Molecular Analysis Of Glucose-6-phosphate Dehydrogenase Deficiency And Its Role Of Hemolysis In Neonatal Hyperbilirubinemia Coincident With Variations In Hepatocyte Bilirubin Transport Factors

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.