异常血红蛋白E（Hemoglobin E，HbE）是最常见的单基因遗传血红蛋白病种类之一。其分子基础是β-珠蛋白基因第26位氨基酸发生点突变，使谷氨酸密码子GAG被赖氨酸密码子AAG替代。HbE杂合子个体（HbEA）一般无明显的临床表现，HbE纯合子（HbEE）个体会产生小细胞低色素贫血，当HbE突变复合其他地中海贫血突变时，临床表现具有较大的异质性。HbE主要广泛分布在东南亚地区，在泰国、老挝、柬埔寨的许多地区，HbE的发生率高达60%，这些地区也被称为“HbE 三角”。在中国，云南是HbE流行率最高的地区，前期研究结果显示云南阿昌族中HbE携带率最高，高达39%。

血红蛋白病的高频地理分布与历史上疟疾在热带和亚热带地区的高流行率具有高度的一致性，因此血红蛋白病的高发生率一直被认为与疟疾自然选择作用有关。但是，与有较多证据支持血红蛋白病中的镰状细胞贫血（HbS）和α-地中海贫血的疟疾选择作用所不同，HbE在东南亚地区的广泛分布与自然选择的关系尚不完全清楚。目前，HbE与疟疾的相关性研究中，在分子生物学机制、流行病学关联分析和群体遗传进化方面的证据不完全一致。而作为HbE携带率最高的中国云南少数民族群体，仍缺乏HbE适应性进化过程中的表型特征以及HbE与自然选择关系的研究。因此，分析HbE的血液学特征，探讨该地区HbE与自然选择压力的关系，对阐明HbE在云南少数民族中高发生率的原因，了解人类基因组适应性进化具有一定意义。

【目的】利用HbE临床样本和HbE高携带率的云南少数民族群体，对HbE的基因型与表型、选择压力和起源进行群体遗传分析，为存在争议的HbE疟疾选择学说提供新的补充内容和证据。

【方法】（1）采用血常规检测、血红蛋白电泳检测和常见α、β地中海贫血突变基因检测对1265例临床HbE突变样本进行基因型与表型分析。（2）利用全外显子测序技术（Whole exome sequencing，WES）对65例阿昌族和65例德昂族群体样本进行基因分型，筛选出包括HbE在内的27个tagSNPs，基因组覆盖范围约744kb，进行以下遗传进化分析：对11号染色体全外显子测序数据利用SHAPEIT软件构建单倍型；对27个tagSNPs利用Plink1.9和Haploview 4.2软件进行哈温平衡（Hardy-Weinberg equilibrium, HWE）检验、连锁不平衡分析（Linkage disequilibrium，LD）；构建的单倍型利用R 3.6.1软件中的rehh包进行扩展单倍型纯合度（Extended haplotype homozygosity，EHH）、整合单倍型分数（Integrated haplotype score，iHS）检验，并根据EHH值和重组率估算HbE突变在人群中的发生时间；利用HbE及附近的4个SNP位点构建的单倍型，通过SplitsTree4 V4.15.1软件NeighborNet方法构建系统发育网络，分析HbE在两个民族中的起源。

【结果】（1）1265例HbE突变共检出26种基因型，最常见的为HbEA（61.82%），其次为β^E/β复合-α^3.7/αα（18.58%）以及HbEE（3.16%）。与HbEA相比，HbEE、β^E/β⁺以及HbEA复合标准型α-地贫中，血红蛋白水平和平均红细胞体积显著降低，β^E/β⁺组Hb F水平显著升高；各个HbE相关的不同基因型中，红细胞均具有小细胞低色素特征。（2）通过遗传进化分析，阿昌族和德昂族27个SNP位点均符合HWE，两个民族LD范围均超过100kb，EHH检验中两个民族携带HbE的单倍型EHH值要高于祖先单倍型，EHH值下降程度比祖先单倍型缓慢，HbE位点iHS值为极大的负值，以上结果均支持HbE受到自然选择作用。（3）通过EHH计算结果对两个民族中HbE突变进行粗略年代估计，得到这一突变发生于大约102代前，以每一代为25年或30年，则认为该突变大约发生于2550～3060年前。（4）HbE与附近的4个SNP构成的单倍型中，携带HbE突变的单倍型在两个民族中超过50%都为同一种单倍型，单倍型系统发育网络中所有携带HbE突变的单倍型具有更近的进化关系，提示云南阿昌族和德昂族的HbE突变可能具有单一的起源。

【结论】（1）云南德宏HbE突变携带率高，与其他遗传性血红蛋白病突变形成的复合突变较为常见，突变谱复杂多样，血液学表型存在异质性，HbE杂合子一般不产生严重血液学表现，HbE纯合子可产生小细胞低色素贫血，HbE杂合子复合β-地贫时，血液学表型加重。（2）云南阿昌族和德昂族中HbE突变受到自然选择作用，结合疟疾流行传播史，该选择作用很可能是疟疾产生的。（3）云南阿昌族和德昂族中HbE突变发生于距今2550～3060年前，是一个近代产生事件。（4）云南阿昌族和德昂族的HbE突变可能具有单一的起源。

Hemoglobin E (HbE) is produced by the substitution of the amino acid in position 26 by lysine. HbE heterozygote (HbEA) usually has no sever clinical manifestations, and HbE homozygote (HbEE) may have a hypochromic microcytic anemia. When the coinheritance of HbE with other types of β-thalassemia, the clinical manifestations usually have great heterogeneity. HbE is widely distributed in Southeast Asia. In many areas of Thailand, Laos and Cambodia, the incidence of HbE is as high as 60%, which also known as the "HbE triangle". In China, Yunnan is the region with the highest prevalence of HbE and the results of previous studies show that the prevalence of HbE in Achang ethnic group is the highest, up to 39%.

Historically, the high prevalence of malaria in tropical and subtropical regions was consistent with the geographic distribution of hemoglobinopathies, so it is generally thought that the high incidence of hemoglobinopathies has been associated with the selection pressure of malaria. At present, a lot of evidences have supported the malaria resistance of sickle cell anemia (HbS) and α-thalassemia, in contrast, the evolutionary interaction between HbE and malaria still remain more controversial. Evidences of HbE-based resistance to malaria in molecular biology mechanism, epidemiology, correlation analysis and population genetic evolution contradict with each other. However, as an ethnic group with the highest prevalence of HbE in  China, there is still a lack of research on the relationship between HbE and malaria selection in this population. Therefore, to explore the relationship between HbE and selection pressure in this region is of great significance to further understand the malaria-adaptive evolution of the human genome.

【Objective】To provide supplementary content and evidence for the controversial theory of HbE malaria hypothesis by the population genetic evolution analysis and phenotype analysis.

【Method】（1）Globin gene mutation detection, blood routine tests and hemoglobin analysis were performed on 1265 individuals to analyze genotype and phenotype. （2）Whole exome sequencing (WES) was used to genotyping on 130 samples of Achang and Deang populations. 27 tagSNPs, including HbE, with genome spanning 744kb, were screened for genetic evolution analysis as follows: Haplotype was phasing from the WES data by SHAPEIT software. Hardy-Weinberg equilibrium(HWE) and linkage disequilibrium (LD) were performed by Plink1.9 and Haploview 4.2 software. Extended haplotype homozygosity(EHH) and integrated haplotype score (iHS) were calculated using rehh package in R 3.6.1 software. The HbE age was estimated according to EHH value and recombination rate. The phylogenetic network was constructed using HbE and four nearby SNP loci through the NeighborNet method of SplitsTree4 V4.15.1 software.

【Result】 (1) A total of 26 genotypes were identified in 1265 cases with HbE mutation, of which the most common was HbEA (61.82%), followed by β^E/β combining with  -α^3.7/αα(18.58%) and HbEE (3.16%). Compared with HbEA, hemoglobin (Hb) and mean corpuscular volume (MCV) levels were significantly reduced in group of HbEE, β^E/β⁺ and HbEA combining with standard α-thalassemia, and the level of Hb F was significantly increased in the β^E/β⁺ group. All genotypes were characterized by hypochromic microcytic anemia or without anemia. (2) Genetic evolution analysis showed that the pattern of LD of HbE extended beyond 100kb, EHH of the ancestral allele decays more rapidly than that of the HbE allele and iHS value of the HbE site was extremely negative, which supported the effect of natural selection on HbE in Achang and Deang populations. (3) Based on the EHH value, the HbE age was roughly estimated to have occurred about 2550～3060 years ago. (4) The haplotype diversity of HbE and surrounding 4 tagSNPs and the phylogenetic network suggested a single origin of the HbE in Achang and Deang populations.

【Conclusion】 (1) HbE has a diversity and complicated mutation spectrum in Dehong, Yunnan. There is heterogeneity in hematological phenotype : HbEA usually has no sever clinical manifestation; HbE homozygotes show a feature of  hypochromic microcytic anemia or without anemia and HbE/β-thalassemia has a more severe anemia  than that of HbEA. (2) There is a nature selection on HbE in Achang and Deang populations, which was probably caused by malaria in combination with malaria epidemic history. (3) HbE have occurred between 2550 and 3060 years ago in Achang and Deang populations, which is a recent event. (4) HbE may have a single origin in Achang and Deang populations in Yunnan.