Rapid Genotyping Of Apolipoprotein E By Denaturing High-performance Liquid Chromatography

Background and ObjectivesHuman apolipoprotein E (apoE, protein; APOE, gene) is a plasma glycoproteinwith a single 299 amino acids polypeptide chain. ApoE has three major isomers of theprotein: apoE2, apoE3, and apoE4. They differ from each other by cysteine-arginineinterchanges at amino acid residues 112(a T/C substitution in nucleotide position17680159, GenBank Accession NT₀11109.15) and 158(a C/T substitution innucleotide position 17680297, GenBank Accession NT₀11109.15). The biosynthesisof each isomer is under the control of three independent co-dominant alleles,ε2,ε3,andε4, located at exon4 of APOE gene, chromosome 19q13.2. It served intransporting cholesterol, triglycerides and phospholipids. The most common allele,ε3,reflects the presence of a cysteine at codon 112 and an arginine at codon 158 and it ispresent in exceed 70% of populations. Theε4 variant determines the substitution ofarginine for cysteine at codon 112 and is present in approximately 10% ofpopulations. The third allele,ε2, codes for cysteines at both loci and is found in lessthan 10% of populations. It has six common genotypes, consisting of threehomozygote (ε2ε2,ε3ε3, andε4ε4) and three heterozygote patterns (ε2ε3,ε3ε4, andε2ε4), depending on the inheritance of any two alleles.APOE gene is a major determinant of variation in plasma lipid and lipoproteinlevels. The APOE genotypes play a key role in the diagnosis of typeâ…¢hyperlipidemia and have also shown to be an important risk factor in the developmentof Alzheimer's and cardiovascular diseases and one of potential targets of associated study on successful aging and personalized therapeutic response. Genotyping of thesetwo known polymorphisms of APOE gene has been previously performed by severaltechniques: restriction fragment length polymorphism (RFLP)-based analysis,single-strand conformation polymorphism (SSCP) analysis, allele-specificoligonucleotide (ASO) blot, amplification refractory mutation system (ARMS),Capillary electrophoretic detection, Real-time multiplex PCR assay, etc. A rapidgenotyping test with high accuracy and reproducibility suitable for the determinationof APOE polymorphisms is needed for population survey and prediction diagnosis ofrelevant disease in both research and clinical setting. The DHPLC technique hasmany advantages when compared with these and other genotyping methods: it is fast,accurate, and cheap. This technique has allowed rapid and accurate genotyping ofvarious mutations/polymorphisms applied to screen large-scale genomic samples. Wehave developed a partially-denaturing DHPLC assay capable of simultaneouslydetermining the two polymorphic sites in APOE gene.Design and method1. A polymerase chain reaction (PCR) was designed to generate sequencescontaining the polymorphic site of interest in the APOE genes. Control samples withdifferent kind of genotype of APOE can be gotten from DNA sequencing.2. The analyze condition can be optimized through the known samples identified bysequencing. The PCR amplicons for each sample were subjected to denaturinghigh-performance liquid chromatography (DHPLC), analysis performed underpartially denaturing conditions as determined by peak pattern and position. We mixedthe PCR amplicons of the samples which can not be discriminate from others withstandard control in 9:1 mole ratio, and analyze the mixture under partially denaturingconditions. The six different genotypes of APOE could be easily determinedsimultaneously by DHPLC profiles.3. To validate the accuracy of the method, a number of samples from each of thegenotype groups detected by DHPLC profiling were selected to perform DNAsequencing. To validate the stability of the method, the retention times for APOE inthree different weeks were compared by ANOVA test and the coefficient of variation (CV) values were analyzed. The gene and genotypic frequency of Han people inGuangzhou city should be analyzed and the Hardy-Weinberg equilibrium wasidentified by chi-square statistic.Result and discussionThe six different genotypes ofε2ε2,ε3ε3,ε4ε4,ε2ε3,ε3ε4 andε2ε4 for APOEcould be unequivocally determined by easily interpreted DHPLC profiles.1. Atotal of 297 genomic DNA samples were tested to validate this assay by blindanalysis. Direct DNA sequencing was performed on samples randomly selected fromeach of the genotype groups detected by DHPLC profiling. The results indicate 100%concordance between the sequencing analysis and the DHPLC detection. Theprecision of DHPLC genotyping was assessed by comparing the retention times andcalculating the coefficients of variation (CV). The coefficient of variation (CV)values ranged from 0.97% to 1.05%, thus further confirming the stability of thisDHPLC method.2. The new PCR/DHPLC assay can rapidly screen two mutations within 10min at acost less than 5 RMBï¿¥. Compared with traditional manual methodologies, theefficiency of our method has been increased and the cost has been reduced.3. The result of the chi-square test showed that the population we selected was inHardy-Weinberg equilibrium. The gene frequency of APOE confirmed the reportpreviously, which also identified the accuracy of the method.From the result we can see the present PCR/DHPLC method is a semi-automated,fast, and straightforward assay that allows accurate detection of the twoAPOE-related polymorphisms simultaneously. It provides a routine tool that can beused in genetic counseling, epidemiology and the investigation about the associationbetween these enzyme polymorphisms and other APOE-related problems.