一种α_1珠蛋白基因突变异常血红蛋白G复合HbH（-α~(4.2)/SEA）病的研究
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摘要
研究背景及目的
异常血红蛋白病(Abnormal hemoglobinopathies)属于常染色体显性遗传遗传疾病，珠蛋白肽链结构异常导致一种或一种以上结构异常的血红蛋白，部分或完全替代了正常的血红蛋白。肽链结构改变可导致血红蛋白功能和理化性质的变化或异常。结构异常可发生于任何一种珠蛋白链，但以β珠蛋白链受累为常见，异常血红蛋白病的蛋白表型均以其基因突变为基础。文献已报道的异常血红蛋白已达1021种，涉及不同的珠蛋白链。多数变种仅为携带者的遗传特性，少数变种则可引起临床症状从而影响患者的健康。血红蛋白G病（Hb G disease）属于异常血红蛋白病范围，于1954年由Edington和Lehamann第一次报导，其正常的血红蛋白为异常的血红蛋白完全或部分替代。血红蛋白G在过去的大规模血红蛋白普查中偶有发现，是一种少见的异常血红蛋白。目前已知HbG病主要是由于α链或β链异常所致，文献报道属α链异常的有HbGTaichung（常称HbQThailand）、HbGAudbaii和HbG Chinese等8种，属β链异常的有HbG Taipei、HbG Coushatta等14种。HbG病的发生与民族、地域有关，中国人以HbGTaichung、HbG Chinese、HbG Taipei和HbG Coushatta为主，HbGCoushatta以北方居多。多数异常血红蛋白G病无临床症状， HbG病分子异质性相当明显，包含多种类型的变异，通常按异常肽链一级结构分析进行分类。我国八、九十年代主要运用肽链氨基酸指纹图谱法进行氨基酸序列分析，目前国外常用高压液相色谱仪、毛细血管电泳等方法进行珠蛋白肽链分析鉴定异常血红蛋白类型。本课题通过血液学检查、高分辨琼脂糖凝胶电泳、珠蛋白α、β基因DNA测序的方法，对血红蛋白G病的血液学表现、分子生物学特点、珠蛋白基因突变进行分析，同时运用Gap-PCR法、反向斑点杂交法进行地中海贫血基因检测。确定本两例血红蛋白G基因突变类型，以及复合地中海贫血的分子基础。为异常血红蛋白G的临床、分子遗传学研究，提供更多的科学实验和理论依据。
材料与方法
研究对象
患者王某某，女，41岁，广东珠海人。从幼年时期反复巩膜黄染，有胆囊炎、胆囊结石病史，体检发现巩膜轻度黄染，肝肋下未及、脾下缘在肋缘下约2cm。检查报告显示：血常规：Hb97.8g/L，MCV63.8fL，MCH18.8pg，MCHC295.0g/L；血液检查：血清铁42umol/L，谷丙转氨酶（ALT）60u/L,谷草转氨酶（AST）57U/L,总胆红素（TBIL）30.9umol/L，结合胆红素（DBIL）10.6umol/L，非结合胆红素（IBIL）20.3umol/L；血红蛋白琼脂糖凝胶电泳：血红蛋白A缺如，血红蛋白F2.0%，血红蛋白A_20.3%，血红蛋白G87.6%，血红蛋白H10.1%。红细胞脆性22.9%。
患者肖某某，女，32岁，广东韶关人。体检发现中度贫血貌，肝脾肋下未及，血常规：Hb84g/L，MCV61fL，MCH19.8pg，MCHC325g/L；血液检查：血清铁21umol/L，谷丙转氨酶（ALT）13u/L,谷草转氨酶（AST）26U/L,总胆红素（TBIL）13umol/L，结合胆红素（DBIL）6.4umol/L，非结合胆红素（IBIL）6.6umol/L；血红蛋白琼脂糖凝胶电泳：血红蛋白A缺如，血红蛋白F1.0%，血红蛋白A_20.5%，血红蛋白G81.0%，血红蛋白H17.5%。红细胞脆性7.9%。
实验方法
血液学检验
红细胞参数分析采用雅培CD-3700全自动血细胞分析仪进行检测，血红蛋白电泳分析采用法国Sebia全自动电泳仪。血清铁、肝功能采用日立7170s全自动生化分析仪。
DNA基因分析
1.采用苯酚∶氯仿∶异戊醇法提取外周血DNA。
2.对α链和β珠蛋白基因进行DNA测序。
3.采用PCR和反向斑点杂交法检测地贫基因。
结果
血液学分析结果
患者王某某，血常规表现为小细胞低色素贫血（Hb97.8g/L，MCV63.8fL，MCH18.8pg,MCHC295.0g/L），红细胞脆性为22.9%，较正常明显下降。琼脂糖凝胶电泳发现异常血红蛋白G87.6%，血红蛋白A缺如，血红蛋白A20.3%，血红蛋白H10.1%。。血清铁偏高42umol/L。谷丙转酶（ALT）60u/L,谷草转氨酶（AST）57U/L,总胆红素（TBIL）30.9umol/L，结合胆红素（DBIL）10.6umol/L，非结合胆红素（IBIL）20.3umol/L。
患者肖某某，血常规表现为小细胞低色素贫血（Hb84g/L，MCV61fL，MCH19.8pg,MCHC325g/L），红细胞脆性为7.9%，较正常明显下降。琼脂糖凝胶电泳发现异常血红蛋白G81.0%，血红蛋白A缺如，血红蛋白A20.5%，血红蛋白H17.5%。血清铁正常21umol/L。谷丙转氨酶（ALT）13u/L,谷草转氨酶（AST）26U/L,总胆红素（TBIL）13umol/L，结合胆红素（DBIL）6.4umol/L，非结合胆红素（IBIL）6.6umol/L。
DNA基因分析结果
采用苯酚：氯仿：异戊醇法提取两例患者外周血DNA，对α、β珠蛋白基因进行DNA测序，均发现α_1珠蛋白基因外显子2第128位G→C，密码子GAC→CAC，即α链N端的第74位天冬氨酸被组氨酸取代（Asp→His），为HbG Taichung（α_274Asp→His β_2）。α1的外显子1、3无异常。β珠蛋白基因外显子1、2和3均无异常。
对两患者DNA进行α地中海贫血SEA、3.7、4.2缺失基因分别进行PCR检测，均检出α地中海贫血SEA缺失杂合子和4.2缺失杂合子。对β地中海贫血基因突变位点：-29，-28、17、βE、41/42、43、71/72/654、Int、14/15、27/28、I-1、I-5、31采用反向斑点杂交(RBD)基因诊断未检出突变基因，发现两患者为中间型α地中海贫血HbH（--/-α~(4.2)）。
结论
1.高分辨琼脂糖凝胶电泳是发现异常血红蛋白G的有效方法。电泳图谱表现出优势的血红蛋白G带并缺乏血红蛋白A带类型，可能是基因突变纯合子或杂合子同时复合地中海贫血基因突变，本两例经分子生物学分析证实为杂合子同时复合血红蛋白H(--/-α~(4.2))基因突变，提示血红蛋白G突变须作进一步DNA基因分析明确其突变类型。
2.分子生物学DNA测序，能准确检测出血红蛋白G基因突变，是确诊异常血红蛋白G类型的直接证据。
3. HbG Taichung是异常血红蛋白G的特殊类型，复合地中海贫血，引发临床症状。
4.血红蛋白G病是一组高度异质性疾病，其血液学、分子生物学、基因突变、临床特点具有多样性。
Background and Objectives
Abnormal hemoglobinopathies are autosomal dominant genetic disease,globin chains structure abnormalities in one or more than one kind of structuralabnormality of the hemoglobin, partially or completely replacing the normalhemoglobin. Polypeptide chain structure changing can lead to change the function ofhemoglobin or abnormality of physicochemical properties. Structural abnormalitiescan occur in any kind of globin chains, but to the beta globin chain involvement iscommon, abnormal hemoglobin disease protein phenotypes are based on its genemutation. Reported abnormal hemoglobin has reached1021types in the literature,involving different globin chains. Most variants only carriers of the geneticcharacteristics, and few variants can cause clinical symptoms and thus affect thepatient's health. Hemoglobin G disease belonging to the abnormal hemoglobin diseaserange and first reported, by Edington and Lehamann in1954, its abnormalhemoglobin completely or partly replaced of normal haemoglobin.Hemoglobin G wasoccasionally found in the past large-scale hemoglobin census,wihch is an uncommonabnormal hemoglobin. Currently known HbG disease is mainly because of the alphaor beta chain replaced by the abnormal chain. The abnormal alpha chainincludes8types in the literature,such as HbG Taichung (It is usually named asHbQThailand),HbGAudbaii、HbGChinese and so on.; The abnormal alpha chainincludes14types in the literature,such as HbG Taipei, HbG Coushatta,and soon.HbG disease is related to nation and region. HbGTaichung, HbG Chinese, HbGTaipei and HbG Coushatta maintly exist,in the Chinese. HbG Coushatta mostly occursin the North area. Most of the abnormal hemoglobin G disease without clinicalsymptoms, The molecular heterogeneity in HbG disease is obvious, containing manytypes, usually analysised as primary structure of in abnormal chains to classify. Wemainly used of peptide amino acid fingerprinting method to analysis amino acidsequence in the eighties or nineties time of20th century, though nowadays peopleusing of high pressure liquid chromatography,capillary electrophoresis to analysistypes of abnormal hemoglobin on abroad, In This topic, haematologic almanifestations,molecular biological characteristics, and globin gene mutations wereanalyzed through blood examination, high-resolution agarose gel electrophoresis, alpha and beta globin gene DNA sequencing method for hemoglobin G disease, at thesame time using Gap-PCR method and reverse dot blot hybridization method forthalassemia gene detection. We will provide more scientific experiments andtheoretical basis for the abnormal hemoglobin G clinical and molecular genetics byidentifying the case of hemoglobin G gene mutations, as well as the molecular basisof compound thalassemia.
Methods
Subject
The Patient Wang XX, female,41years old, ancestral home is the city of Zhuhai.Recurrenting scleral yellow dye from childhood,the patient had fall in cholecystitis,gallbladder lithiasis. Physical examination revealed mild scleral yellow dye, the liversize is normal, The tip of the spleen belows of the costal margin about2cm. Theinspection report shows: Blood routine test：Hb97.8g/L, MCV63.8fL, MCH18.8pg,MCHC295.0g/L; Examination of blood：serum iron42umol/L, ALT60u/L,aminotransferase AST57U/L, TBIL30.9u mol/L, DBIL10.6u/L，IBIL20.3umol/L。Hemoglobin agarose gel electrophoresis: absence of HbA，HbF2.0%, HbG87.6%HbH10.1%. Erythrocyte fragility22.9%.
The Patient Xiao XX, female,32years old, ancestral home is the city of Shaoguan.Physical examination revealed moderate anemia appearance, hepatosplenic no touched,Blood routine test: Hb84g/L, MCV61fL, MCH19.8pg, MCHC325g/L; Examinationof blood: serum iron21umol/L, ALT13u/L, AST26U/L, TBIL13umol/L, DBIL6.4umol/L, IBIL6.6umol/L; Hemoglobin agarose gel electrophoresis: absence of HbA,HbF1%, HbA20.5%,HbG81%, HbH7.5%. Erythrocyte fragility7.9%.
Hematologic Methods
Hematological parameters were measured by ABBOTT CD-3700Full AutomaticBlood Analyzer and quantification of hemoglobin were conducted on the SebiaHydrasys automatic electrophoresis.Serum ferritin, liver function by using HITACHI7170s full automatic biochemical analyzer.
DNA Analysis
1. Extract DNA from the Peripheral blood by a improved phenol: chloroform:isoamyl alcohol method.
2. DNA sequencing on alpha and beta globin chain gene.
3. The PCR and reverse dot blot hybridization assay for detection of thalassemiagene.
Results
Hematological data analysis
The patient Wang XX has small cell low pigment anaemia (Hb97.8g/L,MCV63.8fL, MCH18.8pg,MCHC295.0g/L) through blood routine examination.Erythrocyte fragility is22.9%, obviously falling than normal. Agarose gelelectrophoresis reveals abnormal Hb G87.6%, HbA20.3%，absence of HbA,Hb H10.1%. Serum iron42umol/L, higher than normal value. ALT60u/L, AST57u/L,TBIL30.9u mol/,L DBIL10.6u mol/L,IBIL20.3u mol/L.
The patient Xiao XX has small cell low pigment anaemia (Hb84g/L，MCV61fL，MCH19.8pg, MCHC325g/L) through blood routine examination. Erythrocytefragility is7.9%, obviously falling than normal. Agarose gel electrophoresis revealsabnormal Hb G81%, HbA20.5%，absence of HbA, Hb H17.5%. Serum iron21umol/L. ALT13u/L, AST26u/L, TBIL13u mol/,L DBIL6.4u mol/L，IBIL6.6u mol/L.
DNA Analysis Result
Using phenol: chloroform: isoamyl alcohol to extract DNA in peripheral blood oftwo patients, DNA sequencing on alpha, beta globin gene, found that the128th baseG had changed to the base C in the exon2of Alpha1globin gene. GAC codonchanged to CAC codon, alpha chain N end the seventy-fourth aspartic acid wassubstituted by histidine (Asp changes to His), that is HbG Taichung（α_274Asp→Hisβ_2）. Exon1,3of Alpha1without mutation. Exon1,2and3of Beta globin gene allhave no mutation,too.
The two patients’ DNA on alpha thalassemia,3.7,SEA,4.2missing genes weredetected by PCR, found SEA deletion detection of thalassemia heterozygotes and4.2deletion heterozygote. The beta thalassemia gene mutation: as follows-29,-28,17,E,41/42, P43,71/72/654, Int,14/15,27/28, I-1, I-5,31by reverse dot blot hybridization (RBD) gene diagnosis not detected mutant gene. The patient is theintermediate thalassemia HbH.(--/-α~(4.2)）
Conclusions
1. High-resolution agarose gel electrophoresis is effectively to find the abnormalhemoglobin G. Electrophoresis shows an advantage belt Hb G, and Hb A belt withlack, This Performance may result from homozygous or heterozygous compoundthalassemia, and we confirmed the heterozygous compound hemoglobin H (--/-α~(4.2))gene mutation by Molecular biological methods. It suggests that hemoglobin Gmutation should furtherly analyze the DNA gene.
2. Molecular biological DNA sequencing can accurately detect the hemoglobin Ggene mutation, which is the direct evidence.of diagnosing abnormal hemoglobin Gtype.
3. HbG Taichung is the special type of abnormal hemoglobin G compoundthalassemia, causing clinical symptoms.
4. Hemoglobin G disease is a group of highly heterogeneous disease, havingdiversity of Hematology, molecular biology, gene mutation, clinical characteristics.
异常血红蛋白病(Abnormal hemoglobinopathies)属于常染色体显性遗传遗传疾病，珠蛋白肽链结构异常导致一种或一种以上结构异常的血红蛋白，部分或完全替代了正常的血红蛋白。肽链结构改变可导致血红蛋白功能和理化性质的变化或异常。结构异常可发生于任何一种珠蛋白链，但以β珠蛋白链受累为常见，异常血红蛋白病的蛋白表型均以其基因突变为基础。文献已报道的异常血红蛋白已达1021种，涉及不同的珠蛋白链。多数变种仅为携带者的遗传特性，少数变种则可引起临床症状从而影响患者的健康。血红蛋白G病（Hb G disease）属于异常血红蛋白病范围，于1954年由Edington和Lehamann第一次报导，其正常的血红蛋白为异常的血红蛋白完全或部分替代。血红蛋白G在过去的大规模血红蛋白普查中偶有发现，是一种少见的异常血红蛋白。目前已知HbG病主要是由于α链或β链异常所致，文献报道属α链异常的有HbGTaichung（常称HbQThailand）、HbGAudbaii和HbG Chinese等8种，属β链异常的有HbG Taipei、HbG Coushatta等14种。HbG病的发生与民族、地域有关，中国人以HbGTaichung、HbG Chinese、HbG Taipei和HbG Coushatta为主，HbGCoushatta以北方居多。多数异常血红蛋白G病无临床症状， HbG病分子异质性相当明显，包含多种类型的变异，通常按异常肽链一级结构分析进行分类。我国八、九十年代主要运用肽链氨基酸指纹图谱法进行氨基酸序列分析，目前国外常用高压液相色谱仪、毛细血管电泳等方法进行珠蛋白肽链分析鉴定异常血红蛋白类型。本课题通过血液学检查、高分辨琼脂糖凝胶电泳、珠蛋白α、β基因DNA测序的方法，对血红蛋白G病的血液学表现、分子生物学特点、珠蛋白基因突变进行分析，同时运用Gap-PCR法、反向斑点杂交法进行地中海贫血基因检测。确定本两例血红蛋白G基因突变类型，以及复合地中海贫血的分子基础。为异常血红蛋白G的临床、分子遗传学研究，提供更多的科学实验和理论依据。
材料与方法
研究对象
患者王某某，女，41岁，广东珠海人。从幼年时期反复巩膜黄染，有胆囊炎、胆囊结石病史，体检发现巩膜轻度黄染，肝肋下未及、脾下缘在肋缘下约2cm。检查报告显示：血常规：Hb97.8g/L，MCV63.8fL，MCH18.8pg，MCHC295.0g/L；血液检查：血清铁42umol/L，谷丙转氨酶（ALT）60u/L,谷草转氨酶（AST）57U/L,总胆红素（TBIL）30.9umol/L，结合胆红素（DBIL）10.6umol/L，非结合胆红素（IBIL）20.3umol/L；血红蛋白琼脂糖凝胶电泳：血红蛋白A缺如，血红蛋白F2.0%，血红蛋白A_20.3%，血红蛋白G87.6%，血红蛋白H10.1%。红细胞脆性22.9%。
患者肖某某，女，32岁，广东韶关人。体检发现中度贫血貌，肝脾肋下未及，血常规：Hb84g/L，MCV61fL，MCH19.8pg，MCHC325g/L；血液检查：血清铁21umol/L，谷丙转氨酶（ALT）13u/L,谷草转氨酶（AST）26U/L,总胆红素（TBIL）13umol/L，结合胆红素（DBIL）6.4umol/L，非结合胆红素（IBIL）6.6umol/L；血红蛋白琼脂糖凝胶电泳：血红蛋白A缺如，血红蛋白F1.0%，血红蛋白A_20.5%，血红蛋白G81.0%，血红蛋白H17.5%。红细胞脆性7.9%。
实验方法
血液学检验
红细胞参数分析采用雅培CD-3700全自动血细胞分析仪进行检测，血红蛋白电泳分析采用法国Sebia全自动电泳仪。血清铁、肝功能采用日立7170s全自动生化分析仪。
DNA基因分析
1.采用苯酚∶氯仿∶异戊醇法提取外周血DNA。
2.对α链和β珠蛋白基因进行DNA测序。
3.采用PCR和反向斑点杂交法检测地贫基因。
结果
血液学分析结果
患者王某某，血常规表现为小细胞低色素贫血（Hb97.8g/L，MCV63.8fL，MCH18.8pg,MCHC295.0g/L），红细胞脆性为22.9%，较正常明显下降。琼脂糖凝胶电泳发现异常血红蛋白G87.6%，血红蛋白A缺如，血红蛋白A20.3%，血红蛋白H10.1%。。血清铁偏高42umol/L。谷丙转酶（ALT）60u/L,谷草转氨酶（AST）57U/L,总胆红素（TBIL）30.9umol/L，结合胆红素（DBIL）10.6umol/L，非结合胆红素（IBIL）20.3umol/L。
患者肖某某，血常规表现为小细胞低色素贫血（Hb84g/L，MCV61fL，MCH19.8pg,MCHC325g/L），红细胞脆性为7.9%，较正常明显下降。琼脂糖凝胶电泳发现异常血红蛋白G81.0%，血红蛋白A缺如，血红蛋白A20.5%，血红蛋白H17.5%。血清铁正常21umol/L。谷丙转氨酶（ALT）13u/L,谷草转氨酶（AST）26U/L,总胆红素（TBIL）13umol/L，结合胆红素（DBIL）6.4umol/L，非结合胆红素（IBIL）6.6umol/L。
DNA基因分析结果
采用苯酚：氯仿：异戊醇法提取两例患者外周血DNA，对α、β珠蛋白基因进行DNA测序，均发现α_1珠蛋白基因外显子2第128位G→C，密码子GAC→CAC，即α链N端的第74位天冬氨酸被组氨酸取代（Asp→His），为HbG Taichung（α_274Asp→His β_2）。α1的外显子1、3无异常。β珠蛋白基因外显子1、2和3均无异常。
对两患者DNA进行α地中海贫血SEA、3.7、4.2缺失基因分别进行PCR检测，均检出α地中海贫血SEA缺失杂合子和4.2缺失杂合子。对β地中海贫血基因突变位点：-29，-28、17、βE、41/42、43、71/72/654、Int、14/15、27/28、I-1、I-5、31采用反向斑点杂交(RBD)基因诊断未检出突变基因，发现两患者为中间型α地中海贫血HbH（--/-α~(4.2)）。
结论
1.高分辨琼脂糖凝胶电泳是发现异常血红蛋白G的有效方法。电泳图谱表现出优势的血红蛋白G带并缺乏血红蛋白A带类型，可能是基因突变纯合子或杂合子同时复合地中海贫血基因突变，本两例经分子生物学分析证实为杂合子同时复合血红蛋白H(--/-α~(4.2))基因突变，提示血红蛋白G突变须作进一步DNA基因分析明确其突变类型。
2.分子生物学DNA测序，能准确检测出血红蛋白G基因突变，是确诊异常血红蛋白G类型的直接证据。
3. HbG Taichung是异常血红蛋白G的特殊类型，复合地中海贫血，引发临床症状。
4.血红蛋白G病是一组高度异质性疾病，其血液学、分子生物学、基因突变、临床特点具有多样性。
Background and Objectives
Abnormal hemoglobinopathies are autosomal dominant genetic disease,globin chains structure abnormalities in one or more than one kind of structuralabnormality of the hemoglobin, partially or completely replacing the normalhemoglobin. Polypeptide chain structure changing can lead to change the function ofhemoglobin or abnormality of physicochemical properties. Structural abnormalitiescan occur in any kind of globin chains, but to the beta globin chain involvement iscommon, abnormal hemoglobin disease protein phenotypes are based on its genemutation. Reported abnormal hemoglobin has reached1021types in the literature,involving different globin chains. Most variants only carriers of the geneticcharacteristics, and few variants can cause clinical symptoms and thus affect thepatient's health. Hemoglobin G disease belonging to the abnormal hemoglobin diseaserange and first reported, by Edington and Lehamann in1954, its abnormalhemoglobin completely or partly replaced of normal haemoglobin.Hemoglobin G wasoccasionally found in the past large-scale hemoglobin census,wihch is an uncommonabnormal hemoglobin. Currently known HbG disease is mainly because of the alphaor beta chain replaced by the abnormal chain. The abnormal alpha chainincludes8types in the literature,such as HbG Taichung (It is usually named asHbQThailand),HbGAudbaii、HbGChinese and so on.; The abnormal alpha chainincludes14types in the literature,such as HbG Taipei, HbG Coushatta,and soon.HbG disease is related to nation and region. HbGTaichung, HbG Chinese, HbGTaipei and HbG Coushatta maintly exist,in the Chinese. HbG Coushatta mostly occursin the North area. Most of the abnormal hemoglobin G disease without clinicalsymptoms, The molecular heterogeneity in HbG disease is obvious, containing manytypes, usually analysised as primary structure of in abnormal chains to classify. Wemainly used of peptide amino acid fingerprinting method to analysis amino acidsequence in the eighties or nineties time of20th century, though nowadays peopleusing of high pressure liquid chromatography,capillary electrophoresis to analysistypes of abnormal hemoglobin on abroad, In This topic, haematologic almanifestations,molecular biological characteristics, and globin gene mutations wereanalyzed through blood examination, high-resolution agarose gel electrophoresis, alpha and beta globin gene DNA sequencing method for hemoglobin G disease, at thesame time using Gap-PCR method and reverse dot blot hybridization method forthalassemia gene detection. We will provide more scientific experiments andtheoretical basis for the abnormal hemoglobin G clinical and molecular genetics byidentifying the case of hemoglobin G gene mutations, as well as the molecular basisof compound thalassemia.
Methods
Subject
The Patient Wang XX, female,41years old, ancestral home is the city of Zhuhai.Recurrenting scleral yellow dye from childhood,the patient had fall in cholecystitis,gallbladder lithiasis. Physical examination revealed mild scleral yellow dye, the liversize is normal, The tip of the spleen belows of the costal margin about2cm. Theinspection report shows: Blood routine test：Hb97.8g/L, MCV63.8fL, MCH18.8pg,MCHC295.0g/L; Examination of blood：serum iron42umol/L, ALT60u/L,aminotransferase AST57U/L, TBIL30.9u mol/L, DBIL10.6u/L，IBIL20.3umol/L。Hemoglobin agarose gel electrophoresis: absence of HbA，HbF2.0%, HbG87.6%HbH10.1%. Erythrocyte fragility22.9%.
The Patient Xiao XX, female,32years old, ancestral home is the city of Shaoguan.Physical examination revealed moderate anemia appearance, hepatosplenic no touched,Blood routine test: Hb84g/L, MCV61fL, MCH19.8pg, MCHC325g/L; Examinationof blood: serum iron21umol/L, ALT13u/L, AST26U/L, TBIL13umol/L, DBIL6.4umol/L, IBIL6.6umol/L; Hemoglobin agarose gel electrophoresis: absence of HbA,HbF1%, HbA20.5%,HbG81%, HbH7.5%. Erythrocyte fragility7.9%.
Hematologic Methods
Hematological parameters were measured by ABBOTT CD-3700Full AutomaticBlood Analyzer and quantification of hemoglobin were conducted on the SebiaHydrasys automatic electrophoresis.Serum ferritin, liver function by using HITACHI7170s full automatic biochemical analyzer.
DNA Analysis
1. Extract DNA from the Peripheral blood by a improved phenol: chloroform:isoamyl alcohol method.
2. DNA sequencing on alpha and beta globin chain gene.
3. The PCR and reverse dot blot hybridization assay for detection of thalassemiagene.
Results
Hematological data analysis
The patient Wang XX has small cell low pigment anaemia (Hb97.8g/L,MCV63.8fL, MCH18.8pg,MCHC295.0g/L) through blood routine examination.Erythrocyte fragility is22.9%, obviously falling than normal. Agarose gelelectrophoresis reveals abnormal Hb G87.6%, HbA20.3%，absence of HbA,Hb H10.1%. Serum iron42umol/L, higher than normal value. ALT60u/L, AST57u/L,TBIL30.9u mol/,L DBIL10.6u mol/L,IBIL20.3u mol/L.
The patient Xiao XX has small cell low pigment anaemia (Hb84g/L，MCV61fL，MCH19.8pg, MCHC325g/L) through blood routine examination. Erythrocytefragility is7.9%, obviously falling than normal. Agarose gel electrophoresis revealsabnormal Hb G81%, HbA20.5%，absence of HbA, Hb H17.5%. Serum iron21umol/L. ALT13u/L, AST26u/L, TBIL13u mol/,L DBIL6.4u mol/L，IBIL6.6u mol/L.
DNA Analysis Result
Using phenol: chloroform: isoamyl alcohol to extract DNA in peripheral blood oftwo patients, DNA sequencing on alpha, beta globin gene, found that the128th baseG had changed to the base C in the exon2of Alpha1globin gene. GAC codonchanged to CAC codon, alpha chain N end the seventy-fourth aspartic acid wassubstituted by histidine (Asp changes to His), that is HbG Taichung（α_274Asp→Hisβ_2）. Exon1,3of Alpha1without mutation. Exon1,2and3of Beta globin gene allhave no mutation,too.
The two patients’ DNA on alpha thalassemia,3.7,SEA,4.2missing genes weredetected by PCR, found SEA deletion detection of thalassemia heterozygotes and4.2deletion heterozygote. The beta thalassemia gene mutation: as follows-29,-28,17,E,41/42, P43,71/72/654, Int,14/15,27/28, I-1, I-5,31by reverse dot blot hybridization (RBD) gene diagnosis not detected mutant gene. The patient is theintermediate thalassemia HbH.(--/-α~(4.2)）
Conclusions
1. High-resolution agarose gel electrophoresis is effectively to find the abnormalhemoglobin G. Electrophoresis shows an advantage belt Hb G, and Hb A belt withlack, This Performance may result from homozygous or heterozygous compoundthalassemia, and we confirmed the heterozygous compound hemoglobin H (--/-α~(4.2))gene mutation by Molecular biological methods. It suggests that hemoglobin Gmutation should furtherly analyze the DNA gene.
2. Molecular biological DNA sequencing can accurately detect the hemoglobin Ggene mutation, which is the direct evidence.of diagnosing abnormal hemoglobin Gtype.
3. HbG Taichung is the special type of abnormal hemoglobin G compoundthalassemia, causing clinical symptoms.
4. Hemoglobin G disease is a group of highly heterogeneous disease, havingdiversity of Hematology, molecular biology, gene mutation, clinical characteristics.
引文
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[1]莫桂玲，胡朝晖，张玲.我国罕见异常血红蛋白突变型[β43(CD2)Glu→Lys分析[J].国际检验医学杂志,2010,31(9):918-921.
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[3] VELLAF，AGER JA，LEHMANN H. An abnormal haemoglobin in a Chinese：haemoglobin G[J].Nature，1958，182(4633):460-461.
[4]曾溢滔，黄淑帧，周霞娣，等.血红蛋白G_Chinese的结构分析[J].上海医学,1982,5(3):125-127.
[5] Blackwell RQ,Weng MI,Liu CS,et al.Hemoglobin G Chinese in Chinesesubjects in Taiwan[J].Vox Sang,1972,23(4):363-368.
[6]江西省萍乡矿务局职工医院，上海市儿童医院医学遗传研究室.两例血红蛋白G_Chinese生化分析[J].煤矿医学,1984,6(3):8-9.
[7]张贵寅,曾立志，鲍希桓，等.黑龙江省异常血红蛋白的研究_血红蛋白G_Chinese的结构分析[J].哈尔滨医科大学学报，1984，（2）：6-8。
[8] Blackwell RQ，Yang HJ，Wang CC.Hemoglobin G-Taipei: alpha-2-beta-2-22glu replaced by gly[J].Biochim Biophys Acta，1969，175(2)：237-241.
[9]李厚钧，刘志国，李立，等.新疆地区异常血红蛋白8例报告[J].解放军医学杂志，1987,12(2):97-99.
[10]宋文凤，徐文安，王方年，等.首例纳西族HbG-TaiBei（台北）β22（B4）Glu-Gly的结构分析[J].昆明医学院学报，1991，12（1）：34-36.
[11]刘绍辉，任邦哲.广东省异常血红蛋白研究X：一例HbGTaipei的一级结构与功能研究[J].暨南大学学报，1989，（4）：32-35.
[12] Boissel JP,Wajcman H,Labie D,et al.Hemoglobin G Coushatta (beta22(B4) glu leads to ala) in Algeria: an homozygous case[J].Nouv Rev FrHematol.1979,21(3):225-230.
[13]黄淑帧，曾溢涛，任兆瑞，等.国内发现的HbG_Coushatta纯合子[J].遗传，1983，5（2）：27-28.
[14]王保生，刘勤，杨桂霞，等.我国满族发现的异常血红蛋白G Coushatta一级结构分析研究[J].解放军医学杂志，1989，14（6）:433.
[15]曾溢涛，黄淑帧，周霞娣，等.血红蛋白G-Philadelphia复合α地中海贫血-化学结构和基因组织分析[J].遗传学报，1986，13（3）：238-242.
[16]陈人骏.江西省血红蛋白分子病的种类及分布[M].江西医药,1983，（6）：1-5
[17]北京医学院第一附属医院检验科.北京地区3104人中异常血红蛋白的普查情况[J].北京医学院学报，1984，16（4）：301-304。
[18]杨有利，李厚均，李春玲，等.甘肃武威地区的血红蛋白病患病率调查[J].中华血液学杂志，1997，18（12）：659-660。
[19]王碧玉，罗以琴.湖北省60248人血红蛋白病调查报告[J].同济医科大学学报，1987，（2）：130-133.
[20]李厚均，赵贤宁，李力，等.我国“丝绸之路”地区异常血红蛋白的分布[J].中华医学杂志，1995，75（5）：280-283.
[21]李厚均，李力，余伍忠.我国西北地区49例HbGCoushatta的分布——东北亚古代游牧民族的遗传标志[J].人类学学报，1996，15（3）：250-254。
[22]王碧玉，顾援朝，罗以琴.湖北地区血红蛋白病的研究[J].武汉医学院学报，1982，（1）：1-5.
[2]张俊武,龙桂芳.血红蛋白与血红蛋白病[M].广西科学技术出版社，2003:186-187
[3] Hardison RC,Chui DHK,GiardineB,et al.Hb Var:a relational database of humanhemoglobin variants and thalassemia mutation at the globin gene server[J].Hum Mutat,2002,19(3):225-232
[4]莫桂玲,胡朝晖,张玲，等.我国罕见异常血红蛋白突变型[β43(CD2)Glu→Lys]分析[J].国际检验医学杂志，2010,31（9）:918-921
[5] EDINGTON GM, LEHMANN H. Haemoglobin G:a new haemoglobin found in aWest African[J]. Lancet,1954,267(6830):173-174
[6]李厚均，赵贤宁，李力，等.我国“丝绸之路”地区异常血红蛋白的分布[J].中华医学杂志，1995，75（5）：280-283.
[7]曾溢滔,黄淑帧,周霞娣,等.血红蛋白G_Chinese的结构分析[J].上海医学,1982,5(3):125-127.
[8] Nakanishi T, Miyazaki A, Shimizu A, Yamaguchi A, Nishimura S. Assessment ofthe effect of hemoglobin variants on routine HbA1Cmeasurements by electrosprayionization mass spectrometry[J]. Clin Chim Acta,2002,323:89–101
[9]王碧玉,罗以琴.湖北省60248人血红蛋白病调查报告[J].同济医科大学学报，1987，(2):130-133.
[10]曾溢涛，黄淑帧.血红蛋白Siriraj纯合子的研究[J].遗传学报，1979,6（2）：155-160
[11] Frederick E Chen, M.D.Clara Ooi，Sau Yin Ha,et al. Genetic and ClinicalFeatures of Hemoglobin H Disease in Chinese Patients[J]. The New England journaland medicine,10,343(8):544-550.
[12]伊晓林，张新华,周天红.血红蛋白H病患者的胆石症及其相关因素分析[J].广东医学，2010,31（13）：1720-1721
[13]陈人骏.江西省血红蛋白分子病的种类及分布[M].江西医药,1983，（6）：1-5
[14]杨有利，李厚均，李春玲，等.甘肃武威地区的血红蛋白病患病率调查[J].中华血液学杂志，1997，18（12）：659-660
[15] Blackwell RQ，Yang HJ，Wang CC.Hemoglobin G-Taipei: alpha-2-beta-2-22glureplaced by gly[J].Biochim Biophys Acta，1969，175(2)：237-241.
[16]宋文凤，徐文安，王方年，等.首例纳西族HbG-TaiBei（台北）β22（B4）Glu-Gly的结构分析[J].昆明医学院学报，1991，12（1）：34-36.
[17] VELLAF，AGER JA，LEHMANN H. An abnormal haemoglobin in a Chinese：haemoglobin G[J].Nature，1958，182(4633):460-461.
[18]曾溢涛，黄淑帧，周霞娣，等.在中国发现的一个HbQ Thailand[α74（EF3）Asp→His]的家系研究[J].与生物物理学报,1953，15:23一32
[19]李厚均，李力，余伍忠.我国西北地区49例HbGCoushatta的分布——东北亚古代游牧民族的遗传标志[J].人类学学报，1996，15（3）：250-254
[20] SanchaisuriyaK,Chunpanich S,Fucharoen S,et al.Association of Hb Q‐Thailandwith homozygous Hb E and heterozygous Hb Constant Spring in pregnancy[J].European journal,2005,74:221-227
[21] Yadav AK.Comparative analysis of protein structure of common Hb Q variants.Indian J Pathol Microbiol,2010,53:696-8
[22] Blackwell RQ,Weng MI,Liu CS,et al.Hemoglobin G Chinese in Chinese subjectsin Taiwan[J].Vox Sang,1972,23(4):363-368
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