| Background and Abjectiveβ-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 methods1.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 HbA2 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 HbA2 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.x2 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 x2 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 r2,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 discussionThe 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 HbA2 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.Conclusion1.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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