APOE And MTHFR Gene Polymorphism Analysis In The Research Of Alzheimer Disease’s Genetic Susceptibility

Alzheimer’s disease (AD), a slowly progressive neurodegenerative disease, is the most common form of dementia, which pathological feature mainly manifest as neuritic plaques (NPs) and neurofibrillary tangles (NFTs). The appearance of these pathologic hallmarks in brain tissue are diagnostic standard, but it is hard to be detected.The nosogenesis of AD is unclear, there are some presumptions explaining AD’s progress. One presumption suggests Aβ which is generated from APP by β1-secretase and γ-secretase accumulate and sedimentate because of its unbalanced metabolism. As a result, neuritis plaques come into being and harm the structure and function of brain tissue, consequently cause AD. The other presumption suggests that the phosphorylation of tau protein is the main reasonAD, according to age, can be divided into two types, early-onset and late-onset. That AD incidence before the age of 65 is so-called early-onset, those after the age of 65 is late-onset. The prevalence of AD ranges from 5% to 10% before the age of 65, but doubled every five years after 65, and ranges from15% to 20% at the age of 80 to 90. Genetic predisposition exits in late-onset, and the most typical genetic risk factor is polymorphism of APOE gene. Besides, some factors also trigger AD, for instance, hyperhomocysteinemia is regarded as another significant risk factor. 5,10-methylenetetrahydrofolatefolate(MTHER), an important enzyme which can degrade HCY, catalyzes conversion from MTHFR to 5-methyletrahydrofolate irresistibly, and as a methyl donar,5-methyletrahydrofolate makes HCY convert to methionine with methionine synthase’s catalysis. MTHER activity decreasing leads to HCY level rising, so MTHFR gene has a indirect correlation with occuring of AD.APOE gene has three different alleles, ε2, ε3 and ε4. According to difference in the 112th and 158th nucleotide, these alleles compose 6 genotypes:ε2ε2 (112TT+158TT),ε2ε3(112TT+158T/C), ε2ε4 (112T/C+158T/C),ε3ε3 (112TT+158CC),ε3ε4 (112T/C+158CC) and ε4ε4 (112CC+158CC).ε4 allele is susceptibility gene of AD, ε4ε4 homozygote is more possible leading AD than heterozygote, and AD occurs in ε4 carriers earlier than ε4 non-carriers. However,ε2 gene is regarded as AD protective factor.The MTHFR gene has been mapped to chromosome 1p36.3, the length of code area is 1980bp, encoding the proteins which molecular weight is 74.6kDa. The length of cDNA sequence is 2.2 KB, which contain 10 introns and 11 exons. The length of introns and exons are 250bp-1.5kb and 102-432bp respectively. It has been found that there are more than 30 types of MTHFR gene mutation that changed the enzyme activity. MTHFR C677T mutation is the most common in the crowd, including three kinds of genotypes CC, CT and TT. After gene mutation, the activity of MTHFR decreased, the function of degradation HCY is reduced, therefore, which can lead to increased HCY level.Epidemiological investigation show that there are 35 million people suffering from dementia in the worldwide, more than 75% of which are AD patients at present. AD incidence double every 20 years, it is estimated that there will be 115 million people affected by AD till 2050. It is not only a great pain for patients and their families, but also a huge economic pressure for the whole society. AD have no effective diagnostic indicators at the moment. With the global aging process go faster, AD has become the heavy burden of the society. Therefore, it is particularly important to control risk factors, to intervene in high-risk population, and to delay the occurrence and development of AD. This research aim at study the relation of APOE gene, APOE level, HCY and MTHFR gene, and discusses these factors combining forecast the susceptivity of AD.This research divide into the following three chapters:Chapter one:The relationship of apolipoprotein E (APOE) allelic frequency and serum APOE levels in patients with Alzheimer’s disease.Objective To investigate the relationship of apolipoprotein E (APOE) allelic frequency and serum APOE levels in patients with Alzheimer’s disease (AD).Methods The cases and controls were collected in Guangzhou brain hospital during July 2014 to March 2015. AD group was diagnosed with CCMD-3, with 85 males and 115 females, the average age of which were 76±10. The controls have 70 males and 89 females, the average age of which were 73±10. All of subjects were han Chinese and had not taken lipid-lowing drugs.6～8ml morning fasting venous blood of subjects were acquired, of which 2～3ml venous blood anticoagulant with EDTA and saved at 4±1℃. DNA were extracted within three days. Of which 4～5ml venous blood were collected in vacuum drying tube and tested blood lipid on the same day. DNA microarray was used to detect the APOE genotypes of AD patients (n=200) and age-matched non-demented elderly control subjects (n=159). The accuracy of genotype results were monitored by DNA sequencing. Serum APOE levels was measured by Immunoturbidimetric assay at the same time. Analyze the APOE genotype distribution and the the relationship of apolipoprotein E (APOE) allelic frequency and serum APOE levels. Results In AD group the APOE genotype distribution were 3.0% for ε2ε3,1.5% for ε2ε4,49.0% for ε3ε3,43.5% for ε3ε4 and 3.0% for ε4ε4; the APOE allelic frequencies were 2.25% for ε2,72.25% for ε3, and 25.5% for ε4. While in control group the APOE genotype distribution were 0.6% for ε2ε2,13.8% for ε2ε3,1.3% for ε2ε4,69.8% for ε3ε3 and 14.5% for ε3ε4; the APOE allelic frequencies were 8.18% for ε2,83.96% for ε3, and 7.86% for ε4. The average serum concentrations of APOE were 33.33±10.90mg/L in AD patients and 41.25±11.35mg/L in controls, in dependent of gender and age. In all participants, The average serum levels of APOE were 50.86±6.21mg/L for ε2 carriers, 38.78±12.07mg/L for ε3 carriers and 30.47±7.68mg/L for ε4 carriers. Conclusion The ε2 allele is associated with a higher APOE concentration, whereas with ε3 the concentration is mediate and with ε4 the concentration is lowest (P<0.05). Serum concentrations of APOE show no significant difference between AD patients and the healthy crowd who were with the same genotype (P>0.05). The primary cause of lower serum APOE levels in AD patients is that the APOE ε4 allelic frequencies of them were higher than the healthy persons.Chapter two:Detecting the MTHFR gene polymorphism by DNA microarray technology in patients with different HCY levelsObjective To detect the 677th nucleotide mutation frequency of 5,10- methylenetetrahydrofolate reductase (MTHFR) gene in patients with different levels of homocysteine (HCY) and investigate correlation of MTHFR gene polymorphism and different HCY levels. Methods The cases were collected in Guangzhou brain hospital during February 2015 to September 2015.435 patients, with 363 males and 72 females, the average age of which were 48.92±16.52, whose HCY levels were higher than 15μmol/L were selected to be experimental group and were divided into four subgroups:15 μmol/L<HCY≤20 μmol/L for the first group,20 μmol/L< HCY≤5μmol/L for the second group,25μmol/L<HCY≤30 μmol/L for the third group and HCY>30μmol/L for the fourth group.229 persons whose HCY levels were less than 15μmol/L were selected to be controls, with 173 males and 56 females, the average age of which were 46.38±16.72, and had no difference with experimental group in gender, age and living environment.DNA microarray technology was used to detect the 677th nucleotide mutation of MTHFR gene in all experimental objects. MTHFR genotype and allele distribution of each group were analyzed. Results MTHFR C677T gene mutation was significantly associated with HCY level (χ2=180.16, P<0.0001). An increasing frequency of T allelic gene was associated with a significant rising of HCY levels(χ2=156.67, P<0.0001). However, there was no significant difference in MTHFR genotype and allele distribution between the first group and controls (χ2=0.031, P>0.05). Conclusion MTHFR C677T gene mutation is one of the important factors which makes HCY levels rise markedly and is a major factor for the crowds whose HCY levels are higher than 30μmol/L. The slight increase of HCY may be caused by other reasons, not caused by mutations of MTHFR gene.Chapter three:Multi-predictors combining forecast the susceptivity of Alzheimer’s diseaseObjective To examination the associations between several factors (APOE genotype, MTHFR genotype, APOE and HCY level) and Alzheimer’s disease(AD), and find easy and effective predictors to collaborative calculate the susceptibility of AD. Methods Several factors associated AD were analyzed to identify their influence such as APOE genotype, MTHFR genotype, serum APOE and HCY concentration. DNA microarray was used to detect both APOE and MTHFR genotypes of AD patients (n=200) and non-demented elderly (over 50 years old) control subjects (n=159). The accuracy of genotype results were verified by DNA sequencing. Serum APOE and HCY levels was measured by immunoturbidimetric assay and enzymatic method respectively. Binary logistic regression analysis was carried out for selecting promising predictors. Based on the logistic model, probabilities were gained and applied to establish Receiver-operating characteristic (ROC) curves.Results It was significant that APOE ε4 allele, MTHFR TT genotype, serum APOE level and serum HCY level show effect to AD (P<0.05).Area under the ROC curve (AUC) was 0.828 (P<0.001), Associated criterion was above 0.413. The sensitivity of ROC curve was 77.7%, and the specificity of it was 76.5%. Conclusion APOE ε4 allele, lower APOE level and higher HCY level were independent risk factor of AD, MTHFR genotype was APOE ε4 allele-dependent risk factor of AD. These factors simultaneously influence the occurrence of AD. In addition, we can make use of these multi-factors as predictors to forecast the susceptivity of AD.Summarization:1、In this study, the APOE level in persons with divers APOE genotypes was ε2>ε3>ε4, in accordance with most study.2、The APOE level had no significant difference in the crowd with the same APOE genotype, however, which had significant difference with different APOE genotypes. The APOE level was lower in AD patients than healthy persons.3、The mutation of MTHFR C677T was the important reason which lead to HCY levels increasing dramatically,however,it may be some other factors cause HCY level rising slight.4、From the logistics model,APOE ε4 allele, lower APOE level and higher HCY level were independent risk factor of AD, MTHFR genotype was APOE ε4 allele-dependent risk factor of AD. These factors simultaneously influence the occurrence of AD. In addition, we can make use of these multi-factors as predictors to forecast the susceptivity of AD.