42(-CTTT) mutation in the two selected Zhuang samples and in the two selected Guangxi Han samples(P＞0.05).Among 200 Hainan-Han individuals,2 heterozygotes of CD41-42(-CTTT) mutation were found and the carrier frequency of CD41-42(-CTTT) mutation is 1.00%.No otherβ-thalassemic mutations were found in Hainan Han samples.Except for CD41-42(-CTTT) mutation,13 polymorphisms inβ-globin gene were detected in the populations of Li,Hainan-Han,Guangxi-Han and Zhuang.All 14 polymorphisms are in Hardy-Weinberg equilibrium.There is no statistically significant difference between the allelic frequencies among the populations except for the significant difference among the frequencies of CD41-42 (-CTTT) allele(P＜0.05).
Four Gamete test showed that recombination occurred at most sites of the polymorphisms except for a few sites.A total of 47 haplotypes were found among the populations,with 26 in Li and 20 in Zhuang,20 in Hainan-Han and 27 Guangxi-Han, respectively.Four major haplotypes HP3,HP11,HP30 and HP43 were found, accounting for 89.14%of the chromosomes in the four populations.The haplotypes bearing CD41-42(-CTTT) mutation,HP20 and HP47,arise from haplotypes HP3 and HP43 respectively by one step of deletion.There are no significant differences in the frequencies of the most common haplotypes HP3,HP11,HP30 and HP43 among the four populations(P＞0.05).However,significant differences were found in the frequencies of HP20 and HP47 among the populations(P＜0.05).In the alleles bearing 41-42(-CTTT) mutation,the frequency(95.6%) of HP20 is higher in Li population than in other three populations populations(27.2%,33.3%and 62.5%in Zhuang, Guangxi-Han and Hainan-Han respectively)(P＜0.05).Since both Li and Zhuang originated from "Luo-Yue" group and their divergence occurred about 3000～5000 years ago,we infer that the mutation in southern Chinese is single origin and occurred before the divergence of Zhuang and Li,and is at least 3000 years old based on the almost homogenicity of the haplotypes bearing CD41-42(-CTTT) mutation in Li population and the migration history of Li population.Haplotype HP20 might be the earliest haplotype and the haplotype HP47 was formed through gene conversion in the evolutionary process.
Linkage analysis showed that most sites were in strong linkage disequilibrium except that CD41-42(-CTTT) mutation and a few sites were in linkage equilibrium with other sites.Combined with the results of Four Gamete test,we further confirm our recent finding that gene conversion is involved in shaping the variation patterns inβ-globin gene and the spread of CD41-42 mutation in Chinese population in the evolutionary process.Neutral test showed that there are differences between theπandθ,and Tajima's D was positive in the four populations(P＜0.05),indicating excess of common SNPs exists inβ-globin gene.At the same time,three common lineages of haplotypes were observed in the populations.These results indicated thatβ-globin gene was subject to balancing selection which might be due to malaria selection during evolution.Based on the history of southern Chinese and the results of neutral test and haplotypes,we infer that the CD41-42(-CTTT) mutation might originated from the "Luo-Yue" group living in the malaria endemic region of Guangxi and Guangdong and reached polymorphic due to malaria selection.
In addition to malaria selection,migration and genetic drift might also involved in the spread of CD41-42(-CTTT) mutationin the populations.We observed that the carrier frequency of CD41-42(-CTTT) mutation is significant higher and the haplotypes bearing CD41-42(-CTTT) mutation in Li population are almost HP20 arising from HP3,indicating founder effect is responsible for the spread of the mutation in Li population.Compared with the populations Han and Zhuang,the population size of Li population is small.This might made the ancestral allele of the mutation elevate to higher frequency with rapid expansion of Li population.However,we didn't find founder effect in the haplotypes bearing CD41-42(-CTTT)mutation in the populaions Han and Zhuang.The carrier frequencies of CD41-42(-CTTT) mutation in Guangxi-Han and Zhuang might decline or vary little due to genetic drift since the population size of the populaions Han and Zhuang is large.Historically,the migrations of Han population in ancient time were southward while the local populations in south China almost less migrated northward.The Han population migrated to Guangxi in different periods in ancient time while most of Zhuang population didn't migrate northward.The gene flow between the tow populations might play important role in the spread of the mutation between the two populations. In history,the migration of Hainan Han population to Hainan island was latter than Li's migration and less gene flow between the Han and Li populaion had been historically documented.The differences between the Hainan-Han and Li populations have been observed in the spectra ofβ-thalassemia,the carrier frequency of CD41-42 (-CTTT) mutation and the distribution of haplotypes bearing the mutation,indicaing that the mutation in Hainan-Han population might occurred before their migration to Hainan and gene flow between the two populations might contribute little to the mutation in Hainan Han population.Genetic drift might also involve in the spread of the mutation in Hainan-Han population.
In addition,we analyzed the heterozygosity and differenciation measure(Fst) ofβ-globin gene in the four populations.The results indicate that there is no genetic differentiation inβ-globin gene among the population.
Conclusion
1.It is evident that the CD41-42(-CTTT) mutationin Chinese might be single origin based on haplotype,the LD pattern,Four gamete test,the origin and divergence of Li and Zhuang populations,the migration history of Li population and the rate of recombination or deletion.The mutation might be at least 3000 years old. The haplotype HP20 with CD41-42(-CTTT) mutationmight be the oldest one and the HP47 with CD41-42(-CTTT) mutationwas formed through gene conversion during spreading process in the populations.
2.The results of linkage disequilibrium and nucleotide diversity indicated thatβ-globin gene in the populations was subject to balancing selection in the process of evolution which might be due to malaria selection.Malaria selection might one of the causes responsible for the distribution of CD41-42(-CTTT) mutationin the populations of south China.
3.Founder effect was an important force elevating CD41-42(-CTTT) mutation in the Li population.Genetic drift might be one of the causes reslting in lower carrier frequencies of CD41-42(-CTTT) mutationin the populations Hainan Han,Guangxi Han and Zhang than in Li population.
4.Migration might also play important role in the distribution of CD41-42 (-CTTT) mutation in the populations Hainan Han,Guangxi Han and Zhuang.The CD41-42(-CTTT) mutation in the populations Guangxi Han and Zhuang might be due to gene flow between the two populations.The CD41-42(-CTTT) mutation in Hainan Han population is probablely introduced into the population with their migration to Hainan.Gene flow beween the Han and Li contributed little to the CD41-42(-CTTT) mutation in Hainan Han populaion.
5.The genetic structure ofβ-globin gene is similar in the four populations and there is no genetic differenciation among the populations.
β-地中海贫血(β-thalassemia,简称β-地海)是全球最常见、对人类健康危害最大的单基因遗传病之一,其分子遗传学基础是由于β-珠蛋白基因缺陷,导致血红蛋白分子中β-珠蛋白肽链的合成减少或缺如,从而引起血红蛋白的α/β珠蛋白肽链比例失衡,进而导致血红蛋白不稳定、红细胞破坏而产生以溶血性贫血为主的症状群,在临床上表现为轻重不等的慢性进行性溶血性贫血。β-地贫是我国长江以南各省区人群中发生率最高、危害最为严重的遗传病之一,其中尤以广西、广东、海南等地发生率为甚,广西和广东人群携带率分别为4.8%～6.78%和2.54%～3.36%,海南人群的携带率估计约为2.7%,而海南黎族人群高达8%～11.6%。
流行病学、遗传学和体外实验研究结果表明,人类β珠蛋白基因是受疟疾选择作用最明显的基因之一。流行病学研究结果显示,β珠蛋白基因常见的变异HbS(β6Glu→Val)、HbC(βGlu→Lys)和HbE(β26Glu→Lys)以及β-地贫主要分布在非洲、地中海地区、中东、东南亚和中国南方等热带、亚热带地区,与疟疾的流行区域基本一致。细胞学和流行病学研究发现,血红蛋白病HbS、HbC、HbE以及β-地贫的个体和红细胞均具有抵抗疟原虫感染和降低恶性疟严重程度的作用。最近对β-珠蛋白基因簇单核苷酸多态性的连锁不平衡及单体型的分析发现,HbS、HbC和HbE的发生可能是人类进化史上近5000年内独立发生的事件,提示人类β珠蛋白基因变异与疟疾选择作用密切相关。由于疟疾流行区人群中杂合子优势具有保护人类免患疟疾或减缓恶性疟严重程度的作用。因此,这些具有选择优势的基因在疟疾发生地人群中的高频率有利于人类抵抗疟疾感染而得以生存和繁衍。
β-地贫的基因突变具有高度的异质性,不同种族人群拥有自己一组独特的β-地贫基因突变谱,表现为明显的地域性或群体特异性。到目前为止,在我国南方人群中至少已发现36种β地贫突变类型。其中,常见的突变基因主要有8种,包括CD41-42(-CTTT)、IVS-2-654C→T、CD71-72(+A)、CD17A→T、-28A→G,CD26 G→A(HbE)、-29 A→G和CD43G→T突变,分布在我国东南和西南等南方地区的人群中。值得注意的是,我国人群的β-地贫基因突变类型中,CD41-42(-CTTT)缺失是一种最常见和分布最广泛的突变,在我国南方各省人群中,该突变约占地贫突变的40%,而黎族人中β地贫基因突变几乎全为CD41-42(-CTTT)突变。我们最近的研究说明基因转换(gene conversion)可能是人群中CD41-42(-CTTT)突变扩散的主要分子机制。对于特定人群来说,常见和广泛分布的突变往往与其历史和种群变迁相关,因此,可以通过遗传病突变来追朔其建立者或祖先,进而阐述人群起源和进化史,这也是目前国际上分子进化研究的前沿课题。
壮族是我国人口最多的少数民族,主要聚居在广西壮族自治区的南部和中西部地区,源于古代百越的一支—骆越和瓯越,是我国历史上民族的主体很少迁徙的民族之一。黎族被认为是海南的土著民族,其先民也源于骆越,在海南岛至少有3000年以上的历史,与我国南方属汉藏语系的操壮侗语族诸语言的壮族、侗族、水族、傣族、布依族等民族有密切的渊源关系。尽管β珠蛋白基因CD41-42(-CTTT)突变是包括壮族和黎族人群在内的我国南方地区人群中最常见的β-地贫突变,但是,该突变何时出现?在黎族人群迁徙至海南前出现还是迁徙至海南后出现?在起源上,黎族、壮族和汉族人群的CD41-42(-CTTT)之间有何关系?是否与疟疾选择作用存在相关性?黎族人群为什么有如此高的携带率?到目前为止尚未有确切的分子遗传学依据。本研究根据壮族和黎族密切的历史渊源关系以及黎族在海南有史可证的时代背景,选择在广西和海南当地人群中CD41-42(-CTTT)这一最常见的β-地贫突变为对象,通过群体遗传学分析来阐述中国南方人群中这一具有选择优势的致病突变的起源与扩散,并企图解释人群自然选择的分子进化机制。
材料与方法
1.实验设计和样品:在海南岛采集2291例黎族人随机样品,在广西地区采集1627例壮族人随机样品,用于当地人群的β-地贫基因频率调查。以随机数方式分别从上述海南2291例黎族人和广西1627例壮族人中选出201例黎族人和354例壮族人样品,并分别采集海南岛黎族人居住地的200例汉族人随机样品和广西地区壮族人居住地的262例汉族人样品,用于中国南方人群β-地贫CD41-42(-CTTT)突变的分子进化研究。血液学参数和血红蛋白A_2定量用于筛查样品中的β-地贫携带者,DNA诊断技术用于进行β-地贫携带者突变的分子诊断。经突变分析确诊,并在上述二地的四组样品中排除了其他类型的β-地贫突变样品后,核准用于群体遗传学分析的样品合计907例,分别为:海南黎族人201例,海南汉族人200例,广西壮族人280例和广西汉族人226例。此外,我们还采集了8例β-地贫CD41-42(CTTT)纯合子患者样品用于本研究,分别为海南黎族人1例,海南汉族人3例、广西汉族人2例和广东汉族人2例。
2.方法:用盐析法或磁珠法提取外周血白细胞DNA。用全血细胞计数(FBC)和HbA_2含量测定筛查β-地贫携带者;用等位基因特异PCR和反向点杂交诊断41-42(-CTTT)突变。用PCR扩增β-珠蛋白全长基因及其周围序列(3.5 kb片段),用双向核苷酸序列测定分析β-珠蛋白基因的单核苷酸多态性(SNPs)。用基因记数方法计算出各个位点的基因型频率和等位基因频率,用x~2检验进行基因型Hardy-Weinberg平衡吻合度和各人群的SNP等位基因频率比较,用Fisher's确切概率法检测各个人群之间的各种CD41-42(-CTTT)单倍型的频率有无统计学差异。计算各个人群的单核苷酸多态性杂合度,用方差分析比较各个人群单核苷酸多态性杂合度的差异,并计算人群的分化指数,分析各个人群之间β-珠蛋白基因遗传结构的分化情况。用Phase2.1.1软件进行单倍型分析,用x~2检验进行各个人群单倍型频率的比较。用DnaSP4.10进行Four Gamete分析,进行β-珠蛋白基因中重组事件以及发生位置的评估。用Alequin3.01软件计算各个位点之间的两两相对连锁不平衡系数D'值、两两相对连锁不平衡相关系数r~2值、各个人群β-珠蛋白基因的核苷酸分离位点数θ值和任意两序列之间核苷酸差异的平均数π值,并计算Tajima's D值进行分析对象的中性检验,评估SNP之间的两两不平衡关系及与选择作用的关系。用Net-Work4.1.1.2软件构建样品的单倍型系统树,观察各种单倍型之间的演变关系。参考相关史料来解释和讨论基因突变及其人群起源和进化历史。
结果与讨论
在海南岛黎族人群随机大样品调查得出的β-地贫CD41-42(-CTTT)突变携带率为8.16%,未发现其它突变,随机抽取的201例黎族人CD41-42(-CTTT)突变的携带率为10.45%,抽样样本和人群的CD41-42(-CTTT)突变携带率结果之间没有统计学差异(P＞0.05)。根据血液学参数和HbA_2含量,在1624份广西壮族随机大样品中调查得出β-地贫突变携带率为6.64%。在随机抽取的354份壮族样品和262例汉族样品中,CD41-42(-CTTT)突变携带率的结果分别为3.39%和3.05%,在剔除其它β-地贫突变后,最终入选的280例壮族样品和226例汉族样品中CD41-42(-CTTT)突变携带率的结果分别为3.92%和3.54%,两组壮族样本或两组汉族样本之间CD41-42(-CTTT)突变携带率没有统计学差异(P＞0.05)。200例海南汉族人检出CD41-42(-CTTT)突变携带者2例,未发现其它β-地贫突变,其CD41-42(-CTTT)突变携带率为1.00%。在上述二地的四组人群样品的β-珠蛋白基因中,除了CD41-42(-CTTT)缺失突变外,还存在13个SNP位点,四组人群中的14个多态性位点的基因型均达到Hardy-Weinberg平衡;四组人群中,除了CD41-42(-CTTT)突变的等位基因存在统计学差异外(P＜0.05),其它位点的等位基因频率没有统计学差异。本研究使用这些多态性位点进行单倍型分析。
Four Gamete分析结果显示,四个人群β-珠蛋白基因中,除了少数位点外,大部分位点均有重组事件发生。单倍型分析显示,四组人群中共发现47种单倍型,其中,海南黎族和广西壮族人群分别存在26种和20种单倍型,海南汉族人群和广西汉族人群中分别存在20种和27种单倍型,频率较高的单倍型为HP3、HP11、HP30和HP43,这四种单倍型所占的比例为89.14%。统计学分析表明,频率较高的单倍型HP3、HP11、HP30和HP43在四组人群中的分布没有显著性差异,而CD41-42(-CTTT)突变出现的主要单倍型HP20和HP47在四组人群中的分布存在显著性差异(P＜0.05),他们分别由HP3和HP43发生一次突变衍生而成。在CD41-42(-CTTT)突变的单倍型中,黎族人群中带有CD41-42(-CTTT)突变的单倍型HP20的频率(95.6%)明显高于其他三组人群的单倍型HP20频率(广西壮族27.2%,广西汉族33.3%,海南汉族62.5%)(P＜0.05)。由于黎族人群与壮族人群均起源于骆越,他们的分化时间约在3000～5000年以前,根据黎族人群CD41-42(-CTTT)突变单倍型遗传结构单一的特点,我们推测我国人群中的CD41-42(-CTTT)突变为单一起源,该突变发生在黎族和壮族人群分化之前,至少已有3000年的历史,HP20可能是最早出现的CD41-42(-CTTT)突变单倍型,然后在进化过程中通过基因转换的方式产生HP47。
连锁不平衡分析显示,14个SNP位点中,除了CD41-42(-CTTT)突变及个别位点与其它位点的连锁程度比较弱外,其它位点之间呈较强连锁不平衡。结合Four Gamete分析的结果,我们在大样本研究中进一步证实了我们新近发现的结果,即基因转换是进化过程中β-珠蛋白基因变异的形成以及CD41-42(-CTTT)在人群中播散的主要机制。中性检验显示,四个人群β-珠蛋白基因的π和θ值相差较大,Tajima'D值均为正值(P＜0.05),表明四个人群β-珠蛋白基因的常见SNP位点增多。同时,我们还观察到47种单倍型中存在三种主要单倍型,而且这三种单倍型之间的核苷酸差异比较大,表明这些人群的β-珠蛋白基因存在两个以上频率较高的等位基因,提示β-珠蛋白基因受到平衡选择的作用。根据史料,其结果可能与疟疾选择作用相关。我们推测β-地贫基因CD41-42(-CTTT)突变可能首先发生于长期居住在疟疾流行的广西境内、广东西部等地区的骆越群体中,然后由于受到疟疾的选择作用,在人群中达到多态性水平。
除了疟疾选择作用外,漂变和人群迁徙也可能是造成目前CD41-42(-CTTT)突变分布格局的原因。我们在Net-Work系统树中观察到黎族人群中的绝大部分CD41-42(-CTTT)突变单倍型HP20均由HP3发生4-bp缺失突变衍生而成,其单倍型的起源和结构上的单一性表明,黎族人群的CD41-42(-CTTT)突变的发生和分布符合建立者效应。与汉族和壮族比较,黎族人群体较小,而且是一个相对隔离的人群,建立者效应和人口的快速增长可以解释这一致病基因的频率增长,即使该群体中少数始祖所具有的CD41-42(-CTTT)突变基因逐渐达到较高水平。而在海南汉族、广西汉族和广西壮族人群的CD41-42(-CTTT)突变的单倍型中,我们没有观察到建立者效应。由于汉族和壮族的群体较大,遗传漂变可能导致CD41-42(-CTTT)突变的等位基因频率逐渐降低或变动不大。历史上,我国人群的迁徙主要是自北往南迁,而南方的群体很少往北迁;广西汉族是在不同时期从北方逐渐迁徙到广西的,而壮族基本上没有往北迁徙,汉族与壮族曾发生比较频繁的基因交流。这种基因流的走向可能是形成目前广西汉族和壮族人群中CD41-42(-CTTT)突变分布格局的重要因素。
历史上海南汉族人迁徙到海南晚于黎族人的迁徙,其β-地贫的突变谱、CD41-42(-CTTT)突变的携带率及该突变的单倍型分布与黎族人不同,但与广西、广东地区人群相似,提示海南汉族人群的CD41-42(-CTTT)突变可能是随着汉族迁入海南过程中带入海南汉族群体,该突变在汉族人群迁徙至海南之前已经发生,汉族人群与黎族人群之间的基因交流对海南汉族人群的CD41-42(-CTTT)突变的贡献可能较小。遗传漂变可能也是影响海南汉族人群CD41-42(-CTTT)突变频率的重要因素。
此外,我们还分析了四个人群β-珠蛋白基因单核苷酸多态性的杂合度及分化指数,结果表明β-珠蛋白基因的遗传结构没有分化。
结论
1.根据人群单倍型的特点、LD类型、Four Gamete结果以及重组和突变或缺失发生的机率,结合黎族人群与壮族人群的起源和分化的时间以及黎族迁徙史,我们推测中国人群的CD41-42(-CTTT)突变可能是单一起源,发生在黎族和壮族人群分化之前,至少已有3000的历史,CD41-42(-CTTT)缺失突变单倍型HP20可能是最早出现的单倍型,然后在人群传播过程中,通过基因转换的方式产生HP47。
2.连锁不平衡和遗传多样性分析结果表明海南汉族、海南黎族、广西汉族和广西壮族人群的β-珠蛋白基因在进化过程中受到平衡选择的作用,可能与疟疾选择作用相关。疟疾选择作用可能是造成我国南方地区人群β-地贫携带率高于北方人群的重要原因之一。
3.建立者效应可能是黎族人群CD41-42(-CTTT)突变携带率增高的重要原因。遗传漂变可能是导致海南汉族、广西汉族和广西壮族人群CD41-42(-CTTT)突变携带率低于黎族人群的原因之一。
4.人群迁徙也是影响海南汉族、广西汉族和广西壮族人群CD41-42(-CTTT)突变分布的因素之一。广西汉族人群和广西壮族人群之间的CD41-42(-CTTT)突变可能是由于两个民族之间基因交流的结果,而海南汉族人群的CD41-42(-CTTT)突变是随着汉族迁入海南带入汉族群体,汉族人群与黎族人群之间基因交流的贡献可能较小。
5.四个人群的β-珠蛋白基因的遗传结构非常相似,没有发生分化。
Background and Abjective
β-thalassaemia is one of the most common monogenic diseases of humans, constituting a major global health problem.It results from defective production ofβ-globin chain which arrises from mutations inβ-globin gene,causing imbalanced production ofαandβglobin chains,unstable hemoglobin and chronic hemolytic anemia.The disorder is very common in the south of China,especially in Guangxi, Guangdong and Hainan.The carrier frequencies are 4.8%～6.78%and 2.54%～3.36% respectively in Guangxi and Guangdong.The carrier frequency is estimated to be about 2.7%in Hainan Han population.The carrier frequency is 8%～11.6%in the Li population in Hainan.
Epidemiological,population-based and cellular studies showed strong evidences that humanβ-globin is one of the genes subject to malaria selection.Previous studies showed that the common hemoglobin variants HbS(β6Glu→Val),HbC(βGlu→Lys), HbE(β26Glu→Lys) andβ-thalassaemia are widespread in the tropical and subtropical regions such as Africa,Mediterranean,Middle East,Southeast Asia and the south of China,which is well correlated with the endemic of malaria in the history.Cellular and population-based studies indicated that the some individuals and erythrocytes of HbS,HbC,HbE andβ-thalassaemia confered resistance to the invasion of malaria parasites and increase in protection from life-threatening forms of malaria.Recently, analysis of haplotype structure associated with HbS、HbC,and HbE suggested that these variants are relatively recent and may arise within the past 5000 years, indicating that the variants are strongly associated with malaria selection in the human evolution.Under malaria selection,the variants had risen rapidly in allellic frequency due to heterozygote advantage which protected human from invasion of malaria and reduced the severity of life-threatening forms of malaria.
Mutations causingβ-thalassemia are highly herterozygous and each high-frequency population has different sets of mutations.At least 36 mutations ofβ-thalassemia have been identified in Chinese population,8 of which,CD41/42(-CTTT), IVS-2-654C→T,-28 A→G,CD17 A→T,CD71/72 +A,CD26 G→A(HbE),-29 A→G and CD43G→T,are the most common mutations.It is noteworthy that CD41/42(-CTTT) mutationis is the most frequent and widespread in the population of southern China and accounts for about 40%ofβ-thalassemic mutations,while theβ-thalassemia mutations in Li population is almost CD41/42(-CTTT) mutation. Zhuang is China's largest minority population,with most of its population living in the Guangxi autonomous region,who migrates less in the history.It is thought to be originated from the 'Luo-Yue' and 'Xi Ou' groups.The Li population is considered to be the native population in Hainan Island,which also originated from the 'Luo-Yue' group and lived in Hainan for at least 3000 years.The Li population has close origin to the populations of Zhuang,Dong,Shui,Dai and Buyi whose language belongs to the Zhuang-Dong linguistic subfamily of Han-Tibetan family.Although the CD41/42(-CTTT) mutation is the most common mutation in the populations of southern China,including the Zhuang and Li populations,it is unclear that when the mutation occurred and lack of evidence for the origin and spread of the mutation.Did it happen before the migration of Li population to Hainan or after its migration to Hainan? What is the relationship of the mutation in origin among the Li,Zhuang and Han populations? Is the high requencies of mutation in southern China relative to malaria selection? Why the carrier frequency is higher in the Li population than in other population? Based on the origin of the Li and Zhuang populations from common ancestors and the traceable history of the Li population in Hainan,we analyzed the single nucleotide polymorphisms and haplotypes inβ-globin gene to reveal the origin and spread of the CD41-42(-CTTT) mutation in south of China.
Materials and methods
1.Study design and population sampling:Blood samples from 2291 individuals of Li population in Hainan and 1627 Guangxi Zhuang individuals in Guangxi were randomly collected for screeningβ-thalassemia.201 Li samples and 354 Guangxi Zhuang samples were ramdomly selected from 2291 individuals and 1627 Guangxi Zhuang individuals.At the same time,blood samples of 200 Hainan Han individuals and 262 Guangxi Han individuals were collected for molecular evolution analysis of CD41-42(-CTTT) mutation.β-thalassemia carriers were screened by using haematological parameters and HbA_2 concentration.DNA analysis techniques were used to diagnose thalassemic mutations.After excluding the samples of other mutations and the unknown mutations,a total of 907 samples were finally chosen for further analysis,including samples of 201 Hainan Li,200 Haina Han,280 Guangxi Zhuang and 226 Guangxi Han.In addition,8β-thalassemia homozygous patients of CD 41-42(-CTTT) mutation were recruited for the study,including 1 Li patient,3 Hainan-Han patients,2 Guangxi-Han patients and 2 Guangdong-Han patients.
2.Methods:The DNA was prepared from blood leukocytes by salt-out method or magnetic bead method.β-thalassemia carriers were screened by analysis of peripheral blood cell counts,hemoglobin concentration,mean cell volume of red cell(MCV) and HbA_2 concentration.The thalassemic mutations of all samples were determined by allelic-specific polymerase chain reaction(PCR) or reverse dot blot(RDB).A 3.5-kb fragment ofβ-globin gene and the flanking region was amplified by PCR for all samples.The single nucleotide polymorphisms ofβ-globin gene were detected by direct sequencing bi-directionally for all samples.Nucleotide changes were detected by visual inspection of chromatograms.The allelic frequencies and genotype frequencies were calculated by counting.x~2 test was used to test Hardy-Weinberg equilibrium and to compare the allelic frequencies among the populations.Fisher's exact test was used to test the difference between the frequencies of CD41-42(-CTTT) haplotypes in the populations.The heterozygosity of the SNPs was estimated and the differences between the heterozygosities among the populations were tested by using ANOVA test.The differenciation measures among the populations were estimated to analyze the genetic structrure ofβ-globin gene in the populations. Haplotypes were estimated using program Phase 2.1.1 and the frequencies of haplotypes among the populations were compared by x~2 test.By using DnaSP4.10 program,the four-gamete test was applied to reveal the recombination events and its position in theβ-globin gene.Pairwise |D'| and r~2,the mean number of nucleotide differences(π) and the proportion of segregating sites(θ),for the whole sample and for subpopulations,were calculated by Arlequin3.01 based on the haplotype inference results.Tajima's D was calculated to assess deviations from a neutral equilibrium frequency distribution for all loci.To root the network showing the relationship between each of haplotypes,the network of the haplotyes was constructed by the program NETWORK4.1.1.2.Combined the results with the history data of the ethnics,the origin and spread of the mutation will be discussed.
Results and discussion
The carrier frequency of CD41-42(-CTTT) mutation is 8.16%in 2291 Li individuals and no other mutations were found.In the randomly chosen 201 samples,the carrier frequency of CD41-42(-CTTT) mutation is 10.45%.There is no significant difference between the carrier frequencies in the Li population and in the randomly selected Li samples(P＞0.05).Based on haematological parameters and HbA_2 concentration,the carrier frequencies ofβ-thalassemia is 6.64%in 1624 Guangxi Zhuang individuals.In the randomly selected samples of 354 Guangxi Zhuang individuals and 262 Guangxi Han individuals,the carrier frequencies of CD41-42 (-CTTT) mutation are 3.39%and 3.05%.After excluding the samples of the other mutations and unknown mutations,the the carrier frequencies of CD41-42(-CTTT) mutation 3.92%and 3.54%respectively in the populations of Guangxi Zhuang and Guangxi Han.There are no significant differences between the carrier frequencies of CD41-42(-CTTT) mutation in the two selected Zhuang samples and in the two selected Guangxi Han samples(P＞0.05).Among 200 Hainan-Han individuals,2 heterozygotes of CD41-42(-CTTT) mutation were found and the carrier frequency of CD41-42(-CTTT) mutation is 1.00%.No otherβ-thalassemic mutations were found in Hainan Han samples.Except for CD41-42(-CTTT) mutation,13 polymorphisms inβ-globin gene were detected in the populations of Li,Hainan-Han,Guangxi-Han and Zhuang.All 14 polymorphisms are in Hardy-Weinberg equilibrium.There is no statistically significant difference between the allelic frequencies among the populations except for the significant difference among the frequencies of CD41-42 (-CTTT) allele(P＜0.05).
Four Gamete test showed that recombination occurred at most sites of the polymorphisms except for a few sites.A total of 47 haplotypes were found among the populations,with 26 in Li and 20 in Zhuang,20 in Hainan-Han and 27 Guangxi-Han, respectively.Four major haplotypes HP3,HP11,HP30 and HP43 were found, accounting for 89.14%of the chromosomes in the four populations.The haplotypes bearing CD41-42(-CTTT) mutation,HP20 and HP47,arise from haplotypes HP3 and HP43 respectively by one step of deletion.There are no significant differences in the frequencies of the most common haplotypes HP3,HP11,HP30 and HP43 among the four populations(P＞0.05).However,significant differences were found in the frequencies of HP20 and HP47 among the populations(P＜0.05).In the alleles bearing 41-42(-CTTT) mutation,the frequency(95.6%) of HP20 is higher in Li population than in other three populations populations(27.2%,33.3%and 62.5%in Zhuang, Guangxi-Han and Hainan-Han respectively)(P＜0.05).Since both Li and Zhuang originated from "Luo-Yue" group and their divergence occurred about 3000～5000 years ago,we infer that the mutation in southern Chinese is single origin and occurred before the divergence of Zhuang and Li,and is at least 3000 years old based on the almost homogenicity of the haplotypes bearing CD41-42(-CTTT) mutation in Li population and the migration history of Li population.Haplotype HP20 might be the earliest haplotype and the haplotype HP47 was formed through gene conversion in the evolutionary process.
Linkage analysis showed that most sites were in strong linkage disequilibrium except that CD41-42(-CTTT) mutation and a few sites were in linkage equilibrium with other sites.Combined with the results of Four Gamete test,we further confirm our recent finding that gene conversion is involved in shaping the variation patterns inβ-globin gene and the spread of CD41-42 mutation in Chinese population in the evolutionary process.Neutral test showed that there are differences between theπandθ,and Tajima's D was positive in the four populations(P＜0.05),indicating excess of common SNPs exists inβ-globin gene.At the same time,three common lineages of haplotypes were observed in the populations.These results indicated thatβ-globin gene was subject to balancing selection which might be due to malaria selection during evolution.Based on the history of southern Chinese and the results of neutral test and haplotypes,we infer that the CD41-42(-CTTT) mutation might originated from the "Luo-Yue" group living in the malaria endemic region of Guangxi and Guangdong and reached polymorphic due to malaria selection.
In addition to malaria selection,migration and genetic drift might also involved in the spread of CD41-42(-CTTT) mutationin the populations.We observed that the carrier frequency of CD41-42(-CTTT) mutation is significant higher and the haplotypes bearing CD41-42(-CTTT) mutation in Li population are almost HP20 arising from HP3,indicating founder effect is responsible for the spread of the mutation in Li population.Compared with the populations Han and Zhuang,the population size of Li population is small.This might made the ancestral allele of the mutation elevate to higher frequency with rapid expansion of Li population.However,we didn't find founder effect in the haplotypes bearing CD41-42(-CTTT)mutation in the populaions Han and Zhuang.The carrier frequencies of CD41-42(-CTTT) mutation in Guangxi-Han and Zhuang might decline or vary little due to genetic drift since the population size of the populaions Han and Zhuang is large.Historically,the migrations of Han population in ancient time were southward while the local populations in south China almost less migrated northward.The Han population migrated to Guangxi in different periods in ancient time while most of Zhuang population didn't migrate northward.The gene flow between the tow populations might play important role in the spread of the mutation between the two populations. In history,the migration of Hainan Han population to Hainan island was latter than Li's migration and less gene flow between the Han and Li populaion had been historically documented.The differences between the Hainan-Han and Li populations have been observed in the spectra ofβ-thalassemia,the carrier frequency of CD41-42 (-CTTT) mutation and the distribution of haplotypes bearing the mutation,indicaing that the mutation in Hainan-Han population might occurred before their migration to Hainan and gene flow between the two populations might contribute little to the mutation in Hainan Han population.Genetic drift might also involve in the spread of the mutation in Hainan-Han population.
In addition,we analyzed the heterozygosity and differenciation measure(Fst) ofβ-globin gene in the four populations.The results indicate that there is no genetic differentiation inβ-globin gene among the population.
Conclusion
1.It is evident that the CD41-42(-CTTT) mutationin Chinese might be single origin based on haplotype,the LD pattern,Four gamete test,the origin and divergence of Li and Zhuang populations,the migration history of Li population and the rate of recombination or deletion.The mutation might be at least 3000 years old. The haplotype HP20 with CD41-42(-CTTT) mutationmight be the oldest one and the HP47 with CD41-42(-CTTT) mutationwas formed through gene conversion during spreading process in the populations.
2.The results of linkage disequilibrium and nucleotide diversity indicated thatβ-globin gene in the populations was subject to balancing selection in the process of evolution which might be due to malaria selection.Malaria selection might one of the causes responsible for the distribution of CD41-42(-CTTT) mutationin the populations of south China.
3.Founder effect was an important force elevating CD41-42(-CTTT) mutation in the Li population.Genetic drift might be one of the causes reslting in lower carrier frequencies of CD41-42(-CTTT) mutationin the populations Hainan Han,Guangxi Han and Zhang than in Li population.
4.Migration might also play important role in the distribution of CD41-42 (-CTTT) mutation in the populations Hainan Han,Guangxi Han and Zhuang.The CD41-42(-CTTT) mutation in the populations Guangxi Han and Zhuang might be due to gene flow between the two populations.The CD41-42(-CTTT) mutation in Hainan Han population is probablely introduced into the population with their migration to Hainan.Gene flow beween the Han and Li contributed little to the CD41-42(-CTTT) mutation in Hainan Han populaion.
5.The genetic structure ofβ-globin gene is similar in the four populations and there is no genetic differenciation among the populations.
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