【作者】 李亮；

【导师】 徐湘民；

【作者基本信息】 南方医科大学 ， 细胞生物学， 2009， 博士

【摘要】 葡萄糖-6-磷酸脱氢酶缺乏症是最常见的人类酶缺陷病之一,全世界范围内约有4亿人受累,目前认为发病率较高的原因是由于疟疾选择的结果。因此该病主要分布于疟疾高发的热带亚热带地区,从非洲热带、中东、东南亚到中国南方地区、南美洲某些地区、亚洲热带和亚热带地区、地中海某些地区、巴布亚-新几内亚,而新加坡、夏威夷等因移民原因也成为高发地区。G6PD缺乏症属X连锁不完全显性遗传,G6PD基因定位于Xq28,全长约20kb,含有13个外显子和12个内含子,编码515个氨基酸。G6PD缺乏症的分子基础为G6PD基因的突变导致生化表型的改变并产生临床症状。目前全世界已报道了160多种G6PD基因突变型,大部分是单碱基改变的错义突变。患者中男性半合子G6PD活性显著缺乏,女性杂合子酶活性变异范围较大,可显著性降低,也可在正常范围内。G6PD缺乏症最常见的临床表现是新生儿黄疸和急性溶血性贫血,前者可以导致新生儿核黄疸,从而影响新生儿智力。G6PD缺乏症在我国主要分布在长江以南省份,广东、广西、海南、贵州、云南、四川、台湾等省份,人群中基因携带率约为4～20%,男性发病率高于女性。目前临床上检测G6PD活性方法主要是酶活性检测方法,由于女性杂合子酶活性变异程度较大,该方法常常造成女性杂合子的漏检。本研究试图建立一种准确、经济且适用于检测中国人的常见G6PD基因突变技术体系。此外,本研究还探讨了G6PD缺乏症与新生儿高胆红素血症之间的相关性,研究结果将对G6PD缺乏症的诊断以及新生儿高胆红素血症的预防提供新的诊断方法与有价值的遗传学资料。一、反向点杂交检测中国人常见G6PD点突变方法的研究G6PD缺乏症为X连锁不完全显性遗传,根据莱昂假说,女性杂合子两条X染色体会随机失活一条,所以女性杂合子酶活性有较大的变异度。目前临床上检测G6PD主要是检测酶活性的生化检测法,所以会漏检表型较轻微的女性杂合子,这部分杂合子患者虽然表型轻微,但依然有可能生出一个患病的男孩,而只有分子诊断才能够准确的检测这部分患者,然而,目前存在的G6PD分予诊断方法存在各种各样的问题,难以推广使用。因此,临床上迫切需要一种准确、经济、且能同时检测中国人常见G6PD突变的检测方法。中国人G6PD突变谱现已有25种,但占突变构成比绝大部分的是少数常见突变类型。本研究将多重PCR与反向点杂交技术结合在一起,建立了一种准确、经济,能同时检测中国人G6PD 7种突变的方法。这7种突变类型分别是:c.95A＞G,c.871G＞A,c.1004C＞T,c.1024C＞T,c.1311C＞T,c.1376G＞T和c.1388G＞A,合计占整个突变构成比的85%左右。其基本原理和主要实验步骤如下:反向点杂交技术检测点突变的基本原理是通过位于寡核苷酸探针中部的序列特异性碱基与靶序列DNA的碱基配对和严格条件下洗膜来达到检测基因中少数碱基变化的目的,探针5’端标记有氨基臂（-NH2）,与尼龙膜表面的羧基（-COOH）进行化学反应而共价稳定结合。引物5’端标记了生物素,PCR产物扩增后,产物片段末端即标有生物素,与探针杂交后,用酶标结合蛋白及其酶的底物即可获得特异性杂交斑点信号（颜色反应）,根据显色探针的类型可知所分析标本的基因型。首先,针对G6PD基因和7个突变点设计三对特异性PCR扩增引物和14条特异性探针;然后,优化多重PCR体系以及反向点杂交体系;最后,确定最终反应条件和引物及探针序列。多重PCR引物设计时,尽量使6条引物的Tm值接近以使其复性效率相近从而达到在同一体系中扩增的目的。探针设计时,不但要使探针Tm值接近,还要尽量使突变位点位于探针序列中部,以达到最大的特异性效果。第一步,PCR扩增获得含有7个突变位点的三个G6PD基因片段。第二步,将PCR扩增的产物加入10ml聚丙烯离心管中,管内有10ml的2×SSC-0.1%SDS杂交缓冲液和制备好的膜条,100℃沸水浴变性10分钟,然后置恒温水浴杂交仪中43.5℃杂交过夜。第三步,杂交完全后将膜条转移至另一离心管中（含10ml已预热的0.5×SSC-0.1%SDS洗涤缓冲液）,于43.5℃洗膜10分钟,之后又将膜条转移至另一离心管中（含10ml的2×SSC-0.1%SDS缓冲液）,并加入5μl酶标抗生物素链菌素蛋白,在室温下颠倒振摇15分钟,取出膜条并用2×SSC-0.1%SDS缓冲液洗膜2次（每次各5分钟）后浸入显色液中避光显色10分钟。显色液临用时配制,20ml显色液含0.1mg/ml TMB、2μl的30%H₂O₂、0.1M柠檬酸钠（pH5.0）。阳性结果判定为出现蓝紫色斑点。对于单个患者来说,若相对应的突变位点与野生型位点探针均显色,则为杂合子患者;若只是突变探针显色,而相应的野生型探针不显色,则为女性纯合子或男性半合子,若其相对应的两个突变探针和野生型探针均显色,则为双重杂合子患者。为了评价该方法的特异性,我们选择了209例样品（已用直接测序的方法确定了基因型）对该方法进行双盲实验测试。结果除两例突变类型不在我们待检测突变里的样品不能检测以外,其余样品所检测的结果与测序结果完全相符,证明多重PCR结合反向点杂交技术能对G6PD这7种常见突变的各种基因型组合准确分型。二、G6PD缺乏症与新生儿高胆红素血症相关性研究G6PD缺乏症最严重的后果之一为新生儿高胆红素血症以及进一步导致的新生儿核黄疸。G6PD缺乏症可以导致新生儿黄疸很早就已经被证实,认为是G6PD活性缺乏导致红细胞破坏增多增加了胆红素的生成所致,但后来发现很多G6PD缺乏合并新生儿黄疸的个体并没有很明显的溶血发生,这种现象的原因被认为是G6PD活性缺乏导致溶血增加合并胆红素在肝内结合能力的下降协同作用所致。随后人们发现肝细胞内负责与胆红素结合的尿苷二磷酸葡萄糖醛酸基转移酶1A1（UGT1A1）基因启动子区突变导致酶活性的降低可以进一步增加G6PD活性缺乏新生儿发生高胆红素血症的机率证实了上一观点。由此,我们认为胆红素排泄路径上相关酶和蛋白的SNPs会影响酶和蛋白的功能,从而与G6PD活性缺乏协同作用增加新生儿高胆红素血症发生的机率。胆红素的排泄途径主要分为三步:未结合胆红素由肝细胞膜上的有机阴离子转运多肽C（OATP-C）转运至肝细胞内,在肝细胞内的尿苷二磷酸葡萄糖醛酸基转移酶1A1（UGT1A1）的催化作用下与葡萄糖醛酸结合形成结合胆红素,结合胆红素通过多药耐受相关蛋白2（MRP2）转运到胆管。本研究选择了UGT1A1基因,OATP-C基因和MRP2基因中外显子和启动区中国人频率大于5%的SNPs以及有文献报道的影响三个基因功能且中国人频率大于5%的SNPs列为待分析SNPs。由此,UGT1A1基因-3279T＞G,（TA）.₆TAA-（TA）₇TAA和211G＞A,OATP-C基因-11187G＞A,388A＞G,521T＞C,MRP2基因-24C＞T和1249G＞A 8个SNPs位点被列为待分析SNPs位点。我们应用变性高效液相色谱（DHPLC）对这8个SNPs进行基因分型,应用PHASE2.1.1软件对各个基因单倍型进行构建。本研究设计了病例—对照研究方案,在研究中,病例组为150例G6PD缺乏的新生儿,对照组为178例G6PD正常的新生儿,所有标本中,有其它因素影响新生儿胆红素水平的病例要被排除,包括早产儿、出生后存在窒息、败血症患者、乙肝大三阳患者、胆管阻塞者、ABO和Rh血型不合导致的溶血、携带其它血红蛋白病（如地中海贫血）、严重脱水、严重血管外溶血（如头颅血肿、皮下血肿、肺等部位大出血）、极低体重出生儿、血清胆红素和皮测胆红素值均未测定者、出生7天以后才入院的病例。本研究测量新生儿出生后1至7天每天的血清总胆红素值,把最高的值定为峰值。根据析因设计的方差分析法分别分析病例组与对照组各个SNPs位点和各个基因单倍型与胆红素峰值的相关性,从而找出对血清总胆红素有显著影响的SNPs或单倍型。实验结果中,G6PD缺乏组血清总胆红素峰值水平在各个SNPs的分析中均显著高于G6PD正常组,证实G6PD缺乏确实为新生儿血清胆红素水平的重要影响因素。8个SNPs位点的分析中,UGT1A1 211GG基因型组总胆红素水平为235.12±89.18μmol/L,UGT1A1 211GA/AA基因型组总胆红素水平为272.10±106.70μmol/L,突变组血清总胆红素水平显著的高于正常组（F=11.600,P=0.001）;OATP-C -11187GG基因型组总胆红素水平为238.76±93.04μmol/L,OATP-C-11187GA/AA基因型组总胆红素水平为270.40±102.46μmol/L,突变组血清总胆红素水平显著的高于正常组（F=8.229,P=0.004）,证实携带UGT1A1 211 G＞A和OATP-C-11187 G＞A突变为新生儿高胆红素血症发生的重要风险因素。其余6个SNPs对新生儿血清总胆红素浓度峰值均无显著性差异。在单倍型的分析中,UGT1A1基因的不同双倍型之间和OATP-C基因的不同双倍型之间新生儿血清总胆红素浓度峰值均有显著性差异,MRP2基因的不同双倍型之间新生儿血清总胆红素浓度峰值无显著性差异。我们进一步分析发现,UGT1A1基因的双倍型和OATP-C基因的双倍型与新生儿胆红素浓度峰值相关实际上是UGT1A1 211G＞A和OATP-C-11187G＞A对新生儿血清胆红素浓度峰值影响的间接体现,直接分析相关SNPs对新生儿血清总胆红素水平的影响会比分析单倍型对新生儿血清总胆红素的影响更为直接明了。我们认为G6PD活性缺乏、UGT1A1 211 G＞A突变和OATP-C-11187 G＞A突变为新生儿高胆红素血症发生的三个重要影响因素,且三个因素对新生儿总胆红素浓度具有累积效应,即携带这三个因素数量越多,发生新生儿高胆红素血症的可能性越大,携带三个风险因素个体的优势比为不携带任何风险因素的个体7.233（95%CI:1.416-36.948）倍。三、结论本研究建立基于多重PCR和反向点杂交技术的能同时检测7种中国人常见G6PD突变的方法,该方法不需要昂贵的设备与严格的技术条件,具有准确、低成本等特点,适用于G6PD缺乏症临床诊断的辅助诊断,也可以满足科研研究中对G6PD进行基因分型的需要,尤其适合在欠发达地区的推广使用。此外,本研究探讨了G6PD缺乏与新生儿高胆红素血症之间的关系,得到了胆红素代谢转运通路上的两个SNPs与新生儿高胆红素血症密切相关,该研究可以为临床上G6PD缺乏新生儿以及G6PD正常的新生儿黄疸的预防做一个参考,使易发生黄疸的人群得到更多的关注,最大程度上减少新生儿核黄疸的发生。总之,本研究深入探讨了中国人G6PD缺乏症的分子诊断方法的建立及其严重后果—新生儿黄疸的遗传基础及相关遗传学数据。我们希望以此为基础,提出具体的实施方案,通过预测个体新生儿高胆红素血症发生可能性,避免临床上新生儿高胆红素血症预防的盲目性,从而更大程度上降低医疗费用,减少医生精力的分散。该研究将减少新生儿核黄疸悲剧的发生,从而提高我国出生人口的素质。更多还原

【Abstract】 Glucose-6-phosphate dehydrogenase（G6PD） deficiency is the most common human enzyme defect,affecting more than 400 million people worldwide.The global distribution of this disorder is remarkably similar to that of malaria,which supporting the so-called malaria protection hypothesis.Therefore,the main distribution areas of G6PD deficiency are the tropics and subtropics with high incidence of malaria, extending from the Africa tropic,Middle East,South-east Asia,South China,parts of South America,Asia tropic and subtropic,parts of the Mediterranean and Papua New Guinea.In addition,the Singapore and Hawaii that had substantial immigration from Asia,also have a high incidence.G6PD deficiency is an X-linked,hereditary genetic disorder casued by mutations in the G6PD gene,resulting in protein variants with different levels of enzyme activity.The G6PD gene is located on the Xq28,over 20Kb in length,contains 13 exons and 12 introns,and codes 515 amino acids.G6PD deficiency is hereditary genetic disorder due to mutations in the G6PD gene,which cause functional variants with many biochemical and clinical phenotypes.More than 160 different mutations have been determined,and most of them are missense mutation.The male patients are hemizygotes and have marked deficiency of enzyme activity.The female patients have deviant enzyme activity,from the marked deficiency of enzyme activity to normal enzyme activity.The most frequent clinical manifestations of G6PD deficiency are neonatal jaundice and acute haemolytic anaemia.The neonatal jaundice may develop into kemicterus and cause mental damage.In China,provinces located in the south of Yangtze River have a relatively high incidence of G6PD deficiency,including Guangdong,Guangxi,Hainan,Guizhou, Yunnan,Sichuan and Taiwan with an incidence from 4%to 20%in the population, and the proportion of male patients are higher than the female patients.At present,the main methods to detect G6PD deficiency in clinical are the enzyme activity detection assays.The female patients have normal enzyme activity and can’t be detected by the enzyme activity assay.In this study,we have developed a multiplex polymerase chain reaction（M-PCR）/reverse dot blot assay（RDB） assay for the detection of the common Chinese G6PD gene mutations.In addition,we have analyzed the relationship between G6PD deficiency and neonatal hyperbilirubinemia.The result of our study will provide a new molecular diagnosis assay for G6PD deficiency and valuable genetic data for the prevention of the neonatal hyperbilirubinemia.1.The RDB assay for the detection of common Chinese G6PD mutationsG6PD deficiency is X-linked incomplete dominance.According to the lyon’s hypothesis,one of the two X chromosome will be inactive randomly,so the female patients have deviant enzyme activity.At present,the main methods of the detection of G6PD deficiency in clinical diagnosis are the enzyme activity detection assay,and the female patients with normal enzyme activity can’t be detected by the enzyme activity assay.Although the female heterozygotes have the normal enzyme activity, she can have a G6PD deficient son,so the molecular diagnosis is necessary for the female heterozygotes.Until now the molecular methods to detect the known G6PD point mutations have many disadvantages and are difficult to be extended.Therefore, there is a need for developing a highly accurate,economical and costeffective method to detect Chinese common G6PD mutations.Up to date,there are 25 types of G6PD deficiency mutations found in Chinese population.But a few common mutations account for the majority of G6PD deficiency.In this study,we developed an assay based on the multiplex PCR and RDB that can give an accurate,economical and simultaneous detection for the 7 most common mutations（c.95A＞G,c.871G＞A,c.1004C＞T,c.1024C＞T,c.1311C＞T, c.1376G＞T and c.1388G＞A,account for about 85%of the constitution） among Chinese G6PD mutations.The following part is the main mechanism and procedure of the assay:the mutation detection mechanism of RDB is that the amplified DNA,in combination with relatively short oligonueleotides,could provide sufficient driver to efficiently hybridize with multicopy sequence to provide a detectable signal and the hybridization conditions could be adjusted so that even a single base pair mismatch could be discerned.The 5’ position of the oligonucleotides have an amino（-NH2） which can bind form a covalent bond with carboxyl（-COOH） of the nylon strips.The 5’ position of the primers has a biotin,so the 5’ position of amplification fragments also have a biotin.The amplification fragments hybridize with the oligonucleotide probes,then the streptavidin-POD conjugate and substrate can react with the biotin with a color shown.The genotype of the specimen can be known by the color blot pattern.Firstly,3 pairs of high-specific PCR amplification primers and 14 oligonucleotide probes were designed according to the sequences of G6PD gene and the 7 mutation sites.Secondly,optimize the reaction condition of the multiplex assay and the RDB assay.Finally,confirm the final reaction condition and the sequence of the primers and oligonucleotide probes.When the primers were designed,made sure they had the similar Tm so that they could be amplified in the same condition.The oligonucleotide probes not only had the same Tm,but also had the similar mutation site in the middle of the probes.The first step,amplify the G6PD gene fragments containing the 7 mutations.The second step,the amplified products of the multiplex PCR were added to a screw-top tube with the strip and 10 ml of hybridization solution （2×SSC and 0.1%SDS）,then they were denatured at 100℃for 10 min and incubated at 43.5℃over night.The third step,the strips were collectively washed in a wash solution（0.5×SSC and 0.1%SDS） at 43.5℃for 10 min.After hybridization and washing,strips were incubated at room temperature for 15 min in 10 ml of 2×SSC,0.1%SDS containing 5μl of Streptavidin-POD conjugate.Excess conjugate was removed with two 5-min washes in the same buffer without the conjugate.The color was developed in the dark with 0.1 M sodium citrate,pH 5.0,0.1 mg/ml tetramethyl benzidine and 0.5μl/ml 3%H₂O₂ for 10 min.Positive result of detection should show a blue dot.For an individual to be tested,the heterozygous DNA gave the positive colored-spot for each of probe pair;homozygous or hemizygous for each of a mutant probe;compound heterozygous DNA for one in each of two mutant probes.To evaluate the specificity of this method,a total of 209 genomic DNA samples pre-identified by direct DNA sequencing with various G6PD point mutations were tested in a blind study.The results indicated full concordance between the sequencing analysis and reverse dot blot detection expect that two specimens with a genotype were not in the seven mutations.It was confirmed that this assay could be used to genotype the seven types of common G6PD mutations precisely.2.The study of relationship between the G6PD deficiency and neonatal hyperbilirubinemiaThe most devastating result of G6PD deficiency is neonatal hyperbilirubinemia which may lead to kernicterus.G6PD deficiency can lead to neonatal jaundice has been confirmed for a long time.It is believed that the hemolysis increase the bilirubin load of neonate.However,it is found that there is no evident hemolysis in the neonatal jaundice of the G6PD-deficient infants.So the researchers proposed a hypothesis that the hemolysis dued to the G6PD deficiency increases the bilirubin and the inability of the liver to adequately conjugate bilirubin was the principal cause of neonatal jaundice.This hypothesis is proved when the G6PD deficient infants also inherit the UDP-glucuronosyltransferase 1A1（UGT1A1） which involved with the bilirubin eonjugatation gene promoter polymorphism have a high incidence of neonatal hyperbilirubinemia.Therefore,we believe that the SNPs of the enzyme and protein involved in the bilirubin elimination may influence the function of the enzyme and protein and increase the incidence of neonatal hyperbilirubinemia in the G6PD deficient infants.The elimination pathway of the bilirubin are mainly separated by the three steps:Unconjugated bilirubin may be transported by organic anion transporter polypeptide C（OATP-C） from the blood circulation to the liver,and then it is conjugated with glucuronic acid in the endoplasmic reticulum through a catalytic reaction involving UDP-glucuronosyltransferase 1A1（UGT1A1）.The conjugated bilirubin is excreted into the bile via multidrug-resistance protein 2（MRP2）.In this study we selected the SNPs of UGT1A1 gene,OATP-C gene and MRP2 gene,which have been reported to influence the gene function or with incidence more than 5%in Chinese population.The-3279T＞G,（TA）₆TAA-（TA）₇TAA and 211G＞A of UGT1A1 gene,the-11187G＞A,388A＞G and 521T＞C of OATP-C gene,the-24C＞T and 1249G＞A of MRP2 gene were selected.We used DHPLC assay to genotype the eight SNPs,and used PHASE2.1.1 program analyze the haplotypes of the three genes. The case-control study was designed in our study,the case group is consisted of 150 G6PD deficiency infants,and the control group is consisted of 178 G6PD normal infants.The infants that had some risk factors influencing the serum bilirubin concentration should be excluded.The risk factors include premature birth,asphyxia, sepsis,HBsAg positive,occlusion of bile duct,ABO or Rh incompatibility, hemoglobin disease expect G6PD deficiency,dehydration,cephalohematoma,low birth weight,no record of serum bilirubin and more than 7 days admission after birth. The serum bilirubin was measured from the first day to the seventh day after birth, and set the highest as peak serum bilirubin.We used one-way analysis of variance to analyze the relationship between SNPs and haplotypes of three genes and the peak serum bilirubin,and found the SNPs or haplotypes which had a significant influence on the serum bilirubin.Our results showed that the serum bilirubin of G6PD deficiency group was significantly higher than that of G6PD normal group every SNP analysis.The result confirmed that the G6PD deficiency is definitely a risk factor of neonatal hyperbilirubinemia.In our analysis,the serum bilirubin concentration of UGT1A1 211GG was 235.12±89.18μmol/L,and the serum bilirubin concentration of UGT1A1 211GA/AA was 272.10±106.70μmol/L.The serum bilirubin concentration of UGT1A1 211GA/AA is higher than the one of UGT1A1 211GG（F=11.600,P=0.001）. The serum bilirubin concentration of OATP-C-11187GG was 238.76±93.04μmol/L, and the serum bilirubin concentration of OATP-C-11187GA/AA was 270.40±102.46μmol/L.The serum bilirubin concentration of OATP-C-11187GA/AA was higher than the one of OATP-C-11187GG（F=8.229,P=0.004）.The result showed that the UGT1A1 211G＞A and the OATP-C-11187G＞A were risk factors of neonatal hyperbilirubinemia.The other six SNPs had no significant influence on serum bilirubin concentration of neonate.In the analysis about the relationship between the haplotypes and the serum bilirubin concentration,the serum bilirubin concentration among the UGT1A1 diplotypes had the significant difference,so did the OATP-C diplotypes.The serum bilirubin concentration among the MRP2 diplotypes didn’t have the significant difference.We further found that the significant difference among the different diplotypes of UGT1A1 and the different diplotypes of OATP-C was the indirect reflection of the UGT1A1 211G＞A and OATP-C-11187G＞A.So the direct analysis on the relationship between the SNPs and the neonatal hyperbilirubinemia was clearer than the analysis on the diplotypes.According to our results,the G6PD deficiency,UGT1A1 211G＞A and OATP-C -11187G＞A were risk factors for the neonatal hyperbilirubinemia,and the risk factors have the cumulative effect.The more risk factors the neonates have,the higher incidence the neonates have the hyperbilirubinemia.The odds ratio of neonates with three risk factors is 7.233（95%CI:1.416-36.948） comparing to neonates with no risk factors.3.ConclusionsIn this study,we developed an assay for the simultaneous detection of seven Chinese common mutations based on the multiplex PCR and the reverse dot blot methods.The assay is accurate,cost-effective,and independent of expensive equipments or strict technical conditions,so this assay is especially helpful to the clinical diagnosis of G6PD deficiency in developing regions.In addition,our study analyzed the relationship between the G6PD deficiency and the neonatal hyperbilirubinemia.We identified that two SNPs of the bilirubin elimination pathway were closely related to the neonatal hyperbilirubinemia.Our study provided the valuable information on the neonatal jaundice of G6PD deficient and G6PD normal infants,made the neonate prone to jaundice get more concern and mostly decreased the incidence of neonatal kernicterus.In summary,our study analyzed the development of the Chinese G6PD mutations molecular diagnosis assay and the genetic background of the neonatal jaundice.According to our study,we hope the detailed schedule can be created. Through the schedule,the incidence of the neonatal hyperbilirubinemia can be predicted and the blindness of protection about the neonatal hyperbilirubinemia can be avoided in the clinical practice.The medical cost can be decreased dramatically and the doctor’s effort can be effective.Our study can also decrease the incidence of the devastating tragedy-kemicterus and improve the quality of the population born in our country.更多还原