JP2016178899A - Determination method for risk of onset of esophageal achalasia, and oligonucleotide kits for its determination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish techniques of determining the risk of onset using the detection of an esophageal achalasia susceptibility gene and its SNP.SOLUTION: The invention relates to a determination method for the risk of onset of esophageal achalasia comprising detecting a single-nucleotide polymorphism represented by the rs number registered in National Center for Biotechnology Information (NCBI) SNP database, the single-nucleotide polymorphism being one or more single-nucleotide polymorphisms selected from the group consisting of rs1881420 and rs3795850 in an ALK gene on human chromosome number 2, rs1063646 in PSORS1C1 gene on human chromosome number 6, rs2032582 in ABCB1 gene on human chromosome number 7, rs2526374 in RP5-1171I10.4 or RNF43 gene on human chromosome number 17, rs1127354 in ITPA gene on human chromosome number 20, and rs4898 in TIMP1 gene or SYN1 gene on human X chromosome, and/or a single-nucleotide polymorphism in linkage disequilibrium with the single-nucleotide polymorphism. According to the invention, determining the risk of onset of esophageal achalasia in an early stage is enabled, and giving an early medical instruction to a patient with a high risk or initiating a therapy becomes possible.SELECTED DRAWING: None

Description

  The present invention relates to a method for determining the risk of developing esophageal achalasia including detection of a gene polymorphism, and an oligonucleotide kit for the determination.

  Esophageal achalasia is a type of dysfunction of the lower esophageal sphincter (LES), which is observed in esophageal X-ray examination or upper gastrointestinal endoscopy, and stenosis of the lower esophagus due to abnormal contraction of LES and insufficiency Is a disease characterized by oral expansion. Histologically, there is a decrease or disappearance of the Auerbach plexus.

  In esophageal achalasia, even if food is swallowed, the LES does not relax and the passage is obstructed, so the patient complains of symptoms such as difficulty in swallowing, feeling gripped, and vomiting. In addition, because the motor function of the esophagus itself is also impaired, the esophagus often complains of severe chest pain that is mistaken for myocardial infarction due to abnormal contraction.

  The incidence of achalasia is said to be about 1 per 100,000 population, but its symptoms, especially passage disorders, are a serious problem for patients. However, the cause of its onset has not been elucidated, and it is understood that this is a multifactorial disease involving many causes such as degeneration of esophagus and LES nerve cells, viral infection, stress and the like. In addition to the above external factors, the involvement of internal factors such as gene polymorphism, particularly single nucleotide polymorphism (SNP), has been pointed out.

  Examples of SNPs related to esophageal achalasia include tyrosine phosphatase N22 gene (Non-patent document 1), vasoactive peptide receptor receptor 1 gene (Non-patent document 2), IL23 receptor gene (Non-patent document 3), and IL10 promoter region (Non-patent document 3). SNPs in patent document 4), c-kit gene (non-patent document 5), lymphotoxin-α (LTA) gene, TNF-α locus (non-patent document 6), IL33 gene (non-patent document 7), etc. have been reported. Yes.

  However, since there are patients with esophageal achalasia that do not have the above SNP, it has been suggested that there is a new esophageal achalasia-related gene (Achalasia susceptibility gene) SNP other than these.

Santiago et al., Human immunology, 2007, 68, 867-70. Paladini et al., Neurogastroenterology and mobility, 2009, Vol. 21, 597-602. de Leon et al., Neuroastroenterology and mobility, 2010, Vol. 22, 734-8, e218. Nunez et al., Human immunology, 2011, 72, 749-52. Arahdab et al., Neurogastroenterology and mobility, 2012, 24, 27-30. Wouters et al., Gut, 2014, 63, 1401-9. Latiano et al., Human immunology, 2014, 75, 364-9.

  The object of the present invention is to establish an onset risk determination technique using detection of a susceptibility gene for esophageal achalasia and its SNP.

  By analyzing the presence of genetic polymorphisms in a plurality of patients clinically diagnosed with esophageal achalasia, the present inventors have found a plurality of SNPs associated with the development of esophageal achalasia. Completed.

(1) SNPs indicated by rs numbers registered in the National Center for Biotechnology Information (NCBI) SNP database, rs18881420 and rs3795850 in the ALK gene on human chromosome 2, PSORS1C1 on human chromosome 6 Rs1036646 in the gene, rs2032582 in the ABCB1 gene on the human chromosome 7, RP5-1171I10.4 on the human chromosome 17, or rs2526374 in the RNF43 gene, rs1127354 in the ITPA gene on the human chromosome 20, on the human X chromosome A method for detecting one or more SNPs selected from the group consisting of rs4898 gene in TIMP1 gene or SYN1 gene and / or SNP in linkage disequilibrium with the SNP Including method of determining the risk of developing esophageal achalasia.
(2) Risk of developing esophageal achalasia when the base of rs3795850, rs1036646, rs2032582 and / or rs2526374 is thymine, the base of rs18881420 and / or rs4898 is cytosine, and / or the base of rs1127354 is adenine The determination method according to (1), further including a step of determining that is high.
(3) The determination method according to (1) or (2), wherein the SNP is rs4898.
(4) A kit for determining the risk of developing esophageal achalasia, comprising one or more of the following oligonucleotides a) or b).
a) Oligonucleotide for primer capable of selectively amplifying a base sequence containing the SNP according to any one of (1) to (3) and / or SNP in linkage disequilibrium with the SNP b) (1) The oligonucleotide for a probe which has a base sequence complementary to the base sequence which consists of a continuous at least 10 base pair containing SNP in any one of-(3) and / or SNP which is a linkage disequilibrium with this SNP

  The SNP and / or SNP in linkage disequilibrium with the SNP in the present invention can be used as a marker for determining the risk of developing esophageal achalasia. Thereby, it becomes possible to determine the risk of developing esophageal achalasia at an early stage, and it is expected that medical guidance or treatment can be started at an earlier stage for a person with high risk. Each gene having the SNP and / or SNP in linkage disequilibrium with the SNP can be used as a marker gene for detecting these diseases as an esophageal achalasia susceptibility gene.

The single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) in the present invention is an international standard sequence (GRCh37, http://www.ncbi.nlm.nih.gov/projects/genome/assomely/grc/grc/human). Or hg19, https://genome.ucsc.edu/cgi-bin/hgGateway?db=hg19), which refers to the diversity of single nucleotides present at a frequency of 1% or more in the population. In the present specification, the SNP is specified by an rs number that is a reference number of the NCBI SNP database (http://www.ncbi.nlm.nih.gov/snp). Linkage disequilibrium refers to the case where the frequency of occurrence of a particular allele combination at two closely linked loci differs from the expected value estimated from the respective gene frequencies, and is in linkage disequilibrium in the present invention. SNP refers to an SNP having a linkage disequilibrium coefficient (r ² ) of 0.8 or more with any SNP specified by the rs number in the present invention.

  The present invention relates to rs18841420 and rs3795850 in the ALK gene on human chromosome 2, rs1063646 in the PSORS1C1 gene on human chromosome 6, rs2032582 in the ABCB1 gene on human chromosome 7, RP5 on human chromosome 17 One or more SNPs selected from the group consisting of rs2526374 in the -1171I10.4 or RNF43 gene, rs1127354 in the ITPA gene on human chromosome 20, and the rs4898 gene in the TIMP1 gene or SYN1 gene on the human X chromosome and / or The present invention relates to a method for determining the risk of developing esophageal achalasia, comprising a step of detecting a SNP in linkage disequilibrium with the SNP.

  The ALK gene on the human chromosome 2 is Anaplastic Lymphoma Receptor Tyrosine Kinase, the PSORS1C1 gene on the human chromosome 6 is Psoriasis Susceptibility 1 Candate 1, and the ABCPbette ACBsbetteACBsbetteATB -Family B (MDR / TAP), Member 1, RP5-1171I10.4 on human chromosome 17 is Ribosomal protein, RNF43 gene is Ring Finger Protein 43, and ITPA gene on human chromosome 20 is inosine. Triphosphatase, TIMP1 gene on human X chromosome is TIMP Metallopeptidase inhibitor 1 and the SYN1 gene are genes encoding synapsin I, respectively.

  As described in detail in the Examples below, the SNP is a SNP that has been confirmed to be present at a higher frequency in patients who are clinically diagnosed with esophageal achalasia than healthy individuals. Table 1 shows the position of each SNP on the chromosome, rs number, base substitution, amino acid substitution, frequency and P value in patients or healthy individuals.

  Specifically, the determination method of the present invention is such that when the base of rs3795850, rs1063646, rs2032582 and / or rs2526374 is thymine, the base of rs18881420 and / or rs4898 is cytosine, and / or the base of rs1127354 is adenine. In some cases, the risk of developing esophageal achalasia is high.

  Since all of the SNPs shown in Table 1 are present in the exon region of each gene containing the SNP, it is presumed that some change in the protein encoded by this gene is involved in the risk of developing esophageal achalasia or the onset itself. .

  A particularly preferred SNP in the present invention is rs4898. rs4898 is located in the exon 5 region of the TIMP1 gene. This site is a binding site for snRNP that is essential for pre-mRNA splicing, and therefore rs4898 is presumed to inhibit TIMP1 mRNA maturation. On the other hand, rs4898 is simultaneously located in the intron of the SYN1 gene. SYN1 is a molecule expressed in the nerve terminal synaptic vesicle membrane, controls the movement of the synaptic vesicle to the nerve terminal, and maintains synaptic function homeostasis. It is speculated that rs4898 is involved in the development of esophageal achalasia by affecting the splicing of SYN1 mRNA and decreasing the expression of SYN1 at synapses in the muscular plexus by reducing gene expression.

  The SNPs shown in Table 2 below are also SNPs that have been confirmed to be present at a higher frequency in patients diagnosed clinically as esophageal achalasia compared with healthy individuals. Alternatively, a method for determining the risk of developing esophageal achalasia using a SNP in linkage disequilibrium with the SNP is also an embodiment of the present invention.

  The determination target by the method of the present invention is a human, particularly an Asian race, especially a Japanese. Moreover, you may perform detection of SNP about any nucleic acid of genomic DNA, cDNA, or mRNA. Such nucleic acid may be any biological sample collected from a subject, such as blood, saliva, lymph, airway mucus, bone marrow, urine, semen, peritoneal fluid, tissue cells obtained by biopsy, etc. From the above, it can be extracted, purified and prepared according to a conventional method.

  SNPs identified by the present invention and / or SNPs in linkage disequilibrium with the SNPs can be detected according to conventional methods known to those skilled in the art. Examples of such methods include NASBA method, LCR method, SDA method, LAMP method, TaqMan (registered trademark) PCR and other methods using amplified fragments containing SNP by PCR, SNP using DNA sequencer, etc. A method for directly determining a base sequence, a detection method using a microchip including a DNA chip, a Gene chip, a microchip, a bead array, a chemical cleavage of mismatches (CCM), a primer extension method (CCM: chemical cleavage of mismatches) PEX) or invader method can be mentioned, but is not limited thereto.

  One method of using an amplified fragment is to use a primer set designed so that either a sense primer or an antisense primer hybridizes to that SNP so that an amplified fragment is generated only when the target SNP is present. Can be mentioned. By using such a primer set, it becomes possible to detect the target SNP depending on whether or not an amplified fragment is generated.

  Another method using the amplified fragment is a method using a primer set designed to amplify a region containing the target SNP. SNPs can be detected based on differences in the size, base sequence, higher order structure, etc. of amplified fragments by PCR using such primer sets. For example, the target SNP can be detected from the difference in mobility of amplified fragments when agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like is used.

  It can also be detected using restriction fragment length polymorphism (RFLP). For example, an amplified fragment containing an SNP is prepared using an appropriate primer set, the amplified fragment is cleaved with a restriction enzyme known to produce a fragment of a unique length according to the target SNP, and an agarose gel The target SNP can be detected from the difference in mobility of the cut product when electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis or the like is used.

  In addition, the target SNP may be detected by directly determining the base sequence of the amplified fragment or the cleaved product. PCR-single-stranded conformation polymorphism, wherein the amplified fragment or cleaved product is converted into single-stranded DNA by heat denaturation, and then separated by gel electrophoresis, and the change in mobility due to the change in base sequence is analyzed. The target SNP may be detected by (SSCP).

  SNPs can also be detected by hybridization with a probe specific for one SNP. The probe may be any probe as long as it contains the aforementioned SNP, hybridizes with DNA or the like prepared from a biological sample, and gives a detectable degree of specificity under the detection conditions employed.

  As the probe, for example, an oligonucleotide that can hybridize to a sequence of at least 10 bases, preferably 10 to 100 bases, more preferably 10 to 50 bases, including the SNP can be used. In addition, it is preferable to select the oligonucleotide so that the SNP is present almost at the center of the probe. As long as the oligonucleotide can function as a probe, that is, as long as it hybridizes with the sequence of the gene polymorphism of interest, but does not hybridize with the sequence of other gene polymorphisms, One or more substitutions, deletions, additions may be included. The probe may be labeled with an appropriate means such as a fluorescent substance or a radioactive substance, if necessary.

  Hybridization conditions used in the present invention are conditions sufficient to distinguish gene polymorphisms. For example, when DNA or the like prepared from a biological sample is one allele of a genetic polymorphism, it hybridizes, but when it is another allele, it does not hybridize under conditions such as stringent conditions. is there.

  The probe can also be used as a DNA chip with one end fixed to a substrate. In this case, only a probe corresponding to one allele of the gene polymorphism may be fixed to the DNA chip, or probes corresponding to a plurality of alleles of the gene polymorphism may be fixed.

  A preferable method in the present invention is a method using gene amplification by PCR, particularly TaqMan (registered trademark) PCR, which is easy to operate and highly reliable. Specifically, using a primer oligonucleotide set that can amplify a region containing the target SNP and two types of TaqMan (registered trademark) probes corresponding to SNP base mutations having a sequence complementary to each allele, Perform PCR.

  Many of the methods for detecting SNPs described above require oligonucleotides according to the respective principles, for example, primer oligonucleotides for gene amplification, or probe oligonucleotides for hybridization. These can be appropriately designed and synthesized by those skilled in the art based on the principle of each method and the specific base sequence of the SNP to be detected. Oligonucleotides for detecting specific SNPs according to the present invention are also designed and synthesized by those skilled in the art as appropriate based on the principle of the detection method employed and the specific base sequence of the SNP specified in the present invention. can do.

  The present invention provides a kit for determining the risk of developing esophageal achalasia, which can amplify a base sequence containing a) the SNP and / or a SNP in linkage disequilibrium with the SNP. A primer oligonucleotide, or b) a probe oligonucleotide having a base sequence complementary to a base sequence consisting of at least 10 consecutive base pairs including the SNP and / or a SNP in linkage disequilibrium with the SNP.

  As described in the SNP detection method, the oligonucleotide contained in the kit of the present invention is appropriately selected by those skilled in the art based on the principle of the detection method employed and the specific base sequence of the SNP specified in the present invention. Can be designed and synthesized.

  The kit of the present invention may contain reagents, reaction components and the like suitable for each method for detecting a gene polymorphism in addition to the above-mentioned oligonucleotide. For example, enzyme buffers, dNTPs, control reagents (eg, tissue samples, positive and negative control target oligonucleotides, etc.), labeling or detection reagents, solid supports, instructions, and the like. The oligonucleotide may be included in the kit as a microarray immobilized on a support.

  With the kit for determining the risk of developing esophageal achalasia according to the present invention, the risk of developing the subject can be determined easily and quickly, and appropriate medical treatment can be performed according to the result.

  The following examples further illustrate the present invention.

<Example 1>
From 21 patients diagnosed as esophageal achalasia and 20 healthy persons as esophageal peristaltic waves disappeared by upper esophageal tract endoscopy or x-ray contrast, esophageal dilatation and oral esophageal pressure measurement. Peripheral blood (8 mL) was collected from the sample, and mononuclear cells were separated by Ficoll specific gravity centrifugation. Then, DNA was extracted and purified using a silica gel column. All of the above patients and healthy individuals should comply with the ethical guidelines for human genome / gene analysis research, understand the content of this study that has been reviewed and approved by the ethics committee of Asahikawa Medical University, and agree to research cooperation. It is.

  Genes that have been reported to be associated with inflammatory bowel disease, pancreatic disease, liver disease, blood disease, and metal metabolism disease, and genes related to metabolism, immunity, or signal transduction associated with each disease A total of 1031 genes that were selected and from which duplicate genes and genes for which amplicons cannot be set were deleted were determined as target genes for SNP analysis. A total of 12609 amplicons were divided to cover the entire exon region of the target gene, and primer sets for each were prepared.

  Using 50 ng of DNA as a template, PCR was performed using 5 primer pools to create an amplicon, and then the DNA sequence was analyzed using a high-power sequencer Ion Proton (Life technologies) according to the manufacturer's protocol. SNP analysis on the 1031 gene was performed. In the analysis, a preliminary sequence was performed twice, and it was confirmed that the analysis length and the coverage of each amplicon were sufficient for examination.

  In all samples, the international standard sequence (reference) and the sequence sequence were compared, and a difference of 4750 total references was detected in the patient group and the healthy group. From this difference, the difference in the appearance frequency of gene abnormalities between patients and healthy individuals was regarded as significant when the p-value <0.05 in the Fisher test, and a total of 46 types of SNPs shown in Table 2 were extracted as candidates.

  Among the SNPs shown in Table 2, SNPs having a strand bias of less than 0.55 are extracted as significant polymorphisms from SNPs (circles in the table) located in the exon region of the gene, and Hapmap JPT (http) : //Hapmap.ncbi.nlm.nih.gov/downloads/index.html.ja, a reference in which the gene sequences of 86 Japanese living in Tokyo are accumulated) rs1635498 considered to be a SNP unique to Japanese By removing, 7 types of SNPs shown in Table 1 were selected.

  Furthermore, PCR was performed using the purified DNA as a template to prepare amplified fragments containing each of the seven types of SNPs described above, and sequencing was performed. As a result, all 16 patient individuals in whom SNP was detected with a high-power sequencer were obtained for rs4898. It was confirmed to be homo or hetero and have the same polymorphism. Table 3 shows the relationship between the sexes of the patient group and the healthy person group and the rs4898 allele.

The present invention has applicability as a method for determining the risk of developing esophageal achalasia, and is thereby expected to be able to give medical guidance or start treatment at an early stage for people with a high risk of developing the disease. The

Claims (4)

  A single nucleotide polymorphism indicated by rs number registered in the National Center for Biotechnology Information (NCBI) SNP database, rs18841420 and rs3795850 in ALK gene on human chromosome 2, PSORS1C1 on human chromosome 6 Rs1036646 in the gene, rs2032582 in the ABCB1 gene on the human chromosome 7, RP5-1171I10.4 on the human chromosome 17, or rs2526374 in the RNF43 gene, rs1127354 in the ITPA gene on the human chromosome 20, on the human X chromosome One or more single nucleotide polymorphisms selected from the group consisting of rs4898 gene in TIMP1 gene or SYN1 gene and / or single nucleotide polymorphisms in linkage disequilibrium with the single nucleotide polymorphism Comprising the step of leaving, the determination method of the risk of developing esophageal achalasia.   The risk of developing esophageal achalasia is high when the base of rs3795850, rs1063646, rs2032582 and / or rs2526374 is thymine, the base of rs18881420 and / or rs4898 is cytosine, and / or the base of rs1127354 is adenine. The determination method according to claim 1, further comprising a determination step.   The determination method according to claim 1, wherein the SNP is rs4898. A kit for determining the risk of developing esophageal achalasia, comprising one or more of the following oligonucleotides a) or b).
a) For a primer capable of selectively amplifying a single nucleotide polymorphism according to any one of claims 1 to 3 and / or a nucleotide sequence comprising a single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism Oligonucleotide b) A nucleotide sequence comprising at least 10 consecutive base pairs comprising the single nucleotide polymorphism according to any one of claims 1 to 3 and / or the single nucleotide polymorphism in linkage disequilibrium with the single nucleotide polymorphism. Probe oligonucleotide having a complementary nucleotide sequence