| Part I Genetic analysis of glucose-6-phosphate dehydrogenase deficiency in male children of Guangzhou, ChinaGlucose-6-phosphate dehydrogenase is a kind of metabolism enzyme, which exists in human red blood cells and acts as a key enzyme in the pentose phosphate pathway. In the metabolic process, triphosphopyridine nucleotide(NADPH) protects red blood cells from the threat of oxidizing substances. Glucose-6-phosphate dehydrogenase(G6PD) deficiency[OMIM: +305900] is an incomplete X-linked, dominant enzyme deficiency, affecting more than 400 million people worldwide. It is the most common genetic blood disease, especially in tropical and subtropical regions. In China, it mainly distributes in southern region,especially in Guangdong, Guangxi, Hainan, Yunnan and other provinces. The clinical symptoms of G6 PD deficiency were diverse, since the G6 PD activity in male patient was significantly decreased, while the variation range of enzyme activity in female individual was characteristic different. Some female patient’s G6 PD activity could be decreased significantly,but some’s enzyme activity are normal. Hence, considering the uniformity of the changes in G6 PD activity of male hemizygous, our analysis focused on studying male patients.G6PD gene mutation is the main reason caused G6 PD deficiency. G6 PD gene is the house-keeping gene, locating in the long arm of X chromosome(Xq28), spanned over23182 bp, composed of containing 13 exons and 12 introns. Until 2016, there are 209 kinds of G6 PD deficiency mutations in the Human Gene Mutation Database. Among them, 191 cases of missense/ nonsense mutation, 1 case of regulatory mutation, 3 cases of splicing mutation,10 cases of small deletions, 2 cases of small indels and 2 cases of gross deletions were found.And mutations in the G6 PD gene could destabilize the enzyme and reduce its activity, which makes red blood cells be more vulnerable to damage from exogenous triggers, includinginduced foods, infections, and some drugs, which would lead to RBC lysis and acute haemolytic anaemia(AHA). Accordingly, a third of the neonatal jaundice was caused by G6 PD deficiency. Therefore, G6 PD deficiency is one of the major diseases affecting pediatric health. Considering different genotype induced different phenotype, it needs futher research to explore the ralationship among the G6 PD genotypes, gene expression and G6 PD activity.In China, genotypes of G6 PD Kaiping, G6 PD Canton, G6 PD Gaohe and G6 PD Viangchan are the most common mutations, and the former two accounted for the most important proportion. In this study, using reverse transcription(RT) quantitative real-time PCR(RT-q PCR), which is the most accurate and easy-to-perform technique, we established a G6 PD gene standard curve to quantify G6 PD transcription. Studying the correlation between genotype on G6 PD gene, gene expression and the enzyme activity by quantifying m RNA level enable us have an in-depth understanding of the pathogenic mechanisms underlying G6 PD deficiency. As the most common genotypes Canton(c.1376G>T) and Kaiping(c.1388G>A) are, we mainly focus on research these two mutations in our study. Furthermore,genotype of c.1311C>T has a high prevalence in Chinese, but there is still a dispute on whether it would induce G6 PD activity or not. In addition, the mutations of c.1311C>T,IVSXI+93 T and rs1050757 G exist a linkage disequilibrium. G6 PD c.1311C>T in exon 11 linked IVS+11 93T>C(a silent mutation) and rs1050757 G were found to be associated with in G6 PD deficiency in Asian populations. Our objective was to study whether the haplotype,c.1311C>T/IVS+1193T>C/rs1050757 G was associated with increased risk of G6 PD deficiency or not.[Objective]: We establish a G6 PD standard curve to study G6 PD expression. We investigated whether the glucose 6 phosphate dehydrogenase(G6PD) genotypes of Canton and Kaiping relate to their gene expression and result in different phenotypes. G6 PD c.1311C>T in exon 11 linked IVSXI+93T>C and rs1050757 G, a silent mutation, was associated with G6 PD deficiency in Asian populations. We aim to make clear whether the G6 PD c.1311C>T/IVS XI+93T>C/rs1050757 G would result in splicing abnormalities or not.[Methods]:We performed a genotypic screen for 203 children(180 males, 23 females)collected from Guangzhou, China by direct DNA sequencing of G6 PD gene. The c DNA were analyzed by real-time quantitative RT-PCR to measure the level of G6 PD transcription.Western blot was performed to verify the protein expression. The c DNAs from the subjects with haplotype c.1311C>T/IVSXI+93T>C/rs1050757 G and normal control were furtheranalyzed by direct c DNA sequencing to search for splicing aberration.[Results]: G6 PD enzyme activity has statistically significance(P < 0.05) in different genotypes between Canton and Kaiping. Gene expression has significance(P < 0.05) between normal and patient populations, but not among genotypes(P > 0.05). The Western blot results were consistent with the results. No splicing aberrations were observed in haplotype c.1311C>T/IVS XI+93T>C/rs1050757 G in the c DNA from the patients, although some of the pathogenic mutations were present in 8 out of 10 patients with this haplotype.[Conclusion]: The haplotype of c.1311C>T/IVS XI+93T>C/rs1050757 G on G6 PD gene are not pathogenic mutations. G6 PD gene transcription level was significantly lower in G6 PD deficiency patients than normal subjects. Additionally, clinical phenotype caused by G6 PD Canton is more severe than G6 PD Kaiping.Part II The role of G6 PD gene in unexplained abortionEarly spontaneous abortion is a kind of common pathological pregnancy, and the incidence of abortion is extremely high both in China and abroad. According to the clinical researches, chromosome disorder is the main reason caused abortion. Application of gene chip in clinical provides a quick way to diagnosis the chromosomal abnormality induced abortion; however, it still exist some abortion that we could not explain well.Glucose-6-phosphate dehydrogenase(G6PD) deficiency [OMIM: +305900] has been the most common human enzyme deficiency syndrome, affecting 4 million people worldwide,especially in tropical and subtropical regions. In China, Guangdong, Guangxi and other southern regions have the high incidence of G6 PD deficiency.As a key enzyme in the pentose phosphate pathway, G6 PD could change the G6 P to 6PG and generate NADPH, which is very essential to maintain the redox balance. Previous studies about G6 PD deficiency focus on the red blood cells, while more and more evidence show that G6 PD deficiency would affect the pathophysiology of nucleated cells as well, including regulating cell proliferation and senescence, enhancing the susceptibility of viral infection and damaging the development of embryo. Meanwhile, according to the previous reports, point mutaions account for the mainly G6 PD gene mutations, and large deletional mutations rarely occur. We are interested in whether the large deletional mutations are lethal mutations, further generating negative natural selection. In this study, we screened the fragment deletion and frame shift mutations of G6 PD gene in the fetus chorionic villus samples. We hope to find out the relationship between the G6 PD gene and the development in human early embryo; we also expect to explore some clues relative to clinical early abortion of unknown reasons.[Objective] : To discuss the relationship between G6 PD genotype and unexplainedabortion.[Methods]: DNA-PCR sequencing was performed on G6 PD gene in 56 cases of unexplained abortion villus and 30 cases of normal villus. Through the statistical analysis,we aim to know whether there exist a statistical significance between these two groups.[Results]: In the experimental group, 19 cases were found to be abnormal, including 11 cases of haplotype c.1311C>T/ IVSXI+93T>C, 6 cases of rs3216174, 1 case of SNP rs200111236, 1 case of SNP rs200729823. In addition, 1 case of c.1024C>T mutation was found. In control group, we only found 4 cases of haplotype c.1311C>T/ IVS XI+93T>C.Differences between the two groups have statistical significance(χ2=4.86, P<0.05).[Conclusion]:There are a higher frequence of SNPs in G6 PD gene in the group of unexplained abortion than in the normal. However, no shift code mutation or large fragment deletion can be found in our study. |
PDF Full Text Request