CN111187825A - Method and kit for detecting susceptibility sites of TOMM40 gene and APOE gene of Alzheimer's disease - Google Patents
Method and kit for detecting susceptibility sites of TOMM40 gene and APOE gene of Alzheimer's disease Download PDFInfo
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Abstract
A method for detecting susceptible sites of TOMM40 gene and APOE gene of Alzheimer's disease, said method comprising the steps of: (1) extracting genome DNA of a sample, and amplifying a region containing an rs10119 site in a 3 'UTR of a TOMM40 gene of the sample, a region containing an rs71352238 site in 5' nearGene, and a region containing an rs429358 site and an rs7412 site of an APOE gene to obtain an amplification product; (2) and (3) detecting the genotypes of the rs10119 site, the rs71352238 site, the rs429358 site and the rs7412 site in the amplification product by using an HRM analysis technology and a Tagman analysis technology.
Description
Technical Field
The invention relates to a method and a kit for detecting susceptible sites of Alzheimer's disease.
Background
Alzheimer's disease, also known as senile dementia, is an irreversible neurodegenerative brain disease, the most common cause of dementia affecting the elderly. The genetics of Alzheimer's disease can be divided into Alzheimer's disease and senile dementia. Alzheimer's disease usually occurs before the age of 65. Senile dementia, which occurs after 65 years of age, is a more common form of alzheimer's disease. Senile dementia is genetically complex, involving multiple genes with only moderate familial aggregation. Apolipoprotein e (APOE) is the most well-established gene associated with senile dementia, with people carrying the epsilon 4 allele (APOE 4) at greatly increased risk of developing alzheimer's disease. However, most genetic data for alzheimer's disease, including APOE, are based on studies in caucasian adults. Previous studies have determined that not all of the Alzheimer's disease-susceptible gene loci found in Caucasian have the same association in the Han population, suggesting that the mechanism of Alzheimer's disease in the Han population may have different genetic risk factors than those in Caucasian. The relationship between the genotype of APOE and the genotype of SNP site is shown in the following table
In recent years, increased research has found that mitochondrial dysfunction plays an important role in neurological diseases. It has been reported that TOMM40 (mitochondrial outer membrane transferase 40), which is a protein transfer required to maintain normal mitochondrial function, plays an important role in the pathogenesis of related diseases. TOMM40 is a compound formed by outer membrane Transferase (TOM), a subunit formed during import of proteins into important entry channels of mitochondria, which is essential for transport of proteins to mitochondria. With the increasing research on the association of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease, some researchers studied the association of polymorphism of TOMM40 gene located at 19q13 with Alzheimer's disease, and reported that TOMM40 gene may affect the integrity of white matter of brain and is related to the age of Alzheimer's disease, and confirmed that TOMM40 polymorphism plays an important role in the pathogenesis of AD in Han population. Some studies suggest that TOMM40 is associated with the pathogenesis of alzheimer's disease, as SNPs in TOMM40 directly lead to mitochondrial dysfunction, such as dysfunction in energy metabolism, oxidative stress injury, and the like. However, the relationship between specific SNP and Alzheimer's disease is not deeply researched at present, so that finding a susceptible site highly related to Alzheimer's disease in TOMM40 gene and applying the site in combination with APOE is a problem to be solved in the prior art.
Disclosure of Invention
In the prior study, 830 cases of patients with alzheimer's disease and 630 cases of healthy elderly without alzheimer's disease were analyzed, and it was found that the site of SNP rs10119 (hereinafter, referred to as "site rs 10119") and the site of SNP rs71352238 (hereinafter, referred to as "site rs 71352238") in TOMM40 gene have strong correlation with alzheimer's disease, and they not only act on alzheimer's disease independently of APOE gene, but also act on alzheimer's disease in cooperation with APOE, and this analysis comparison is performed by using rxcx2 test, statistical analysis is performed by SPSS software, and the analysis results after sex and age correction are shown in the following table:
meanwhile, the relevance of the site of SNP rs429358 and the site of SNP rs7412 in the APOE gene to Alzheimer's disease is also determined in the research. Based on the discovery, in order to solve the problems in the prior art, the invention adopts the technical scheme that:
in order to solve the problems, the technical scheme provided by the invention is as follows: a method for detecting susceptible sites of TOMM40 gene and APOE gene of Alzheimer's disease, said method comprising the steps of: (1) extracting genome DNA of a sample, and amplifying a region containing an rs10119 site in a 3 'UTR of a TOMM40 gene of the sample, a region containing an rs71352238 site in 5' nearGene, and a region containing an rs429358 site and an rs7412 site of an APOE gene to obtain an amplification product; (2) and (3) detecting the genotypes of the rs10119 site, the rs71352238 site, the rs429358 site and the rs7412 site in the amplification product by using an HRM analysis technology and a Tagman analysis technology.
The method for detecting the susceptibility sites of the TOMM40 gene and the APOE gene of the Alzheimer disease further comprises the step that the genotype of rs10119 of the TOMM40 gene carries TT, and the genotype of rs71352238 of the TOMM40 gene carries CC.
Further, when the genotype of the rs10119 site of the TOMM40 gene carries TT, the susceptibility of the tester of the sample of the TOMM40 gene to Alzheimer's disease is higher than that of the tester without TT; when the genotype of the rs71352238 locus of the TOMM40 gene carries CC, the susceptibility of the tester of the sample of the TOMM40 gene to Alzheimer's disease is higher than that of the tester without CC.
Furthermore, when the genotype of the rs429358 and rs7412 locus of the APOE gene has epsilon 3/epsilon 4 or epsilon 4/epsilon 4, the susceptibility of a tester of a sample to which the APOE gene belongs to the Alzheimer's disease is higher than that of a tester of a wild genotype epsilon 3/epsilon 3.
The method for detecting the susceptibility sites of the TOMM40 gene and the APOE gene of the Alzheimer's disease further comprises the steps of amplifying a region containing rs10119 and rs71352238 in the TOMM40 gene of a sample and a region containing rs429358 site and rs7412 in the APOE gene by using specific primers; the specific primer is a primer pair capable of specifically amplifying DNA fragments containing rs10119 site, rs71352238 site on TOMM40 gene and rs429358 site and rs7412 site on APOE gene.
Further, the specific primer has the sequence shown in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11 and SEQ ID NO: 12.
Further, the primer pair of the amplified DNA fragment containing the SNP site is directly tested for genotype by using HRM analysis technique and Tagman analysis technique.
Further, a primer pair of the amplified DNA fragment containing the SNP site is transferred to an analysis apparatus, and then the genotype of the primer pair is analyzed by HRM analysis technique.
Further, the method for detecting the susceptible site of the Alzheimer's disease is applied to in vitro diagnostic detection of the susceptibility of the Alzheimer's disease.
The invention also provides a kit for detecting the susceptible site of the Alzheimer disease, which is characterized in that the kit is used for carrying out amplification, HRM technical analysis and Tagman technical analysis on a region containing the rs10119 site in the 3 'UTR of the TOMM40 gene, a region containing the rs71352238 site in the 5' nearGene, a region containing the rs429358 site in the APOE gene and a region containing the rs7412 site.
Further, the kit comprises specific primers for specifically amplifying the region containing rs10119 and rs71352238 in the TOMM40 gene and the region containing rs429358 site and rs7412 site in the APOE gene.
Further, the specific primer has the sequence shown in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11 and SEQ ID NO: 12.
Further, the components and contents of the kit comprise 300ul 2X HRM Analysis RreMix, 3ul 100uMPrimer Mix, 100ul more than 10ng/ul DNA and 197ul H2O, having the sequence of SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 5. SEQ ID NO: 6, and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 11. SEQ ID NO: 12, or a primer specific to the nucleotide sequence shown in figure 12.
The invention also provides application of the method and the kit in predicting susceptibility of Alzheimer's disease.
The invention also provides application of the method and the kit in detecting the SNP loci of the genetic risk factors of Alzheimer's disease.
In the prior study, the rs10119 and rs71352238 genotypes of a large number of patients and healthy people are analyzed, and the genotype of the rs10119 of the TOMM40 gene is TT, and the genotype of the rs71352238 of the TOMM40 gene is CC, so that the gene has strong correlation with the onset of Alzheimer's disease, and the gene not only acts on the Alzheimer's disease independently of the APOE gene, but also has a promoting effect on the onset of the Alzheimer's disease in cooperation with the APOE. Based on the above findings, the method and kit provided by the present invention: HRM analysis technology is adopted to carry out genotype analysis on the areas of the amplified TOMM40 gene containing the rs10119 site and the rs71352238 site and the APOE gene containing the rs429358 site and the rs7412 site.
Drawings
FIG. 1 is a screen shot of genotyping results of Taqman analysis technique showing the result of SNP genotype at site rs 10119;
FIG. 2 is a screen shot of the genotyping results of Taqman analysis technique showing the result of SNP genotype at site rs 71352238;
FIG. 3 is a screen shot of genotyping results of Taqman analysis technique showing the result of SNP genotype at locus rs 429358;
FIG. 4 is a screenshot of the genotyping results of Taqman analysis technique showing the result of SNP genotype at locus rs 7412.
FIG. 5 is a sectional view showing the result of genotyping at the rs10119 locus by HRM analysis technique;
FIG. 6 is a screen shot of the result of genotyping by HRM analysis technique, showing the result of SNP genotype at locus rs 71352238;
FIG. 7 is a sectional view showing the result of genotyping the rs429358 site SNP in HRM analysis technique;
FIG. 8 is a sectional view showing the result of genotyping the rs7412 site SNP.
Detailed Description
The invention will be further elucidated with reference to the following specific examples. Experimental procedures for the specific conditions not specified in the examples below are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A Laboratory Manual (New York: Cold spring harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
The invention provides a method and a kit for detecting susceptible sites of Alzheimer's disease, wherein the method comprises the following steps:
1) extracting genome DNA of a sample, and amplifying a region containing rs10119 in the 3 'UTR and a region containing rs71352238 in 5' nearGene of the TOMM40 gene of the sample to obtain an amplification product;
2) the genotype of the site rs10119, rs71352238, and rs429358 and rs7412 in the amplified product is detected by using a High-Resolution Melt curve (HRM) analysis technique. The detection method and the kit for the Alzheimer disease susceptible locus can also be used for predicting the susceptibility of Alzheimer disease.
The rs10119 is located in the 3' UTR region of TOMM40 gene (fragment contig: NT _011109.17 position 17662542G > A), wherein the sequence number of Deoxyribonucleotide (DNA) is as follows: the rs10119 position is based on SEQ ID NO: 1 or SEQ ID NO: 17; primer 1 is based on SEQ ID NO: 2; primer 2 is based on SEQ ID NO: 3; the amplification product is based on SEQ ID NO: 7.
The rs71352238 is located in the 5' nearGene region of TOMM40 gene (fragment contig: NT _011109.17 position 17650205T > C), wherein the Deoxyribonucleotide (DNA) sequence number: rs71352238 position based on SEQ ID NO: 4 or SEQ ID NO: 18; primer 3 is based on SEQ ID NO: 5; primer 4 is based on SEQ ID NO: 6; the amplification product is based on SEQ ID NO: 8;
the rs429358 is positioned in an exon region of the APOE gene (fragment contig: NT-011109.17 position 17667810T > C), wherein the sequence number of Deoxyribonucleotide (DNA) is as follows: rs429358 position is based on SEQ ID NO: 7 or SEQ ID NO: 19; primer 3 is based on SEQ ID NO: 8; primer 4 is based on SEQ ID NO: 9; the amplification product is based on SEQ ID NO: 15;
rs7412 is located in exon region of APOE gene (fragment contig: NT-011109.17 position 17667948C > T), wherein, the sequence number of Deoxyribonucleotide (DNA) is as follows: rs7412 position is based on SEQ ID NO: 10 or SEQ ID NO: 20; primer 3 is based on SEQ ID NO: 11; primer 4 is based on seq id NO: 12; the amplification product is based on SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof
The research proves that the TOMM40 gene and the APOE gene are closely related to Alzheimer's disease, and the new functions of the single nucleotide polymorphism sites rs10119 and rs71352238 of TOMM40 are found: the change of the genotypes of the single nucleotide polymorphism site rs10119 in the 3' UTR region of TOMM40 gene and the single nucleotide polymorphism site rs71352238 in the 5' nearGene region of TOMM40 gene will lead to the increased risk of developing Alzheimer's disease, wherein the results of correlation studies show that there is a very significant difference in the distribution of TOMM40 gene rs 10119G > A and TOMM 40T > C in case and control groups (P < 0.01). .
The detailed sequence of the TOMM40 gene can be found in the nucleotide sequences of accession nos. rs10119 and rs71352238 (see the websites http:// www.ncbi.nlm.nih.gov /)
The detailed sequence of the APOE gene can be found in the nucleotide sequences of accession nos. rs429358 and rs7412 (see the websites http:// www.ncbi.nlm.nih.gov /).
Example 1 fluorescent PCR assay
First, experimental material
The LightCycler480 fluorescent quantitative PCR instrument was purchased from Roche, Switzerland, and the polymerase chain reaction solution (HRMAnalysis RreMix) was custom-synthesized by Tiangen Biochemical technology, Inc. (Beijing).
Secondly, primer and probe design and synthesis:
primers were analyzed using Primer Premier5 software using partial sequences of the 3 'UTR region and 5' nearGene region of TOMM40 gene and APOE exon region as templates, and custom-synthesized by Wuxi Jingji Biotech, Inc.
Detection primers:
rs10119 upstream primer sequence of TOMM40 gene:
5’-TGGTGCTCAGAATCCTGCG-3’（SEQ ID NO：2）
rs10119 downstream primer sequence of TOMM40 gene:
5’-CGCTTCGGGATTCTGAGTAGC-3’（SEQ ID NO：3）
primer sequence upstream of rs71352238 of TOMM40 gene:
5’-CCTGAAATGTCCTTTTGTATTGGTT-3’（SEQ ID NO：5）
downstream primer sequence of rs71352238 of TOMM40 gene:
5’-CTGCGAGCCAATGGGAAG-3’（SEQ ID NO：6）
the sequence of an APOE gene rs429358 upstream primer:
5’-GGCTGTCCAAGGAGCTGC-3’（SEQ ID NO：8）
primer sequence of rs429358 downstream of APOE gene:
5’-CCGAGCATGGCCTGCA-3’（SEQ ID NO：9）
the sequence of the primer at the upstream of the APOE gene rs 7412:
5’-CCTCCGCGATGCCGA-3’（SEQ ID NO：11）
primer sequence at the downstream of APOE gene rs 7412:
5’-ACGCGGCCCTGTTCCA-3’（SEQ ID NO：12）
thirdly, sample detection:
the experiment totally detects 200 cases of Alzheimer's disease and 200 normal control crowds, each sample of blood sample is collected by about 2mL, genome DNA is extracted by phenol/chloroform, and the extraction result is detected by a micro ultraviolet/visible spectrophotometer (the thermo Fisher company).
Fluorescent PCR amplification was performed as follows, followed by scanning with Roche LC480 Software 1.5 and cluster analysis
Fourthly, detecting the genotype:
detection results of genomic DNA extraction:
the genomic DNA of all samples met the detection requirements (260/280 >1.8, concentration >10 ng/ul)
Example 2 Tagman method for detecting genotypes of sites rs10119, rs71352238, rs429358 and rs7412 of Alzheimer's disease
The Tagman method is used for detecting rs10119 and rs71352238 of the genetic risk gene TOMM40 gene of the Alzheimer disease and rs429358 and rs7412 sites of the APOE gene. 200 samples of the Alzheimer disease cases, namely control group samples, are selected to carry out Tagman typing experiments, and the genotypes of rs10119, rs71352238, rs429358 and rs7412 are judged.
First, experiment method
The Tagman probe method uses the Tagman probe and Tagman genotypingMasterMix of applied biosystems, USA.
PCR amplification was performed as follows, and finally scanned and cluster analyzed by Roche LC480 Software 1.5
Second, experimental results
The screenshots of the genotyping results of the Taqman analysis technique are respectively shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4.
The screenshots of HRM analysis technique genotyping results are shown in FIG. 5, FIG. 6, FIG. 7 and FIG. 8, respectively.
Finally, the results of Tagman typing in 400 cases were in complete agreement with the results of HRM analysis by Light Cycler480 fluorescent PCR.
Third, correlation analysis of genotype of rs10119 and rs71352238 of TOMM40 gene and APOE gene rs429358 and rs7412 and susceptibility of Alzheimer's disease
Comparison of the distribution of rs10119 and rs71352238 gene TOMM40 and rs429358 and rs7412 locus of APOE gene in Alzheimer's disease patients and controls the results of statistical analysis using SPSS software using the R × C × 2 test are shown in the following table:
example 3 detection kit for susceptible sites of Alzheimer's disease
The detection kit provided by the invention can be used for amplifying a region containing an rs10119 site in a 3' UTR of a TOMM40 gene, a region containing an rs71352238 site in a 5' nearGene, a region containing an rs429358 site in an exon of an APOE gene and a region containing an rs7412 site, carrying out HRM technical analysis and Tagman technical analysis, can be used for detecting the susceptibility of Alzheimer's disease, and comprises specific primers capable of amplifying a region containing an rs10119 site in a 3' UTR of a TOMM40 gene, a region containing an rs71352238 site in a 5' nearGene, a region containing an rs429358 site and an rs7412 site in an exon of an APOE gene and other PCR-HRM corresponding reagents, wherein the contents of components for PCR amplification and HRM analysis are the same.
The detection kit is applied to 50 person detection, and-20oC, storing in dark, wherein the components and the content comprise:
300ul 2X HRM Analysis RreMix
6ul 100uM Primer Mix
100ul DNA larger than 10ng/ul
197ul H2O
Has the sequence shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 5. SEQ ID NO: 6, and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 11. SEQ ID NO: 12.75 ul each of the primers specific for the nucleotide sequence shown in fig. 12.
The invention has practical exemplification:
and detecting the genotypes of the rs10119 and rs71352238 loci in the TOMM40 gene and the rs429358 and rs7412 loci in the APOE gene of the individual so as to judge whether the risk of the individual suffering from the Alzheimer's disease is higher than that of the general population.
The rs10119 and rs71352238 sites in the TOMM40 gene and the rs429358 and rs7412 site polymorphism in the APOE gene can be directly used for the prevention guidance of Alzheimer's disease.
The detection of the genotypes of the rs10119 and rs71352238 locus in the TOMM40 gene and the rs429358 and rs7412 locus in the APOE gene can also be used for the personalized treatment of patients with Alzheimer's disease.
All documents referred to herein are incorporated by reference into this application as if each were incorporated by reference.
While the technical content and technical features of the invention have been disclosed above, those skilled in the art may make various substitutions and modifications based on the teaching and disclosure of the invention without departing from the spirit of the invention. Therefore, the protection scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the invention, which are encompassed by the claims of the present patent application.
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Claims (10)
1. A method for detecting a susceptible site of TOMM40 gene and APOE gene in Alzheimer's disease, which comprises the steps of: (1) extracting genome DNA of a sample, and amplifying a region containing an rs10119 site in a 3 'UTR of a TOMM40 gene of the sample, a region containing an rs71352238 site in 5' nearGene, and a region containing an rs429358 site and an rs7412 site of an APOE gene to obtain an amplification product; (2) and (3) detecting the genotypes of the rs10119 site, the rs71352238 site, the rs429358 site and the rs7412 site in the amplification product by using an HRM analysis technology and a Tagman analysis technology.
2. The method for detecting the susceptible site of TOMM40 gene and APOE gene of Alzheimer's disease according to claim 1, wherein when the genotype of the rs10119 site of TOMM40 gene has TT, the susceptibility of the subject to Alzheimer's disease of the sample to which the TOMM40 gene belongs is higher than that of the subject whose genotype does not have TT; when the genotype of the rs71352238 locus of the TOMM40 gene carries CC, the susceptibility of a tester of a sample to which the TOMM40 gene belongs to Alzheimer's disease is higher than that of a tester of which the genotype does not have CC; when the genotype of the rs429358 and rs7412 sites of the APOE gene has epsilon 3/epsilon 4 or epsilon 4/epsilon 4, the susceptibility of a tester of a sample to which the APOE gene belongs to the Alzheimer's disease is higher than that of a tester of a wild genotype epsilon 3/epsilon 3.
3. The method for detecting the susceptibility site of TOMM40 gene and APOE gene of Alzheimer's disease according to claim 1 or 2, wherein the method comprises amplifying the region containing rs10119 and rs71352238 in TOMM40 gene and the region containing rs429358 site and rs7412 in APOE gene of the sample by using specific primers; the specific primer is a primer pair capable of specifically amplifying DNA fragments containing rs10119 site, rs71352238 site on TOMM40 gene and rs429358 site and rs7412 site on APOE gene.
4. The method according to claim 3, wherein the specific primers have the sequences of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11 and SEQ ID NO: 12.
5. The method of detecting the susceptibility sites of TOMM40 gene and APOE gene of Alzheimer's disease according to claim 4, wherein the genotype of the primer set of the amplified DNA fragment containing said SNP sites is directly detected by HRM analysis technique and Tagman analysis technique.
6. The use of the method for detecting a susceptible site of Alzheimer's disease as defined in any one of claims 1 to 5 for the in vitro diagnostic detection of a susceptibility to Alzheimer's disease.
7. A kit for detecting susceptibility sites of Alzheimer's disease is characterized in that the kit is used for carrying out amplification, HRM technical analysis and Tagman technical analysis on a region containing an rs10119 site in 3' UTR of a TOMM40 gene and a region containing an rs71352238 site in 5' nearGene, and a region containing an rs429358 site and an rs7412 site of an APOE gene.
8. The kit as claimed in claim 7, wherein the kit comprises specific primers for specifically amplifying the region of TOMM40 gene comprising rs10119 and rs71352238 and APOE gene comprising rs429358 site and rs7412 site, the specific primers having the amino acid sequences shown in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11 and SEQ ID NO: 12.
9. The kit of claim 8, wherein the kit comprises 300ul 2X HRMAnalysis RreMix, 3ul 100uM Primer Mix, 100ul more than 10ng/ul DNA, 197ul H2O, having SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 5. SEQ ID NO: 6, and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 11. SEQ ID NO: 12, or a primer specific to the nucleotide sequence shown in figure 12.
10. Use of the method according to claims 1 to 5 and the kit according to claims 6 to 9 for predicting susceptibility to alzheimer's disease.
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