【作者】 梁卫华；

【导师】 邢福祺； 徐湘民； Grant Wu；

【作者基本信息】 第一军医大学 ， 人体解剖与组织胚胎学， 2005， 博士

【摘要】 葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症(glucose-6-phosphate dehydrogenase deficiency)是最常见的人类酶缺陷病之一,世界范围内约有4亿人受累。患者临床表现变化大,从无症状到新生儿黄疸、药物或感染造成的急性溶血、蚕豆病和重症慢性非球形细胞血溶性贫血,严重者导致新生儿期重症核黄疸,造成死亡或永久性神经系统的损伤。该病的及早诊断和预防是我国优生优育工作的一个重要组成部分,检测是否存在G6PD缺乏,给患者合理的忠告,避免使用可能引起溶血的药物,合适处理由此引起的新生儿高胆红素血症,是该病的重点。 G6PD缺乏症的分子基础是基因突变,G6PD基因位于Xq28,由13个外显子及12个内含子组成,全长20114bp,是一典型的看家基因。随着G6PD基因结构的阐明和分子检测技术的发展,不断发现新的突变类型,迄今世界上已报道140多种。G6PD基因突变具有以下特点:多为单个碱基置换的错义突变;大多数基因突变属于转化型突变,常发生在CpG二核苷酸内的C-T的转换;具有更多还原

【Abstract】 Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is one of the most common human enzyme deficiency in the world affecting an estimated 400 million people. It extends from the Mediterranean basin and parts of Africa, through the Middle East, the Indian subcontinent, Southeast Asia and parts of south America. Clinical expression is ranged wildly and most people are not anemic and asymptomatic.This defect also may cause neonatal jaundice,from mild hemolytic anemic to chronic nonspherocytic hemolytic anemia with attacks of severe anemia induced by infections,specific drugs or following consumption of fava beans.It can even lead to kernicterus.So it is important to have an accurate sensitive method to know if a deficiency is present.The gene encoding G6PD is on the long arm of the X-chromosome(band Xq28).It consists of 13 exons and spans approximately 20Kb encoding a protein of 515 amino acids.Over 140 mutations are characterized at DNA lever from different ethnic populations world wild.In China, at least 17 types of point mutations have been identified in the human G6PD gene. They are 95G, G392T, G487A. A493G. T517C, C519T, C592T, A835G, A835T, G871A, C1004T, C1024T, G1360T. G1376T. G1381A, C1387T and G1388A .The points underlined involve in the major G6PD-deficient mutations of the southern China account for more than 80% of all known mutations.In the past few decades, a panel of molecular approaches have been applied to identify and visualize G6PD-deficient mutations dependent on various designs for allele discrimination, such as, amplification refractory mutation system ARMS, single-strand conformation polymorphism (SSCP) followed by DNA sequencing, denaturing gradient gel electrophoresis (DGGE), PCR-based restriction enzyme method, and very recently reported DNA microarray and MALDI-TOF MS assay. Each of these approaches has its advantages and limitations, but the technical aspects and the cost of the operations have not made them routine in most laboratories.Rapid and accurate genotyping of G6PD deficiency mutations is badly needed in meeting the requirements for genetic counseling, survey in genetic epidemiology, and clinical molecular diagnosis of this disorder.As a useful tool for mutational analysis, denaturing high performance liquid chromatography (DHPLC) assay has been extensively applied to scan unknown mutations in relative large genes using heteroduplex analysis strategy or detect known mutations in small genes or in mutational ’hot spots’ using multiplex primer extension (PE)/ fully-denaturing DHPLC strategy. We established optimal conditions for simultaneous detection of the above ten G6PD-deficiency mutations and C1311T polymorphism, which are the most frequent alleles in Southern China. The mutant and wild-type alleles are distinguished as the separation of oligonucleotides by both size and sequence dependent under DHPLC fully-denaturing conditions.Chapter 1 Establishment and Evaluation of PE/DHPLC for detection of G6PD gene mutations.1.1 ExperimentalThe eleven mutations tested were distributed in six exons in the G6PD gene, amplified in three PCR amplicons and divided into two groups of multiplex PE-based detections that can simultaneously detect six mutations (95G, G392T, C592T,C1311T, G1376T, G1388A) or five mutations (G487A, A493G, G871A, C1024T, G1360T).To evaluate the specificity and sensitivity of PE/DHPLC for detection of G6PD gene mutations, we used conventional DNA sequencing to validate the present method in a blind analysis. The DNA sequencing results were unknown until the genotypes obtained by PE/DHPLC were scored. A total of 209 blood samples unrelated from Liuzhou and Guangzhou subjects of both sexes were recruited to test the specificity of this assay by blind analysis. Blood samples were diagnosed to G6PD-deficiency using standard method for measurement of G6PD/6PGD ratio.Human genomic DNA was isolated from leukocytes in peripheral blood using standard DNA extraction methods. Eleven DNA samples with different known genotypes that included the above eleven G6PD-deficient mutations or polymorphism were used to validate this application. Each of these known samples was previously characterized by direct sequencing.1.2 Results195 of the 209 samples tested were found mutations by DHPLC with 99.5%(194/195) concordance to the DNA sequencing data except for one case having an additional false positive (genotype of 1376/N) instead of 1376/1388. With this assay, we can accurately define the genotypes of G6PD-deficient mutant or normal alleles from hemizygote in deficient males and from normal males, both female heterozygotes and mutant homozygotes even although the latter states are rarely detected in population.Chapter 2 Genetic assays to 209 G6PD deficiency from Guangzhou and Liuzhou rigions.2.1 Experimental209 G6PD deficiency from Guangzhou and Liuzhou rigions diagnosed by G6PD/6PGD ration were defined genotype by PE/DHPLC .The unknown samples by PE/DHPLC assay were defined by DNA sequencing to all the encoding exons in order to find out novel mutations.2.2 ResultsThe 195 positive samples included all of the 10 mutations and 1 polymorphism . Among these mutations, 1388, 95 and 1376 were most commonly found in the populations ,which were 79.17%.There were no different between males and females. 30 genotypes was reported for the first time,including 115 hemizygotes(12 genotypes), 47 heterozygotes(7 genotypes) and 33 homozygotes(ll genotypes) of G6PD deficiency with eleven different known mutations.14 unknown samples was characterized by DNA sequence analysis for all exons in which we found 4 cases of A202G (a synonymous mutation) and 2 cases of A835T (a missense mutation). The substitution of C1311T existed in exon 11 has already been described as a silent mutation worldwide. Since this variation has an association with many G6PD-deficient variants, it may be clinically relevant. To investigate the relationship between complex C1311T mutation in exon 11 and T93C in intron 11 of G6PD gene,we used DNA sequencing to identify the 16 samples of C1311T by PE/DHPLC.The result showed they were all 1311 mutation together with 93 mutation.This complex mutation may be the cause of enzyme reduced activity of G6PD.Chapter 3 Phenotype-genotype correlation in G6PD deficiency from analysis on 105 Chinese patients3.1 ExperimentalTo investigate the genotype distribution of G6PD deficiency in Iiuzhou region of southern China and if measuring the G6PD/6PGD ratio is associated with different genotypes of G6PD-deficient individuals. 105 unrelated samples from Liuzhou subjects of both sexes were recruited to this study, who were all detected as the positive tests with G6PD deficiency by measurement of G6PD /6PGD ratio and followed by genotyping of these positive subjects using PE/DHPLC assay for detecting the eleven G6PD-deficient mutations and the polymorphism of the Southeast Asian countries. The subjects with unknown mutations were identified by DNA sequence analysis.3.2 ResultsNinety-eight subjects (93.3%) were detected by DNA analysis and six known G6PD-deficient mutations and one silent polymorphism, with total of seventeen G6PD-deficient genotypes have been characterized from 105 G6PD subjects, including the six genotypes in 57 hemizygotes from males, the four genotypes in 14 heterozygotes, the two genotypes in 6 homozygotes and the five genotypes in 14 compound heterozygotes from females, respectively. Statistic analysis shows that there were significant correlations between the G6PD/6PGD ratio parameters and different G6PD-deficient genotypes consisted in three groups of hemizygotes, heterozygotes, and homozygotes when the three groups were analyzed separately, in addition to a significant difference existed between normal control group and the G6PD-deficient group. So It has been concluded G6PD/6PGD values is a valuable parameter for predicting different genotypes of G6PD deficiency in hemizygous, heterozygous, and homozygous individuals.Chapter 4 Prevalence survey and molecular characterization of G6PD in Liuzhou city4.1 ExperimentalTo investigate the gene frequencies and mutation patterns of G6PD deficiency in Liuzhou city, we used cluster sampling method. A total of 2351 of healthy young people when receiving pre-marriage consultation were recruited for G6PD deficiency prevalence survey. Those samples with MHb<75% and G6PD/6PGD<1.5 were identified genotype by PE/DHPLC.A11 the samples were tested for the state of earring G6PD alleles.4.2 Results168 of samples were defined to be G6PD deficiency by G6PD/6PGD ratio with a detection rate 7.15%. 152 samples were charactered the genotype by PE/DHPLC with 6.47%.8 genotypes were found including 6 mutations and 1 polymorphism of eleven tested mutations.6 well-known types of G6PD were detected with gene contributions of 33.55%(1388),30.92%(1376),19.08%(95),7.24%(1311), 6.58%(1024), 1.32%(392, 871). Conclusionsl.The present method is a semi-automatic, fast, accurate and straightforward assay that allows detection of the major Chinese G6PD-deficient mutations. Our results,along with the relative low reagent costs (approximately $5.0/genotype) and short processing time(one day for 65 sampled testing for 11 mutations),suggest a potential use of this technology as a quality control applied in genetic service center for screening programs.2.We analyze the general gene mutations and genotypes of 209 samples from Guangzhou and Liuzhou regions. 11 mutations, 1 polymorphism and 1 synonymous mutation were defined.30 genotypes were defined and almost of them were reportedfor the first time.3. All 1311 mutation is together with 93 mutation.This complex mutation may be the cause of enzyme reduced activity of G6PD.4. G6PD/6PGD values is a valuable parameter for predicting different genotypes of G6PD deficiency in hemizygous, heterozygous, and homozygous individuals.5.Through PE/DHPLC, we get gene frequencies and mutation spectrum in Liuzhou. Date from this prevalence survey is useful for genetic cousultation and diagnosis of this disease.更多还原