KR101933847B1 - APOE promoter SNP associated with the risk of Alzheimer's disease and the use thereof - Google Patents

Abstract

The present invention relates to single nucleotide polymorphisms (SNPs) that can be used to predict the risk of Alzheimer ' s disease. The SNP is rs405509, located on chromosome 19 at 44905579, based on the human gene map version of GRCh38.p7. The present invention suggests that the T allele of rs405509 plays a role in further increasing the risk to the risk of dementia of APOE E4 / E4. In addition, the comparison of cerebral cortical thickness and hippocampus volume between APOE genotypes revealed that E4 / E4 shrinkage was greater than E3 / E3 in Asian population. In addition, it has been found that in the Cocarsus with the E4 / E4 genotype, the cerebral cortex of the rs405509 T / T genotype is more markedly attenuated. Thus, the present invention could be used to diagnose or predict the risk of Alzheimer ' s disease and / or Alzheimer ' s disease by identifying the rs405509 T / T mutation with APOE E4 / E4.

Description

APOE promoter single base mutation associated with Alzheimer's risk and its use APOE promoter SNP associated with the risk of Alzheimer's disease and the use thereof [

The present invention relates to early detection of Alzheimer's disease and single base mutation for predicting the risk of Alzheimer's disease. The present invention also relates to an early diagnosis of Alzheimer's disease using a single base mutation and a method for predicting the risk of Alzheimer's disease.

Alzheimer's disease is a progressive neurodegenerative disease characterized by declining cognitive ability and senile plaques in the brain. It is the most common cause of dementia in older people, accounting for 60-80% of dementia (Barnes and Yaffe, 2011). It is estimated that approximately 13% of elderly people aged 65 and over and about 45% of elderly people aged 85 or older are Alzheimer's patients. Alzheimer's disease (AD) is a complex neurodegenerative disease that causes structural changes in the brain as well as memory loss and other cognitive disorders (Ballard et al., 2011; Forero et al., 2006). AD patients exhibit increased amyloid plaques, reduced hippocampal volume in the posterior cingulate cortex (PCC), the medial prefrontal cortex, and the hippocampus of the brain. Patients also show decreased levels of beta-amyloid and increased levels of tau and p-tau in CSF (Jack, 2012; Trojanowski et al., 2010).

Alzheimer's dementia is under development, but there is no clear treatment until now, and there is a high need for early diagnosis and early prediction. Alzheimer's dementia diagnostic techniques include neuropsychological tests, MRI brain imaging tests, clinical diagnosis by specialist findings, and pathologic examination with cerebrospinal fluid and florbetaben-based amyloid-PET. Clinical diagnosis can diagnose the onset of dementia or mild cognitive impairment, but it may be difficult to distinguish it from other brain diseases, and it is not suitable for the purpose of early diagnosis because it can be diagnosed only after the onset of symptoms. In the case of cerebrospinal fluid examinations, quantitative analysis such as beta amyloid protein and tau protein analysis is performed and the testee's rejection level is very high due to reliable diagnosis of dementia scale and invasive cerebrospinal fluid collection. Pathologic testing with amyloid-PET is more reliable but costly. In the case of MRI brain imaging, cerebral cortical atrophy, hippocampal atrophy, and brain damage associated with dementia are identified and techniques for early diagnosis are under development, but the diagnosis is not known to be early. In addition, the development of diagnostic technology for dementia through blood is actively under way, but there is a limit to the size and accuracy of the experimental group.

In genetic factors, the amyloid precursor protein, presenilin-1 and presenilin-2, are the primary factors for the early onset of Alzheimer's disease due to family history (Blennow et al In the APOE, the e4 allele is the strongest risk factor for the onset of sporadic Alzheimer's disease (Bu, 2009; Corder et al., 1993; Huang and Mucke, 2012). The APOE gene has three polymorphisms, e2, e3 and e4 globally with frequencies of 8.4%, 77.9%, and 13.7%, respectively (Farrer et al., 1997). The frequency of e4 in Alzheimer's disease dramatically increases to ~ 40% (Farrer et al., 1997). APOE is located in the long arm of chromosome 19 (19 q13.2) and has three alleles e2, e3 and e4 formed due to the change of amino acid 112 (Cys / Arg) and 158 (Arg / Cys) There are 6 genotypes (E2 / E2, E2 / E3, E3 / E3, E2 / E4, E3 / E4, E4 / E4) polymorphism by combination. Of these, APOE e4 is usually found in more than 50% of AD patients, but only in less than 15% of controls with normal cognitive ability (Ward et al., 2012). Previous studies have reported that APOE e4 can delay the onset of AD 5 to 15 years (Corder et al., 1993; Gomez-Tortosa et al., 2007). In addition, although the effect of APOE e4 on cognitive decline has been reported, some reported decreased cognitive performance by APOE e4, while others reported no effect on cognitive performance, (Jones et al., 2002), which suggests that cognitive decline is slowed by e4 (Anstey and Christensen, 2000; Beaudreau et al., 2013; Caselli et al., 2009; Craft et al. , 2007; Kleiman et al., 2006; Mayeux et al., 2001; Van Gerven et al., 2012). Despite the consequences of this discussion, healthy individuals with APOE e4 homozygotes showed decreased hippocampus volumes, whereas heterozygotes with e4 showed differences from subjects who did not have e4 in the healthy elderly group (Crivello et al., 2010; Farlow et al., 2004; Lemaitre et al., 2005). APOE e4 has also been shown to be involved in the conversion of healthy elderly and mild cognitive impairment to Alzheimer's disease (Wang et al., 2011). The risk of e4 alleles was reported to differ between ethnic groups (Brainerd et al., 2013; Farrer et al., 1997; Heun et al., 2010; Hsiung and Sadovnick, 2007; Hsiung et al , 2004; Rose, 2005). But. The prediction based on APOE e4 can be explained only within 20% of the genetic impact on dementia. As such, the risk of e4-mediated and possible inducers of Alzheimer's disease remains unclear. On the other hand, Korean Patent Registration No. 10-1335021 describes the association of T / G heterozygosity of APOE rs405509 with patients with Alzheimer's disease or mild cognitive impairment.

Thus, the present inventors have studied genetic mutations that may affect the risk of dementia of APOE E4 as well as APOE E4 genetic mutations around the APOE gene. In addition, the APOE E4 / E4 homozygote was identified by ethnic differences in the risk of dementia, and analyzed to analyze the cause. As a result, it has been surprisingly confirmed that the single base mutation located in the APOE promoter not only explains the risk difference of the APOE E4 / E4 homozygotes by race, but also shows the difference in cortical thickness according to alleles or alleles Respectively. The present inventors also confirmed the effect of APOE polymorphism on the brain structure by analyzing the cerebral cortex thickness and hippocampus volume.

The present invention provides a single nucleotide polymorphism (SNP) genetic variation for predicting the risk of dementia caused by Alzheimer's disease and / or Alzheimer's disease.

Another object of the present invention is a method of detecting a single nucleotide polymorphism (SNP) genetic variation to predict the risk of dementia by Alzheimer ' s disease and / or Alzheimer ' s disease.

This incidence provides single nucleotide polymorphism (SNP) that can be used to predict the risk of dementia due to Alzheimer ' s disease and / or Alzheimer ' s disease. More specifically, the SNP is rs405509, which is located on chromosome 19 at 44905579 on the basis of the human gene map GRCh38.p7 version, and its alleles are T and G. There was a difference in minor allele frequency (MAF) between races,

In one embodiment of the present invention, the present invention provides a method of isolating and purifying genomic DNA from a sample isolated from a subject, such as a cell, tissue, blood, or body fluid, analyzing the rs405509 single base mutation polymorphism and APOE genotype of the APOE promoter And identify and predict the risk of dementia.

In another embodiment of the present invention, there is provided a method of providing information about predicting Alzheimer's dementia using APOE epsilon genetic variation (rs429358 and rs7412) and rs405509 genetic variation.

In another embodiment of the present invention, genetic mutations indicative of a mild cognitive impairment or Alzheimer's disease (AD) in a sample separated from a subject comprising a nucleic acid are detected to diagnose a mild cognitive impairment or Alzheimer's disease, (A) contacting the sample with a reagent capable of detecting the presence or absence of the genetic mutation in a gene selected from a gene encoding the APOE E4 allele or a gene product thereof; And (b) contacting the sample with a reagent capable of detecting the presence or absence of the genetic mutation selected from a gene encoding a rs405509 T allele of the APOE promoter or a gene product thereof, wherein the APOE E4 / E4 and rs405509 T / T genetic mutations are indicative of the subject having a mild cognitive impairment or Alzheimer's disease, a mild disorder or a high risk for Alzheimer ' s disease Or to predict the risk for these diseases. Here, the method for predicting the risk may be by amplifying a nucleic acid isolated from the subject, and the nucleic acid may be genomic DNA or RNA.

Another embodiment of the present invention further includes obtaining an image of a subject's cerebral cortical thickness, for example, a magnetic resonance (MR) brain image, wherein the atrophy of the cerebral cortex indicates that the subject has a longitude Cognitive disorder or Alzheimer's disease, a disorder that is mild, or a high risk for Alzheimer ' s disease.

Yet another embodiment of the present invention may further comprise performing one or more of the above methods in a method comprising a neuropsychological test, a CSF test and an amyloid-PET test.

In one embodiment of the present invention, a method for detecting the presence or absence of said genetic mutation comprises: obtaining a nucleic acid in a single stranded form in a sample separated from said subject, forming a complex with a complementary primer set, Is the method of analyzing the presence or absence of the mutation. The presence or absence of genetic mutations in the complex may be detected by polymerase chain reaction, nuclease digestion, hybridization, Southern blotting, restriction enzyme fragment polymorphism ), Sequencing, primer extension, single-stranded conformation polymorphism, or the methods described above.

Another embodiment of the present invention is directed to a method of screening for the treatment of mild cognitive impairment or Alzheimer ' s disease, which is capable of detecting the presence or absence of said genetic mutation in a gene selected from a gene encoding the APOE E4 allele or a gene product thereof, And may further comprise a gene encoding the rs405509 T allele of the APOE promoter or a gene product thereof. Thus, in one embodiment of the present invention, the kit can be a kit comprising a primer set and a reaction enzyme complementary to the APOE single nucleotide polymorphism APOE E4 and the APOE promoter single nucleotide polymorphism rs405509 T / T, T / G and GG have.

The gene product in the present invention is understood to include RNA, such as tRNA, mRNA, rRNA, miRNA and siRNA, polypeptides and proteins as a result of gene expression.

In another embodiment of the present invention, the kit includes an assay method for analyzing genetic variation, a method for amplifying a nucleic acid, or a kit including a reference control standard.

In another embodiment of the present invention, there is provided a complementary primer set capable of detecting the presence or absence of said genetic mutation in a gene selected from a gene encoding APOE E4 allele or a gene product thereof, wherein the APOE promoter A gene encoding the rs405509 T allele or a gene product thereof may be provided. Thus, in one embodiment of the invention, the primer set may be a primer set complementary to APOE single nucleotide polymorphism APOE E4 and APOE promoter single nucleotide polymorphism rs405509 T / T, T / G and GG.

In another embodiment of the present invention, the primer set can be a label, for example, a radioisotope or a set of primers attached with a fluorophore or chemical derivative.

According to the present invention, the risk of Alzheimer's dementia can be more accurately diagnosed by examining the genetic variation of rs405509 in addition to the APOE genotype, particularly APOE E4 / E4, as a predictive genetic variation in sporadic Alzheimer's dementia.

Figure 1 shows a strategy for screening polymorphic candidates to modulate the risk of APOE e4 / 4 (Figure 1A). Figure 1B shows a schematic representation of the APOE gene structure. SNPs of rs449647 (-491 A / T), rs405509 (-219 T / G), rs440446 (+113 G / C), rs429358 and rs7412 were shown in panel B. Figure 1C shows the distribution of genotype frequencies for rs405509, rs449647, rs440446 for whole case-control subjects or e4 homozygotes from East Asian, Corsican and African descent. Here, the frequency of the genotypes was calculated as an average frequency in each case and a control group.
Figure 2 is a map showing cortical thickness reduction in East Asian and Caucasians (Figure 2A). We applied a general linear model and estimated the point-wise cortical thickness difference using APOE e4 / 4 and e3 / 3 as fixed factors and using age, sex and field strength as covariates. The numbers represent statistical differences. ( p < 0.05). The black circles represent brain atrophy in the medial olfactory and hippocampal regions, and the red circles represent brain atrophies in the anterior region (Fig. 2A). The mean cortical thicknesses in the medial temporal cortex (medial olfactory and hippocampal side regions) (Fig. 2B) and sprouting (Fig. 2C) were compared according to APOE genotypes in East Asian and Caucasian. Data were normalized to e3 / 3 and expressed as% with error bars with 95% confidence intervals. The bar graph compares the hippocampal volumes between the APOE genotypes in East Asian and Caucasian populations (Fig. 2D).
Figure 3 shows that the rs405509 T-allele decreases APOE protein expression. Human serum is Western blotted with anti-APOE and anti-TF (transferrin) to investigate serum expression of rs405509-dependent APOE protein (Figure 3A). Serum samples are selected for the rs405509 (G / G, G / T, and T / T) genotypes in the APOE e3 / 3 homozygotes. The bar graph shows the relative intensity of APOE expression (Figure 3B). The GG genotype was used as a reference. Transferrin (TF) was used as a normalized control. Data are mean ± SEM. (* p < 0.05, *** p < 0.001).
Figure 4 shows the susceptibility of Asians to the onset of APOE e4 / 4-mediated Alzheimer &apos; s disease. The ozone ratio of APOE e4 / 4 with reference to e3 / 3 for Alzheimer's disease was compared between different ethnic groups. The bar graph represents the odds ratio of e4 / 4 to Alzheimer's disease (Figure 4A) and neuropathologically confirmed series (Figure B) based on clinical diagnosis. * Data set was analyzed by logistic syllabus, and † is cited from previous research results.

In the present invention, we have analyzed APOE e4-mediated AD susceptibility to other racial groups through large scale studies. As a result, e4 homozygotes were more vulnerable to Alzheimer's disease in East Asian populations compared to Caucasian and African descent. In addition, the present inventors confirmed such racial differences by a neuropathologically related study on patients diagnosed with Alzheimer &apos; s disease and controls. A small number of studies have reported APOE e4 allele distribution in different populations (Farrer et al., 1997; Singh et al., 2006). The high frequency of the e4 allele is in Africa, Europe and Asia order, which is contrary to the risk sequence of e4 for Alzheimer's disease.

It has been reported that APOE e4 promotes the reduction of cortical thickness within the innervated cortex, parahippocampal cortex, and precuneus (Donix et al., 2010; Foley et al Thompson et al., 2011). Another study of subjects with normal cognitive abilities showed that individuals with e4 also had reduced cortical thickness (Fouquet et al., 2014). In addition, individuals with the e4 allele exhibited severe atrophy and memory impairment in the hippocampus, which plays an important role in memory function (Alexopoulos et al., 2011). In accordance with the present invention, the present inventors have found that the APOE e4 homozygotes exhibit a significant cortex in the inner olfactory cortex, the hippocampal cortex and the laryngeal region, as compared with the APOE e3 homozygotes in both East Asian and Caucasians (Foley et al., 2010; Thompson et al., 2011), which is associated with decreased thickness and hippocampal atrophy (Donix et al. Surprisingly, these cortical and hippocampal atrophies observed in e4 / 4 subjects were found to be more severe than in Caucasians in East Asia. The present invention suggests that East Asian people with APOE e4 / 4 have an increased risk of Alzheimer's disease as well as decreased cortical thickness and hippocampal atrophy in the brain, as compared to Caucasians.

We hypothesized that differences in e4 / 4-mediated AD risk among population groups would be due to ethnic differences in the genetic background, particularly polymorphism around the APOE gene. APOE e4 is not the only APOE polymorphism associated with Alzheimer's disease. It has been reported that polymorphisms within the APOE promoter and intron regions regulate the effect of APOE e4 on AD outbreaks (Bertram et al., 2007; Lambert et al., 2002; Lambert et al., 1998b; Lescai et al. 2011). Two SNPs (rs449647 and rs405509) in the promoter and one SNP in the intron (rs440446) were evaluated to modulate the effect of APOE epsilon mutations on Alzheimer's disease. Lambert et al., 1998a; Lambert et al., 1998b; Limon-Sztencel et al., 1998), and the -491AA and 219TT genotypes have been reported to increase AD risk independently of the APOE epsilon genotype (Lambert et al., 2002; , 2016; Wang et al., 2000). It has also been reported that rs405509 has a synergistic effect with APOE e4 in influencing cognition (Ma et al., 2016). The present inventors have analyzed that these SNPs exhibit different allelic frequencies in different ethnic populations. In the case of rs405509 among these SNPs, East Asian people, including Koreans and Japanese, have a higher frequency of the risk allele rs405509-T in both the whole population and the e4 isozyme-conjugated group compared to the Caucasian and African descent . This supports the fact that the e4 homozygotes have a high risk for Alzheimer's disease in East Asian populations compared to other ethnic groups. Likewise, Caucasians have a higher allele frequency for rs405509-T compared to African descent, and Caucasians have also been found to have higher e4 / 4 homozygote odds ratios compared to African descent .

Several neuroimaging studies have reported the effects of APOE e4 homozygotes at brain atrophy, but the effects of APOE promoter polymorphism have not yet been elucidated. Previous studies have reported the effect of the rs405509 polymorphism in the APOE promoter region on the human brain in the vascular aging process in the Chinese population. It was found that individuals with rs405509-TT with APOE e4 exhibit age-dependent cessation of cortical thickness compared to individuals with the rs405509 G-allele (Chen et al., 2015a). We have found that individuals of the rs405509-TT genotype are highly associated with Alzheimer &apos; s disease and result in a stronger atrophy than cortical thickness and hippocampal volume compared to individuals with the G-allele among e4 homozygotes. In particular, this cortical thickness reduction pattern was observed in the medial temporal cortex (the olfactory and hippocampal side) and the precuneus, while the atrophy in the hippocampal region was observed in previous studies (Chen et al., 2015a ). The present invention confirmed severe hypocellular thickness reduction and hippocampal atrophy in the olfactory cortex, hippocampal cortex and laryngeal region in the individuals with the APOE e4 homozygote and rs405509-TT. In the present invention, the inventors have confirmed that the racial differences in Alzheimer's disease and brain atrophy e4 / 4-mediated risk are due to the racial diversity of the rs405509 polymorphism.

In addition, the present inventors have demonstrated that rs405509-TT induces reduced APOE expression in human brain and serum. In an analysis of reporter genes using allelic substitution at the rs405509 T or G allele, we demonstrated that the promoter was less expressed in the APOE gene with the rs405509-T allele, which reduced the risk including rs405509-T RTI ID = 0.0 &gt; APOE &lt; / RTI &gt;

Thus, the present inventors have found that the APOE e4 homozygotes in East Asia are more vulnerable to Alzheimer's disease than Caucasian and African descent. The different allele frequencies in racial group APOE promoter polymorphism will make individuals with APOE e4 differentially susceptible to the onset of Alzheimer's disease. Taking into account the relevance of rs405509 to the modulation of APOE protein levels, we show that reduced APOE levels in e4 APOE individuals with rs405509-TT induce an increased risk for Alzheimer's disease.

Hereinafter, the present invention will be described based on examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Materials and methods

(1) Samples and data

In the present invention, full-length genome information, MRI brain imaging information, and clinical diagnosis information were utilized. (Mean ± SD), mean age 71.65 ± 5.65 (mean ± SD) of patients with Alzheimer 's disease, mean age of the normal person was 73.45 ± 5.30 (62.4% of women, 61.5% of women in Alzheimer's disease), and obtained brain image information through MRI scans (3T MRI, Skyra, Siemems) of each subject. Each subject was subjected to neuropsychological tests (K-MMSE and Seoul Neuropsychological screening battery) necessary for the diagnosis of dementia, and as a result, normal, mild cognitive impairment or diagnosis of dementia was confirmed by a dementia clinic specialist. 1145 were normal and 930 were demented. We also used a database of the National Reserch Center for Dementia (NRCD) in Gwangju, Korea. All experimental and study protocols of the present invention were approved by the Board of Directors of Chosun University Hospital. All volunteers and their relatives submitted a written consent before participating in the experiment. East Asian subjects consisted of 2,309 subjects with normal cognitive ability and 1,886 subjects with AD. The genomes of the Caucasians were obtained from the ADNI (Alzheimers Disease Neuroimaging Initiative; http://adni.loni.usc.edu/) database, and the brain images of the Caucasians were also obtained from the ADNI database. 515 normal subjects and 320 AD patients.

The clinical diagnosis of AD was made according to the AD criteria of the National Institute of Neurological and Communication Disorders and Stroke / Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) (McKhann et al., 1984b). Mild cognitive impairment (MCI) was diagnosed according to currently agreed criteria (Winblad et al., 2004). The cognitively normal group is a group that has no neurological disease, has no impairment of cognitive function, or has no problems in daily life. Subjects with mental illnesses that may affect local lesions, head trauma careers, or their mental health on brain MRI were excluded. Subjects with mild medical abnormalities (eg, essential hypertension, diabetes without serious complications, or mild hearing impairment) were included.

The Caucasoidal genome used for the rds405509 odds ratio was National Institute of Aging-late onset Alzheimer's disease (NIA-LOAD), National Cell Repository for Alzheimer's Disease (NCRAD) and Alzheimers Disease Neuroimaging Initiative (ADNI) Respectively. 2-species data sets or 3-species data sets were combined and analyzed. A total of 395 individuals with E4 / E4 genotypes were analyzed using NIA-LOAD, NCRAD and ADNI. Among them, 330 patients with Alzheimer's dementia And 65 normal persons were used.

(2) Brain amyloid Imaging

¹⁸ F-Fluorbetaben PET imaging was performed according to conventional methods (Osama Sabri, 2015, Clin Trans Imaging, Barthel, 2011, Lancet-neurology). Amyloid pathology was determined as brain amyloid-beta plaque load (BAPL). The four brain regions (frontal cortex, posterior cingulate, posterior cingulate cortex) were classified according to the set RCTU (regional cortical tracer uptake) scoring system (1 = no uptake, 2 = minor uptake, 3 = pronounced uptake) ( ¹⁸ F) PET images of the lateral temporal cortex, parietal cortex). The RCTU scores for the frontal cortex, the posterior cortex, the lateral temporal cortex, and the parietal lobe were then compared to a single pre-established 3-grade scoring system for each PET scan, a brain amyloid-beta plaque loading (BAPL) score (1 = no amyloid load, 2 = minor amyloid load, and 3 = significant amyloid load. In vivo amyloid imaging was performed with positron emission tomography (PET) using Pittsburgh compound B (PiB). The PiB deposition to the region of interest (ROI) was determined using the FreeSurfer version 5.1 software (Martinos Center for Biomedical Imaging) and the corrected standardized uptake value ratio (SUVR) Area. Mean cortical SUVR was calculated as FreeSurfer in the frontal cortex, the posterior cortex, the lateral temporal cortex, and the parietal lobe. The cerebellum cortex was used as a reference area. PiB positivity was defined as SUVR 1.42, corresponding to the previously defined mean cortical binding potential of 0.18 for PiB positivity. Cocacy &apos; s amyloid-PET data for both ¹⁸ F-florbetapir and ¹¹ C-PiB were obtained from the ADNI database ( http://adni.loni.usc.edu ).

(3) Data generation and analysis

SNP Genotyping

Genomic DNA was extracted from isolated peripheral blood leukocytes isolated from whole blood cells collected in EDTA tubes. Blood samples were centrifuged at 1500 xg for 10 minutes to remove plasma and blood leukocytes were used for DNA extraction. The samples were genotyped using Affymetrix genome-wide genotyping arrays (Affymetrix® Axiom KORV 1.0) optimized for Koreans. The KORV 1.0 was designed by the National Institute of Health's Genome Science Center and was obtained from the Korean chip consortium. In addition, genotyping analysis via Taqman or SNP type (Fluidigm) method was performed to evaluate the accuracy in chip-based SNP genotyping. Genotyping data for ADNI was obtained from the ADNI database ( http://adni.loni.usc.edu ).

Genome Wide  data Imputation

The study data set from NRCD consisted of 6,413 individuals. Data sets from ADNI consisted of 818 from ADNI-1, 432 from ADNI-GO / 2 and 818 from ADNI-WGS data set. The quality control for the sample is based on individual call rate <95%, gender mismatch, ± 3SD from mean, redundancy or twins, SNPs with call-rate <95%, SNPs with HWE P-value <10 ^-6 and MAF < 1%. Each data set was separately implanted using the HRC reference panel version 1.1. The HRC assessment from the NRCD was conducted with pre-phased study haplotypes as the reference populations of East Asian populations. The ADNI dataset was selected and run separately as a reference population for Europeans in the HRC Imationation Server with the pre-paged haplotypes. After inflation, low-quality implanted SNPs (info <0.5) were excluded from further studies (McCarthy et al., 2016; Nagy et al., 2017; Surakka et al., 2016).

(4) Imaging  protocol

From a Korean subject MR image  Obtain

For 2443 Korean subjects, an approximate 0.9 mm axial MPRAGE (Magnetization prepared rapid gradient-echo) image of the entire brain was imaged using a 1.5 T MR scanner (Magnetom Avanto, Siemens) (TR = 1800 ms; TE = 3.43 ms; TI = 1100 ms; 15 flip angle; FoV = 224 x 224; matrix = 256 x 256; number of slices = 176). Close 0.8 mm sagittal MPRAGE volume was measured with a 3 T MR scanner (Skyra, Siemens) (TR = 2300 ms; TE = 2.143 ms; TI = 900 ms; 9 flip angle; FoV = 256 x 256; 320; number of slices = 178.volumes). All images were acquired by MR scanner of Chosun University Hospital, Gwangju, Korea.

From ADNI MR image

Download baseline 1.5T and 3T MR images from 463 well-informed subjects, 796 mild cognitive impairment subjects, and 310 AD patients from the ADNI database (ADNI, http://adni.loni.usc.edu/) Respectively. All MR images for the subject were obtained with 1.5T or 3T GE, Philips and Siemens equipment according to the standard ADNI MPRAGE protocol. In a 1.5T scanner, the nominal parameters for the MPRAGE protocol are as follows: sagittal plane, TR = 2300 to 2400 ms for multi-coil phased-array head coil (TR = 3000 ms for bird cage or volume head coil, minimum full TE, TI = 1000 ms, flip angle 8 °, FOV = 240 x 240 mm, 192 x 192 in-plane matrix and slice thickness 1.2 mm. In a 3T scanner, the MPRAGE protocol is as follows: sagittal plane, TR = 2300 or 3000 ms, minimum full TE, T1 = 853-900 ms, flip angle 8-9˚, FOV = 256-260 x 240 mm, 256 x 256 in-plane matrix and slice thickness 1.2 mm (Jack et al., 2008). AD subjects were included on a MMSE (Burns et al., 1998) score of 20 to 26, and CDR = 0.5 or 1 (Morris, 1993). In addition, the selected AD group met the NINCDS / AARDA criteria for possible AD. For healthy controls, CDR 0 and individuals without dementia were included (Wolz et al., 2011).

(5) Image data processing and analysis

MR image  Data processing

Image processing was performed using the common software FreeSurfer software (FreeSurfer 5.3.0) (FreeSurfer, http://surfer.nmr.mgh.harvard.edu/) used for brain structure analysis (Dale et al., 1999; Fischl and Dale, 2000; Fischl et al., 2004). Using DICOM-formatted MR images in an automated pipeline for FreeSurfer processing, we constructed a three-dimensional model of the brain cortical surface (Dale et al., 1999; Dale and Sereno, 1993; Fischl et al., 1999a) . In the above processing, the intensity of the image was normalized and the skull removed from the normalized image. The cortical subdivision processing is then initiated with a connected components algorithm, and any found holes represent a single-pilled volume of white matter for both brain cortex hemispheres Respectively. In addition, tessellation and surface smoothening reduced any metric distortions. Next, the gray / white boundary was represented by a refinement process, followed by initial surface model construction. Next, the surface was deformed outward to form a pial surface. First, the shortest distance from each point on the gray matter / white matter surface and from the point on the smoke surface to the surface of the smoke film was calculated to obtain the thickness measurement of the brain cortex and the shortest distance to the gray matter / white matter was calculated. The final cortical thickness was then obtained from the average of these two values at a particular point. The cortical thickness was measured at 163,842 vertices in each hemisphere with a triangular grid about 1 ㎜ away from other positions representing points or vertices in the cortical mantle. The cortical folding pattern can be used to smoothen the map into 0, 5, 10, 15, 20 and 25 mm full-width-half-maximum Gaussian kernels and use non-rigid high- and a rigid high-dimensional spherical averaging method. The adjusted map can detect a difference of less than ㎜ mm between subjects and is not limited by voxel resolution. Subcutaneous volume was also obtained by automated volume measurement using FreeSurfer (Dale et al., 1999; Fischl and Dale, 2000; Fischl et al., 1999b).

(6) Image statistical analysis

Cortical thickness differences between groups with different APOE alleles were compared using the Surfstat (http://www.math.mcgill.ca/keith/surfstat/) toolbox for MATLAB (R2012a, The Mathworks, Natick, Mass., USA) And analyzed statistically. We statistically tested each unmasked point on the soft surface. Using the general linear model (GLM) as a covariate, using the APOE allele (e4 / 4 vs e3 / 3) as a fixed factor and sex, age, education level and field strength as covariates, (point-wise) cortical thickness difference (Fan et al., 2010). To evaluate the rs405509 allele difference, subjects with a flow type of T / T-E4 / 4 and G / T-e4 / 4 + G / G-e4 / 4 were compared separately with subjects with e3 / 3 . In the GLM of rs405509, the inventors used the APOE allele (rs405509-e4 / 4 flow type vs e3 / 3) as a fixative and used the covariance of the point-wise cortical thickness (Chen et al., 2015b). In all cortical thickness analyzes, random field theory (RFT) -based corrections for multiple PoIy-Wise cortex thickness comparisons were applied at the cluster level and clusters that passed the false discovery rate (FDR) correction value of p <0.05 (Taylor and Adler, 2003) ( http://www.math.mcgill.ca/keith/surfstat/ ). In addition, hippocampal volumes and anatomical observation regions were statistically compared for groups bearing APOE alleles in the R program (R version 3.3.1). The hippocampus volume difference was determined by using the APOE allele (e4 / 4 vs e3 / 3, rs405509-e4 / 4 flow type vs e3 / 3) as a fixed factor and the sex, age, education level, (Hickie et al., 2005; Zierhut et al., 2013) using covariance (ANCOVA) analysis as the covariate. Next, differences in observation area (ROI) between different groups (e4 / 4 vs e3 / 3, rs405509-e4 / 4 flow type vs e3 / 3) were compared by age, sex, Respectively. In the analysis, the significance level was p <0.05 was considered in, FDR correction is p <0.05 level if the results were considered significant (Stricker et al, 2013;. . Ye et al, 2016).

(7) Statistical analysis

The proportion of subjects in the control and AD groups was compared by chi-square tests. The odd ratio of events was calculated using a logistic regression analysis with a 95% confidence interval for both adjusted (gender and age) unadjusted analyzes (Basaria et al., 2010). All statistical tests were performed using both SPSS (version 23.0) and R program (version 3.3).

(8) From a human blood sample APOE  Gene expression

Western blotting

Serum was first recovered from whole blood (obtained from NRCD). SDS-PAGE and western blotting were performed according to the manufacturer's instructions with slight modifications (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with 5% skim milk powder in PBS with 0.1% Tween 20 for 1 hour and subsequently incubated with primary antibody for 1 hour at room temperature. The primary antibodies were diluted as follows: 1: 1,000 rabbit anti-APOE (D719N; Cell Signaling); 1: 1,000 rabbit anti-transferrin (ab109503; Abcam). HRP (Horseradish peroxidase) -conjugated secondary antibody was used at 1: 5,000. The immunological activity was measured with EZ-Western Lumi Plus (DoGen).

(9) Reporter gene analysis

The APOE gene (positions 1,983 to +935) promoter region was amplified from genomic DNA using the following primers from blood cells obtained from normal and AD patients: dislocation, 5'-GGGGTACCGAAAGCAGCGGATCCTTGAT3 '(SEQ ID NO: 1); Inverse, 5'-CCCCTCGAGCTTCCTGCCTGTGATTGGC3 '(SEQ ID NO: 2). The amplified DNA was digested with KpnI and XhoI and ligated into pGL3.basic vector (Promega). The following primers were used to perform G → T and T → G allele substitutions using a PCR-based position-specific mutation of rs405509 (219G / T): T → G potential, 5'-GAGGAGGGTGTCTGGATTACTGGGCGAG-3 ' No. 3); Inverse, 5'-CTCGCCCAGTAATCCAGACACCCTCCTC3 '(SEQ ID NO: 4); G? T potential, 5'-GAGGAGGGTGTCTGTATTACTGGGCGAGG-3 '(SEQ ID NO: 5); 5'-CCTCGCCCAGTAATACAGACACCCTCCTC-3 '(SEQ ID NO: 6). The analysis was performed using PfuUltra High-Fidelity DNA Polymerase (Agilent).

(10) Luciferase ( Luciferase ) analysis

HEK 293T cells were cultured in 12-well plates. After 24 hours, cells were transfected with 0.25 [mu] g pGL3 with 0.25 [mu] g firefly luciferase reporter gene (Promega) and 0.25 [mu] g pCMV-beta-galactosidase (Clontech) using TransFectin® Lipid Reagent for 24 hours Co-transfected. Transfected cells were lysed with reporter lysis buffer (Promega). Luciferase and [beta] -galactosidase activity were quantified using the GloMax ™ Luminometer (Promega) and the Epoch microplate spectrophotometer (BioTek), respectively. Luciferase activity was normalized to? -Galactosidase activity. The results represent three independent experiments.

(11) Total field dielectric information

The full-length genome information was quantified after extracting gDNA using a QIAGEN DNA mini kit using normal buffy coats isolated from the blood of a subject, Alzheimer's disease cognitive impairment patients and Alzheimer's demented subjects. ND-1000 spectrophotometer and Quant-iT PicoGreen dsDNA Reagent and Kits. Next, the DNA quality was confirmed by electrophoresis on agarose 1.0% TBE (Tris-borate-EDTA, Invitrogen) gel, and microarray-based genotyping analysis was performed on the samples that passed the DNA quality control. The Axiom Korean chip from Affymetrix was used according to the manufacturer's instructions to obtain information on each genotype. For SNPs not microarrayed, genotyping was performed by imputation method. 1000 genomes ( https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ ) were used for full-field genome implantation, using plink, EIGENSTRAT, Shapeit, impute2, GTOOL, EAGLETOOL, , And Michigan imputation server ( https://imputationserver.sph.umich.edu/index.html) as reference panels.

result

APOE  The association of e4 / 4 with Alzheimer's disease

In relation to Alzheimer's disease, we also analyzed the risk of APOE mutations in different ethnic groups of East Asian, Caucasian, and African descent. In all racial groups, including controls and clinically diagnosed with Alzheimer's disease, distribution of APOE genotypes and allele frequencies were significantly different in control and Alzheimer's patients. These results were more prominent in subjects with Alzheimer's disease than subjects with e4. Although APOE e4 is an important risk factor for Alzheimer's disease, racial differences in e4-mediated risk have not been identified. In an analysis using patients who were clinically diagnosed with a high likelihood of Alzheimer's disease and controls, a very high odds ratio was observed for e4 / 4 subjects in East Asia compared to Caucasians (odds ratios: e4 / 4 = 33.40 And 15.86, East Asian and Caucasians) (Table 1). In Table 1, the odds ratio was analyzed by logistic transformation with reference to e3 / 3.

Diagnosis Population N OR (95% C.I .) Ref. e3 / 4 e4 / 4 Clinically diagnosed East Asian East Asian *
( NRCD ) 4195 3.94
(3.4-4.6) 33.40
(17.0-65.8) Korean † 336 2.9
(1.7-5.2) 24.7
(2.8-214.5) Kim KW, 1999 Japanese † 2313 5.6
(3.9-8.0) 33.1
(13.6-80.5) Farrer LA, 1997 European ancestry Caucasian *, ‡
( ADNI ) 835 3.81
(2.73-5.3) 15.86
(8.17-30.77) Caucasian †
(Clinic / Autopsy) 6305 3.2
(2.8-3.8) 14.9
(10.8 -20.6) Farrer LA, 1997 Caucasian †
(population-based) 4858 2.7
(2.2-3.2) 12.5
(8.8 - 17.7) Farrer LA, 1997 France † 1447 2.2
(1.5-3.5) 11.2
(4.0 -31.6) Bickeboller H, 1997 Norway † 937 4.2
(3.0-5.9) 12.9
(6.6 -26.7) Sando SB, 2008 African ancestry African-Americans † 474 1.1
(0.7-1.8) 5.7
(2.314.1) Farrer LA, 1997 Nigeria † 582 1.33
(0.8-2.1) 1.68
(0.73.7) Gureje O, 2006 Tunisia † 129 2.9
(1.3-6.6) 5.4
(1.421.5) Rassas AA, 2012 Neuropathologically confirmed East Asian *, §
( NRCD ) 883 5.26
(3.7-7.4) 125.10
(16.7-935.2) Caucasian *, ¶
( ADNI ) 1067 6.48
(4.78.9) 20.92
(10.248.9)

† The right column shows a previous study suggesting an OZBI as a reference; • Data set from ADNI was used for the risk analysis of Alzheimer's disease for Caucasians; § evaluating amyloid burden for neuropathological diagnosis of NRCD subjects; ¶ Amyloid accumulation in Caucasians was obtained from ADNI.

It was a surprising result that the e4 / 4-mediated risk was race-dependent, in the order of East Asian and Caucasian. The results of the present invention were consistent with previous studies analyzing the association of the APOE genotype with Alzheimer's disease (Table 1). Table 2 below shows subjects in the Alzheimer &apos; s disease and control group based on clinical diagnosis.

Population Total Cognitively normal controls Alzheimer's disease cases n Female (%) Age †
(mean ± SD) n Female (%) Age ‡
(mean ± SD) East Asian§
(NRCD) 4195 2309 1423 (61.6%) 74.6 ± 5.7
1886 1288 (68.3%) 73.6 ± 5.6 Caucasian
(ADNI) 835 515 265 (51.4%) 74.38 + - 5.58
320 139 (43.4%) 75.65 + - 6.84

* Aged 60 to over 90; † The age of the control group is the age at the time of the survey; ‡ Age of Alzheimer's disease is age at onset; § East Asian dyadic set obtained from NRCD; ¶ The Caucasus data set was obtained from ADNI.

To identify ethnic differences for e4 / 4-mediated outbreaks, we analyzed associations using a set of neuropathological diagnostic data from Koreans and Caucasians (Table 3).

Population Total Aβ (-) † Aβ (+) † n Female (%) Age ‡
(mean ± SD) n Female (%) Age§
(mean ± SD) East Asian¶
(NRCD) 883 557 321 (55.6) 72.3 ± 6.3 306 162 (52.9) 73.4 ± 6.0 Caucasian
(ADNI) 1067 475 203 (42.7) 72.8 ± 6.5 592 266 (44.9) 74.3 ± 6.3

* Older than 60 years old under 90 years old; † Ab (-) indicates amyloid-negative, Ab (+) indicates amyloid-positive; ‡ The age of the control group is the age at the time of the survey; § The age of patients with Alzheimer's disease is age at onset; East Asian is a Korean who participated in amyloid - PET scanning; The Caucasian data set for amyloid abnormalities was obtained from ADNI.

In both racial groups, more e4 holders were identified than in the amyloid-positive group than in the amyloid-negative group (Table 4).

APOE n Genotypes frequencies (%) Allele frequencies (%) Genotype e2 / 2 e2 / 3 e2 / 4 e3 / 3 e3 / 4 e4 / 4 e2 e3 e4 East Asian
(NRCD) Aβ (-) * (%) 577 0.3 10.4 0.5 74.3 14.2 0.2 5.8 86.6 7.6 Aβ (+) * (%) 306 0.6 2.6 1.6 42.8 42.5 9.8 2.8 65.4 31.9 γ2  (P) 180.60 (4.010 ^-37 ) 178.33 (1.810 - ³⁹ ) Caucasian
(ADNI) Aβ (-) (%) 475 0.4 13.7 1.1 66.1 17.1 1.7 11.5 73.5 15.0 A? (+) (%) 592 0 2.5 2.9 33.4 47.4 13.2 3.5 54.8 41.7 γ2  (P) 203.05 (4.51x10 ^-46 ) 122.16 (2.1 x 10 ^-28 )

* Ab (-) indicates an amyloid-negative subject, and Ab (+) indicates an amyloid-positive subject.

In a study of association between APOE genotype and amyloid accumulation, e4 / 4 subjects in Koreans showed significantly higher odds ratios compared to Caucasian (odds ratios for e4 / 4 = 125.1 and 20.92 for Korean and Caucasian) (Table 1 and Table 5). These results suggest that East Asian e4 homozygotes are more susceptible to Alzheimer's disease than other ethnic groups.

Population n e3 / 3 † e2 / 2 + e2 / 3 e2 / 4 e3 / 4 e4 / 4 OR ‡
(95% CI) P OR
(95% CI) P OR
(95% CI) P OR
(95% CI) P East Asian
(NRCD) 883 Ref 0.49
(0.2-0.9) 0.049 5.33
(1.2-22.8) 0.024 5.26
(3.7-7.4) 1.910 ^-21 125.10
(16.7-935.2) 3.010 ^-6 Caucasian
(ADNI) 1067 Ref 0.38
(0.2-0.7) 0.0015 6.27
(2.4-19.7) 0.0005 6.46
(4.7-8.9) 2.810 ^-30 20.92
(10.2-48.9) 1.310 ^-14

* Analysis of the association between APOE genotype and amyloid-based neuropathological diagnosis in different ethnic groups by logistic regression; † e3 / 3 is used as a reference genotype; ‡ Adjusting the odds ratio according to sex and age.

APOE  Polymorphism screening around genes

We estimated that the risk of different e4 / 4 risk of racial differentiation among East Asian, Caucasian, and African descendants was due to the polymorphism around APOE. To investigate the causative mechanism, we screened SNPs within the 40 kb region of the APOE peripheral gene related to the allele frequency between different racial groups (FIG. 1A). Of the 630 implanted SNPs spanning 40 kb, 72 SNPs were selected based on criteria such as order of frequency of alleles of baseline, East Asian, Caucasian, and African descent sequence, or vice versa. Next, we removed SNPs according to the significant association and minor allele frequency with Alzheimer &apos; s disease without APOE modulation. As a result, three SNPs, rs449647, rs405509, and rs440446 were derived. These polymorphisms were located in the APOE promoter and intron region (FIG. 1B). These showed large differences in the frequencies of alleles (rs449647-A, rs405509-T, and rs440446-G) in East Asian, Caucasian and African descent (FIG. Among them, rs405509 showed significant differences between ethnic groups (Based on 1000 genome databases, T-allele frequency = 0.67, 0.48, and 0.24 for East Asian, Caucasian, and African ancestry, respectively). In particular, the e4 homozygotes from East Asian were 90.4% TT-genotypes, while the Caucasian and African were 64.2% and 21.3%, respectively, and the difference in rs405509 allele frequency was found in other ethnic groups May provide a difference in susceptibility to AD onset of e4 / 4 in the liver. rs449647 and rs440446 showed different allelic frequencies among different racial groups, but their frequency in e4 / 4 was not significantly different between the ethnic groups.

with rs405509  The relevance of Alzheimer's disease

To investigate whether the TT genotype of rs405509 confers high risk of Alzheimer's disease at e3 / 4 and e4 / 4, we tested the relevance of rs405509 in e3 / 4 and e4 / 4 subjects. As expected, rs405509-TT showed a significantly higher odds ratio in both the 3/4 and e4 / 4 subject groups from the NRCD data set (East Asian) (Table 6) Racial differences. The e4 homozygotes in the East Asian population with a high rs405509-TT frequency showed higher susceptibility to Alzheimer's disease than Caucasian and African descent.

Group Total
(n) APOE  e3 / 4
(n) rs405509  in e3 / 4
( TT  vs GT + GG ) APOE  e4 / 4
(n) rs405509  in e4 / 4
( TT  vs GT + GG ) OR †
(95% C.I.) P OR †
(95% C.I .) P NRCD NP 14322 2272 1.40
(1.19-1.65) 6.59x10 ^-05 140 3.59
(1.94-6.62) 4.48 x 10 ^-05 AD 2079 855 197

† The rs405509-TT genotypes were analyzed by logistic regression using the rs405509-GT + GG genotypes in e3 / 4 and e4 / 4 genotype subjects; • NP is a normal population control; § AD is an Alzheimer's disease patient.

 In addition, in order to confirm the effect of rs405509 on e3 / 4 and e4 / 4-mediated Alzheimer's risk, the present inventors found that the association of e3 / 4 and e4 / 4 in other rs405509 genotype backgrounds, rs405509-TT, GT, (Table 7). As a result, subjects with e3 / 4 and e4 / 4 showed higher odds ratios for the rs405509-TT genotype than the rs405509-G allele background in both NRCD and ADNI data sets.

Dataset rs405509 n e3 / 3 e3 / 4 e4 / 4 Odds ratio
(95% C.I .) P Odds ratio
(95% C.I .) P NRCD TT 8773 ref 4.23 (3.71, 4.81) 2.55E-105 15.53 (12.14 19.87) 1.39E-105 GT 7212 ref 3.69 (3.08, 4.41) 3.90E-46 7.50 (3.62, 15.57) 6.24E-8 GG 7183 ref 2.09 (1.03, 4.23) 0.041 4.36 (0.18 107.99) N / A ADNI TT 265 ref 6.79 (3.67, 13.03) 2.80E-9 22.83 (7.89 85.42) 1.54E-7 GT 482 ref 5.03 (3.27, 7.85) 3.75E-13 11.85 (4.22, 42.72) 1.83E-5 GG 197 ref 5.09 (2.09, 13.63) 5.73E-4 N / A N / A

* With reference to the e3 / 3 ratio of the APOE genotypes e3 / 4 and e4 / 4 to the e3 / 3 for Alzheimer's disease, the logistic times in each rs405509 genotype data set (rs405509-TT, rs405509-GT, and rs405509- Respectively.

APOE  The relationship between e4 / 4 and brain atrophy

To determine whether the brain atrophy mediated by APOE e4 / 4 is indicative of race-to-race differences, the present inventors used a general linear model for East Asian and Caucasians to identify points between e4 / 4 and e3 / 3 subjects The point-wise cortical thickness was analyzed. Both East Asian and Caucasian e4 / 4 subjects exhibited a thinner cortical area compared to e3 / 3 (Figs. 2A, 2B, and 2C). The e4 / 4-dependent constricted areas were the inferior parietal lobule, the entorhinal-parahippocampal-fusiform, the precuneus, and the lobulated parietal lobule. (P <0.05). Surprisingly, the reduction in cortical thickness in these areas was much stronger in East Asia compared to Caucasians. Covariance analysis (ANCOVA) was performed to measure the racial differences in e4 / 4-induced cortical atrophy. The relative mean cortical thickness in the cortical atrophy region was calculated for e4 / 4 subjects with reference to e3 / 3. E4 / 4 and e3 / 3 in the mean cortical thickness within the medial temporal cortex, including both the olfactory cortex and the parahippocampal cortex and the precuneus, both East Asian and Caucasian (* P <0.05, ** P <0.01), respectively. In addition, the East Asian population showed a larger difference between e4 / 4 / and e3 / 4 than the Caucasian (Figs. 2B and 2C). A direct comparison (relative to e3 / 3) of the relative cortical contraction of e4 / 4 between East Asian and Caucasians also showed significant results (* P <0.05, ** P <0.01). In addition, the present inventors analyzed the hippocampal volume between e4 / 4 and e3 / 3 subjects in East Asian and Caucasian. The hippocampal atrophy was more prominent in e4 / 4 than in e3 / 3 in both East Asian and Caucasian, but compared to e3 / 3, e4 / 4-induced hippocampal dilation was higher in East Asian than in Caucasians (** P < 0.01) (Figure 2D). These results suggest that East Asian people are more vulnerable to APOE e4 / 4-mediated brain atrophy, including decreased cortical thickness and hippocampal contraction. To investigate the effect of rs405509 on brain atrophy in e4 / 4 homozygotes, we used combinations of e4 / 4 and rs405509 (e4 / 4-TT, e4 / 4-GT and e4 / 4-GG) Were analyzed with reference to e3 / 3 in Caucasian subjects (Figs. 2E, 2F, and 2G). e4 / 4 subjects with rs405509-TT showed significant clusters within the medial temporal cortex (the inner olfactory cortex and the hippocampal cortex) and the larynx. (P <0.05), whereas e4 / 4 subjects with the rs405509-G allele showed no such clusters. Quantitative comparative analysis with ACNOVA showed that e4 / 4 with r405509-TT genotype was significantly higher in the medial temporal cortex (F = 8.38, P <0.01) and in the anterior part (F = 10.707, P < ), Whereas e4 / 4 with rs405509-GG showed no significant difference in the cortical area (Figs. 2F and 2G). We also tested hippocampal atrophy in e4 / 4 subjects with rs405509-TT and rs405509-GG in Caucasians. The hippocampal contraction of e4 / 4 with rs405509-TT was stronger than that of e3 / 3, whereas the contraction of e4 / 4 with rs405509-GG was not significant (Fig. 2H). These neuroimaging results support that the TT genotype in rs405509 affects not only the risk of Alzheimer 's disease but also the brain contraction of e4 / 4 subjects. Table 8 shows the subjects participating in neuroimaging.

East Asian *
(NRCD) Caucasian †
(ADNI) n 1684 831 Age § (y, mean ± SD) 72.77 ± 5.12 74.70 ± 6.59 Female (n,%) 1028 (61.0%) 358 (43.0%) Years of education (y, mean ± SD) 9.01 5.10 16.05 + - 2.78 MMSE (mean SD) 26.01 + - 3.84 27.43 + - 2.54

* East Asian is a participant of NRCD at Chosun University in Gwangju city, Korea; † The Caucasus is a participant in the ADNI cohort; § The elderly are between 60 and 90 years of age.

APOE's  Expression

According to the present invention, rs405509 is associated with the onset of Alzheimer &apos; s disease and brain contraction in the e4 homozygote. Therefore, we analyzed the biological relevance of rs405509 to the modulation of APOE. To investigate whether the rs405509 genotype modulates the expression of the APOE protein, we performed western blotting of human sera of rs405509-TT, GT, and GG genotypes in e3 / 3 subjects (FIG. 3A). In the subject with rs405509-TT, APOE protein levels were reduced to a surprising level compared to the level of subjects with rs405509-GG and GT (Fig. 3A). These results were consistent with previous studies suggesting that reduced levels of APOE are associated with increased risk of Alzheimer's disease. In addition, the effect of rs405509 on the transcriptional activity of APOE was confirmed by reporter gene analysis. The assays were performed using APOE promoter fragments from individuals with AD cognitive ability with rs405509-T allele and rs405509-G and rs405509-T allele. The rs405509 allele in each promoter region was changed to an alternative-allele, followed by a luciferase-based reporter gene assay (Fig. 3C and 3D). Substitution from G to T resulted in a dramatic reduction of promoter activity (66% compared to the control), while a single base mutation from T to G at the rs405509 position resulted in a surprising increase in APOE promoter activity (160% compared to the control). This suggests that the rs405509-T allele reduces APOE transcription compared to the G allele. These results suggest that rs405509 modulates the effect of e4 / 4 by modulating the level of APOE protein.

The present invention provides a method for more accurately predicting the risk of dementia by analyzing the genetic variation of rs405509 in the APOE promoter as well as the APOE genotype in genomic DNA obtained from blood or biological samples. The present invention can contribute to complex clinical and pathological information such as MRI brain image, neuropsychological test, CSF test and amyloid-PET test, as well as future diagnosis technology based on dementia big data.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> APOE promoter SNP associated with the risk of Alzheimer's disease          and the use thereof <130> CSU17P-0003-KRP <150> KR 10-2017-0060225 <151> 2017-05-15 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer - APOE promoter <400> 1 ggggtaccga aagcagcgga tccttgat 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer - APOE promoter <400> 2 cccctcgagc ttcctgcctg tgattggc 28 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer - APOE rs405509 allele replacement <400> 3 gaggagggtg tctggattac tgggcgag 28 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer - APOE rs405509 allele replacement <400> 4 ctcgcccagt aatccagaca ccctcctc 28 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer - APOE rs405509 allele replacement <400> 5 gaggagggtg tctgtattac tgggcgagg 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer - APOE rs405509 allele replacement <400> 6 cctcgcccag taatacagac accctcctc 29

Claims (15)

A method for detecting a genetic mutation indicative of Alzheimer's disease (AD) in a sample separated from a subject containing nucleic acid and providing information necessary for predicting the risk of developing Alzheimer's disease,
(a) contacting the sample with a reagent capable of detecting the presence or absence of the genetic mutation in a gene selected from a gene encoding the APOE E4 allele or a gene product thereof; And
(b) contacting the sample with a reagent capable of detecting the presence or absence of the genetic mutation selected from a gene encoding the rs405509 T allele of the APOE promoter or a gene product thereof,
Wherein the presence of APOE E4 / E4 and rs405509 T / T genetic mutations indicates that said subject is an Alzheimer &apos; s disease patient or has a risk of developing Alzheimer &
A method of providing information necessary for predicting the risk of developing Alzheimer's disease. The method of claim 1, wherein the method for detecting the presence or absence of a genetic mutation in the APOE E4 / E4 and rs405509 T / T comprises: obtaining a single stranded nucleic acid from a sample isolated from the subject, And analyzing the presence or absence of said genetic variation in said complex. 3. The method of claim 2, wherein analyzing the presence or absence of a genetic variation of APOE E4 / E4 and rs405509 T / T in the complex comprises polymerase chain reaction, nuclease digestion, hybridization hybridization, Southern blotting, restriction enzyme fragment polymorphism, sequencing, primer extension, single-stranded conformation polymorphism, And analyzing them together. 2. The method of claim 1, wherein the nucleic acid obtained in the sample isolated from the subject is amplified. 5. The method of claim 4, wherein the nucleic acid obtained in the sample separated from the subject is genomic DNA. 5. The method of claim 4, wherein the nucleic acid obtained in the sample separated from the subject is RNA. 2. The method of claim 1, further comprising obtaining an image of a subject's cortical thickness, wherein the atrophy of the cortical thickness indicates that the subject is an Alzheimer's disease patient or is at risk for developing Alzheimer's disease In method. 8. The method of claim 7, wherein the image of the cortical thickness is an MR brain image. The method according to any one of claims 1, 7, and 8, further comprising performing one or more of the following tests: a neuropsychological test, a cerebrospinal fluid (CSF) test, and an amyloid-PET test . A gene coding for the rs405509 T allele of the APOE promoter, or a gene product thereof, which detects the presence or absence of the APOE E4 / E4 genetic mutation in the gene selected from the gene encoding the APOE E4 allele or a gene product thereof, Wherein the rs405509 T / T genetic mutation is selected from the group consisting of: &lt; RTI ID = 0.0 &gt; rs405509 &lt; / RTI &gt; 11. The kit for diagnosis or prediction of Alzheimer's disease according to claim 10, comprising a primer set and a reaction enzyme complementary to the APOE E4 allele and the APOE promoter rs405509 T allele. 12. The kit of claim 10, comprising an assay method for analyzing genetic variation. 11. The kit according to claim 10, comprising a method for amplifying a nucleic acid. 11. The kit of claim 10, wherein the reference control standard is included.
The method according to claim 7 or 8, wherein the cortical thickness is the cortical thickness in the inner olfactory cortex, hippocampal cortex and larynx.

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