JP2016149988A - Method of examination of autism spectrum disorder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method of examination of autism spectrum disorder.SOLUTION: The invention was completed by finding out that a single-nucleotide polymorphism of a base existing on a BST-1 gene and a single-nucleotide polymorphism of a base in linkage disequilibrium with the base are involved in autism spectrum disorder.SELECTED DRAWING: None

Description

  The present invention relates to an inspection method for an autism spectrum.

(Autism spectrum)
The autism spectrum is an autistic disorder, Asperger's disorder, Rett syndrome, pediatric disintegration disorder, and unspecified pervasive developmental disorder as a component of a pervasive developmental disorder. . The main characteristics (disorders) of the spectrum include qualitative obstacles to interpersonal interactions, significant abnormalities in communication or obstacles to their development, and marked localization of activities and interests.

  The present inventors previously reported a mouse deficient in the gene for the membrane protein molecule CD38 having ADP ribosyl cyclase activity as an animal model of autism spectrum disorder (ASD) (see Non-Patent Document 1). . In this CD38-deficient mouse, disorder of social behavior, such as decreased social memory and child-rearing behavior is observed. As a mechanism for the occurrence of such behavioral abnormalities, the present inventors have found that a decrease in cyclic ADP ribose production due to CD38 gene deficiency impairs calcium-induced calcium release, and oxytocin, a posterior pituitary hormone necessary for social recognition, Experimental data indicating that secretion is reduced were obtained (see Non-Patent Document 1). Since then, there have been reports showing the relationship between single-nucleotide polymorphism (SNP) of human CD38 gene and ASD (see Non-Patent Document 2 and Non-Patent Document 3). Rs3796863 (C> A), an SNP of the human CD38 gene, is a sample of high-functioning autism derived from the Autism Genetic Resource Exchange (AGRE). Although a significant correlation was shown in the sample, no correlation was observed in the sample of a high-functioning autistic person in Japan (see Non-Patent Document 2). Another SNP rs1800561 (g.4693C> T; p.R140W) was found in 3 probands of 29 ASD individuals, and was also found in 10 ASD and ASD prone in 3 families (see Non-Patent Document 2). The ASD person with this R140W type polymorphic change had a lower plasma oxytocin concentration than the ASD person who had the multitype R140 as a homozygote (see Homozygous) (see Non-Patent Document 2).

(BST-1)
The CD157 molecule is also called bone marrow stromal antigen-1 (BST-1) and belongs to the NAD degrading enzyme / ADP ribose cyclase family (see Non-patent Documents 4-12). This family of molecules also includes the CD38 molecule. CD157 and CD38 have 36% similarity in amino acid sequence, and both act as an ectoenzyme that metabolizes nicotinamide adenine dinucleotide (NAD). It is also known to act as a signal transduction molecule for biological information, leading to deflection, migration and extravasation of human granulocytes. CD157 / BST-1 was originally isolated as a cell membrane surface molecule that promotes the growth of pre-B lymphocytes (see Non-patent Documents 7 and 13), but humoral immune reaction, neutrophil tissue infiltration It plays various roles such as hematopoietic stem cell support (see Non-Patent Documents 7, 9, and 14-16). In addition, CD157 / BST-1 is involved in the formation of various pathological conditions including the survival of B lymphocytes in rheumatoid arthritis, the progression of leukemia, and the metastasis of human ovarian cancer (see Non-Patent Documents 7 and 12). 17-19).

Recent genetic studies have identified CD157 / BST-1 present in the human chromosome 4p15 region as a disease susceptibility marker for Parkinson's disease (see Non-patent Documents 20-29).
In addition, recently, the present inventors have shown that mice lacking CD157 / BST-1 have anxiety-like behavior and depression-like behavior (Reference: Non-Patent Document 30).
However, these documents do not disclose or suggest that “the three single nucleotide polymorphisms on CD157 / BST-1 specified in the present invention are associated with autism spectrum disorder”.

Furthermore, the following prior patent documents exist.
Patent Document 1 discloses “a method for examining an autism spectrum characterized by determining whether rs35062132 on the oxytocin receptor gene is major or minor”.
Patent Document 2 discloses a “method for confirming genetic predisposition to autism or autism spectrum disorder in a subject, or the presence thereof, including identifying the presence or absence of a genetic mutation in a JARID2 nucleic acid molecule”. Yes.
However, these patent documents do not disclose or suggest that “the three single nucleotide polymorphisms on CD157 / BST-1 specified in the present invention are related to autism spectrum disorder”. .

JP 2014-171392 A Special Table 2013-538589

Nature 2007, 446 (7131), 41-5. Neurosci Res 2010, 67 (2), 181-91. Autism Res 2010, 3 (6), 293-302). FEBS letters 1994, 356 (2-3), 244-8. Biochemical and biophysical research communications 1994, 203 (2), 1309-17. Immunogenetics 1999, 49 (7-8), 597-604. Chemical immunology 2000, 75, 235-55. The FEBS journal 2005, 272 (18), 4590-7. Molecular medicine (Cambridge, Mass.) 2006, 12 (11-12), 334-41. Physiological reviews 2008, 88 (3), 841-86. The Journal of biological chemistry 2012, 287 (38), 31633-40. Cytometry. Part B, Clinical cytometry 2013, 84 (4), 207-17. Proceedings of the National Academy of Sciences of the United States of America 1994, 91 (12), 5325-9. Blood 2004, 104 (13), 4269-78. The Journal of biological chemistry 2005, 280 (7), 5343-9. Critical reviews in biochemistry and molecular biology 2013, 48 (4), 397-408. The Journal of clinical investigation 1998, 102 (3), 606-18. J Natl Cancer Inst 2010, 102 (15), 1160-77. Frontiers in bioscience 2014, 19, 366-78. Nature genetics 2009, 41 (12), 1303-7. Lancet 2011, 377 (9766), 641-9. BMC Med Genet 2011, 12, 104. Human molecular genetics 2011, 20 (3), 615-27. Eur J Hum Genet 2011, 19 (6), 655-61. Curr Opin Neurol 2011, 24 (4), 318-23. PLoS genetics 2012, 8 (3), e1002548. Neurology 2012, 79 (7), 659-67. Parkinsonism & related disorders 2012, 18 (8), 958-63. Nature genetics 2014, 46, 989-993. Frontiers in behavioral neuroscience 2014, 8, 133.

  An object of the present invention is to provide a novel test method for an autism spectrum.

  As a result of intensive studies to solve the above problems, the present inventors have found that a single nucleotide polymorphism of a base present on CD157 / BST-1 and a single nucleotide polymorphism of a base in linkage disequilibrium have autism spectrum disorder And the present invention has been completed.

The present invention comprises the following.
“1. A single nucleotide polymorphism of a base present on CD157 / BST-1 in a sample obtained from a subject or a single nucleotide polymorphism of a base in linkage disequilibrium is detected. Inspection method.
2. The single nucleotide polymorphism of the base is any one or more from rs4301112, rs28532698 and rs10001565, and the single nucleotide polymorphism of the base in linkage disequilibrium is from rs11931532, rs12645693, rs4698412, rs4538475, rs11724635 and rs4273468 2. The inspection method according to item 1, wherein any one or more.
3. 3. The inspection method according to item 1 or 2, wherein the single nucleotide polymorphism of the base and the single nucleotide polymorphism of the base in linkage disequilibrium are determined to be major or minor.
4). An autism spectrum test kit comprising one or more of the following probes.
(1) rs4301112 minor type recognition probe (2) rs28532698 minor type recognition probe (3) rs10001565 minor type recognition probe (4) rs11931532 minor type recognition probe (5) rs12645693 minor type recognition probe (6) rs4698412 Minor type recognition probe (7) minor type recognition probe of rs4538475 (8) minor type recognition probe of rs11724635 (9) minor type recognition probe of rs12502586 (10) minor type recognition probe of rs4273468 "

  Single nucleotide polymorphisms of nucleotides present on CD157 / BST-1 used in the test method of the present invention and single nucleotide polymorphisms of nucleotides in linkage disequilibrium are statistically significantly higher in patients with autism Since it has been confirmed that it exists in the group, it can be used in a method for examining an autism spectrum.

1 is a list of single nucleotide polymorphisms analyzed in Example 1. FIG. Structure of human CD157 / BST-1 and position of SNP {GenBank accession numbers Based on the data of NM_004334 and NC_000004, the exon intron structure is illustrated. Black and white squares (boxes) mean a translated region (protein coding region) and an untranslated region, respectively. Asterisk means SNP, previously reported as a marker for Parkinson's disease}. CD157 / BST-1 linkage disequilibrium plot {number inside square means D 'value. Asterisk means SNP, previously reported as a marker for Parkinson's disease}.

  The present invention detects a single nucleotide polymorphism of a base present on CD157 / BST-1 derived from a subject (hereinafter sometimes referred to as “SNP”) and a single nucleotide polymorphism of a base in linkage disequilibrium. Provides a method for examining an autism spectrum (hereinafter, sometimes referred to as “test method of the present invention”).

(Testing method for autism spectrum)
The method for examining an autism spectrum of the present invention is characterized by detecting a single nucleotide polymorphism on CD157 / BST-1. More specifically, it is characterized by determining whether the single nucleotide polymorphism is major or minor.
Single nucleotide polymorphism is included in genetic polymorphisms and means a single nucleotide difference between individuals. A gene polymorphism is an individual difference in the sequence of DNA constituting a gene. In the present specification, the rs number, which is a number representing SNPs, is obtained by taking into account human genetic information published in the NCBI SNP database dbSNP.

  In the method for examining an autism spectrum according to the present invention, an index for determining that the patient is an autism spectrum patient or is suspect (has a genetic predisposition to the autism spectrum) It may also be referred to as “)”. From the results of Example 1 below, due to the difference in alleles, it is suspected that the patient is an autism spectrum patient, has an autism spectrum patient (has a genetic predisposition to the autism spectrum), or It can be determined that there is no.

(Single nucleotide polymorphism on CD157 / BST-1)
Based on the result of Example 1 below, the genotype of rs4301112 is A / A (adenine and adenine homozygous type), and A / G (adenine and guanine heterozygous type) and G / G (heterozygous type). The case of guanine and guanine homozygous) is defined as minor. If the rs4301112 genotype is minor (especially adenine and guanine heterozygous), it is likely that the patient is an autism spectrum patient, or has a genetic predisposition to the autism spectrum. It is determined that it has.
Based on the result of Example 1 below, the case where the genotype in rs28532698 is A / A (homozygous type of adenine and adenine) is taken as a major, and A / G (heterozygous type of adenine and guanine) and G / G ( If the rs28532698 genotype is minor (especially heterozygous between adenine and guanine), it is a patient with autism spectrum. Or have a genetic predisposition to the autism spectrum.
Based on the result of Example 1 below, the genotype in rs10001565 is C / C (cytosine and cytosine homozygous) as the major, C / T (cytosine and thymine heterozygous) and T / T (heterozygous). The case of thymine and thymine homozygous) is defined as minor. If the rs10001565 genotype is minor (especially a heterozygous cytosine and thymine), it is likely that the patient has an autism spectrum, is a patient with an autism spectrum, or has a genetic predisposition to the autism spectrum. It is determined that it has.

(Single nucleotide polymorphism in linkage disequilibrium)
When the frequency of combination of specific alleles at two closely linked loci in a biological population is different from the expected value estimated from each allele frequency, this is called linkage disequilibrium.
From the results of Example 1 below, the single nucleotide polymorphisms in linkage disequilibrium with the three single nucleotide polymorphisms described above can be exemplified as follows (see: FIG. 3). If you have any one or more of the following single nucleotide polymorphisms in linkage disequilibrium, you are likely to be a patient with autism spectrum, or a genetic predisposition to autism spectrum It is determined that it has.
rs11931532: In the case of minor type, it is judged as a risk allele of patients with autism spectrum.
rs12645693: In the case of a minor type, it is determined as a risk allele of a patient with autism spectrum.
rs4698412: In the case of minor type, it is determined as a risk allele of patients with autism spectrum.
rs4538475: In the case of minor type, it is judged as a risk allele of patients with autism spectrum.
rs11724635: In the case of minor type, it is judged as a risk allele of patients with autism spectrum.
rs12502586: In the case of minor type, it is determined as a risk allele of a patient with autism spectrum.
rs4273468: In the case of minor type, it is judged as a risk allele for patients with autism spectrum.

(Inspection process of the present invention)
An example of the inspection process of the inspection method for the autism spectrum of the present invention is as follows.
(1) A step of determining a genotype for a single nucleotide polymorphism of a base present on CD157 / BST-1 and a single nucleotide polymorphism of a base in linkage disequilibrium in a sample obtained from a subject (2) If the genotype is minor, the step of determining that the patient is an autism spectrum patient, is likely to be an autism spectrum patient, or has a genetic predisposition to the autism spectrum

(Inspection target)
The inspection method of the present invention is expected to be effective for yellow races, preferably East Asian races, particularly preferably Japanese.

(subject)
The test subject of the present invention refers to an individual having autism spectrum symptoms, suspected of having autism spectrum symptoms, or having a high genetic predisposition to the autism spectrum, In addition to humans, chimpanzees and the like are included, but humans are most preferable. In the above, the sample obtained from the subject is not particularly limited as long as it is derived from the subject. For example, blood, lymph, semen, urine, saliva (including oral mucosal cells), other body fluids, and biological tissues In particular, blood (lymphocytes) is most preferable in terms of easy handling such as collection and low invasiveness. The method for obtaining the sample from the subject is not particularly limited, and can be a method known per se.

(Sample processing method)
The sample obtained from the subject can be processed in advance to detect each single nucleotide polymorphism. The method for extracting DNA or mRNA from the sample is not particularly limited, and can be a method known per se. For example, when DNA is extracted, salting out, PCI (phenol / chloroform / isoamyl alcohol extraction) method, a method using a commercially available DNA extraction kit or the like, or other known methods can be used. For example, when extracting mRNA, it is possible to use a method in which the solution is acidified in the PCI method and extracted from the aqueous layer, a method using an oligo dT column or the like, a method using a commercially available RNA extraction kit, or other known methods. .

  When the amount of polynucleotide such as DNA or mRNA obtained from the sample is small, these can be amplified by a known method, if necessary. The method for amplifying the polynucleotide is not particularly limited, and a method known per se can be adopted. When the target of amplification is DNA, for example, PCR (polymerase chain reaction) method or LAMP (loop-mediated isothermal amplification) method is used. When the target of amplification is mRNA, RT (reverse transcription) -PCR method, RT-LAMP method, etc. can be used.

  A region containing a single nucleotide polymorphism can be amplified from DNA or mRNA obtained from the sample. The amplified nucleotide fragment may be of a measurable size according to the method for determining the allele type described below. Nucleotide fragments of measurable size can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more.

(Primer used in the inspection method of the present invention)
The primer for amplifying the region containing each single nucleotide polymorphism specifically hybridizes with the antisense strand and the 3 ′ end of the sense strand of the polynucleotide of the region containing each single nucleotide polymorphism. , Forward (sense) primers and reverse (antisense) primers. These primers can be appropriately designed according to the base sequence of the region containing each single nucleotide polymorphism.

  The size of the primer is not particularly limited as long as it can amplify a region containing a single nucleotide polymorphism, but it may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp. The primer may be labeled with a labeling substance. Examples of the labeling substance include fluorescent molecules, radioactive substances, biotin, digoxigenin, enzyme labels, and the like. The label can be bound to the polynucleotide according to a method known per se.

  Specific primers and probes in the present invention include oligonucleotides having the following base sequences.

(Rs4301112)
Forward primer for region amplification containing rs4301112:
5'-GAATTCACTGAAGTTCTCCTAAACACTTTCAATGGGACC-3 '(SEQ ID NO: 1)
Reverse primer for region amplification containing rs4301112:
5'-TCAGAGCCATGTCTATAGAGTTAAACCATTAACAGCTGAT-3 '(SEQ ID NO: 2)
G type minor probe:
5'-CCGTGTCCACTCTAGAAAAACCTTCTTAAAAAAAAATAAAGAGCTTTTTGAG-3 '(SEQ ID NO: 19)
Type A major recognition probe:
5'-ACCACCGCTTGAATACAAAACATTCTTAAAAAAAAATAAAGAGCTTTTTGAA-3 '(SEQ ID NO: 20)
Common probe:
5'-GTTTTTCTTCTTTAGCTTTTATTTTTAGGTTACTTTTTACCGTCGCTAATAATCGGATATGG-3 '(SEQ ID NO: 21)

(Rs28532698)
Forward primer for region amplification containing rs28532698:
5'-TATGGGCAATAAAGAAATGGGAGCTTCCTCATTTCTTTAC-3 '(SEQ ID NO: 3)
Reverse primer for region amplification containing rs28532698:
5'-TACCACTTGGTGAGCAGCTATGGGCCAGACACTTTAT-3 '(SEQ ID NO: 4)
G type minor probe:
5'-CCGTGTCCACTCTAGAAAAACCTCATGGTTTGTATATTTTTCTGAACG-3 '(SEQ ID NO: 22)
Type A major recognition probe:
5'-ACCACCGCTTGAATACAAAACATCATGGTTTGTATATTTTTCTGAACA-3 '(SEQ ID NO: 23)
Common probe:
5'-TAGATTATATTTCCAGAAAAGGACATTTGCCTTGTTTCTAGCAATCTTTAACCCAGACGG-3 '(SEQ ID NO: 24)

(Rs10001565)
Forward primer for region amplification containing rs10001565:
5'-TATGGGCAATAAAGAAATGGGAGCTTCCTCATTTCTTTAC-3 '(SEQ ID NO: 5)
Reverse primer for region amplification containing rs10001565:
5'-TACCACTTGGTGAGCAGCTATGGGCCAGACACTTTAT-3 '(SEQ ID NO: 6)
T type minor recognition probe:
5'-ACCACCGCTTGAATACAAAACATTGGGCTGAGTTATAATTGCT-3 '(SEQ ID NO: 25)
C type major recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTGGGCTGAGTTATAATTGCC-3 '(SEQ ID NO: 26)
Common probe:
5'-GGGCCAGAGCCCAGGTCTCCGCAGAGTGAGTTTCATTTAGGCG-3 '(SEQ ID NO: 27)

(Rs11931532)
Forward primer for region amplification containing rs11931532:
5'-TGGAGTTTCACACTTGTTGACTGGGCTGGAGTGCA-3 '(SEQ ID NO: 7)
Reverse primer for region amplification containing rs11931532:
5'-GTGTGTATTTTAAATTTTGATATGTTCTGAGAAATTTTGATATGT-3 '(SEQ ID NO: 8)
Type A minor recognition probe:
5'-ACCACCGCTTGAATACAAAACATTGGGCTGAGTTATAATTGCT-3 '(SEQ ID NO: 28)
G-type major recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTGGGCTGAGTTATAATTGCC-3 '(SEQ ID NO: 29)
Common probe:
5'-CTGTCATCAGGTTGTGTACAAAGTCCTTGGGCCTCAACTTATTGTCATTGGCG-3 '(SEQ ID NO: 30)

(Rs12645693)
Forward primer for region amplification containing rs12645693:
5'-ACAGCTGTTCTTATATGCTACATTCTCTAAAATATTGCCAATA-3 '(SEQ ID NO: 9)
Reverse primer for region amplification containing rs12645693:
5'-TCTGGAGTCATCATCTCATTTTACTCCAACCACATTCC-3 '(SEQ ID NO: 10)
G type minor probe:
5'-ACCACCGCTTGAATACAAAACATGGGTACACTTACTGGTGGG-3 '(SEQ ID NO: 31)
Type A major recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTGGGTACACTTACTGGTGGA-3 '(SEQ ID NO: 32)
Common probe:
5'-AGTAGAATATAGCCCTTAAAGACAATGGTAGGTGCGTTATAATCTATTCCCGGGC-3 '(SEQ ID NO: 33)

(Rs11724635)
Forward primer for region amplification containing rs11724635:
5'-CTTTTCTCTTTCTCTAAGTACCCAAAAGTCTTATGCTGCT-3 '(SEQ ID NO: 11)
Reverse primer for region amplification containing rs11724635:
5'-CAAAAAACAGTAGACATCACTTCTCTGGGACAGCTG-3 '(SEQ ID NO: 12)
T type minor recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTAYGSCACACCTTGTTATAAAT-3 '(SEQ ID NO: 34)
G-type major recognition probe:
5'-ACCACCGCTTGAATACAAAACATAYGSCACACCTTGTTATAAAG-3 '(SEQ ID NO: 35)
Common probe:
5'-CTTCCCTTGTACCCTCCCTGGCCGTCCGTAAGAAGCTTTTGGGAC-3 '(SEQ ID NO: 36)

(Rs4698412)
Forward primer for region amplification containing rs4698412:
5'-ACTTCTCTGGGACAGCTGGGCTGGTTTCAGTCGGA-3 '(SEQ ID NO: 13)
Reverse primer for region amplification containing rs4698412:
5'-ATTCGGGAAAAAAGTGAAGTTATGTTTAGTTTTTAAAAA-3 '(SEQ ID NO: 14)
Type A minor recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTCCAGCCAAGGCGCA-3 '(SEQ ID NO: 37)
G-type major recognition probe:
5'-ACCACCGCTTGAATACAAAACATCAGCCAAGGCGCG-3 '(SEQ ID NO: 38)
Common probe:
5'-TCTCTTAGACCAAGCTTGGAAAACACAGTGTCTGCAGTGATGTCGTTGGTAGAATA-3 '(SEQ ID NO: 39)

(Rs4273468)
Forward primer for region amplification containing rs4273468:
5'-ATAAAAACAAATGGATCTGGAACTAAGTAAGGCTTCGAGG-3 '(SEQ ID NO: 15)
Reverse primer for region amplification containing rs4273468:
5'-TATCATTGCCTCCTGACTGATCTCTCTGCTTCTGCTT-3 '(SEQ ID NO: 16)
C type minor type recognition probe:
5'-ACCACCGCTTGAATACAAAACATCACTCAGGACCCAAGC-3 '(SEQ ID NO: 40)
T-type major recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTCACTCAGGACCCAAGT-3 '(SEQ ID NO: 41)
Common probe:
5'-GTAGGACATTGCTCTGGCAGCATTGTCCGTCGCTAATAATCGGATATGG-3 '(SEQ ID NO: 42)

(Rs4538475)
Forward primer for region amplification containing rs4538475:
5'-ATAAAAACAAATGGATCTGGAACTAAGTAAGGCTTCGAGG-3 '(SEQ ID NO: 17)
Reverse primer for region amplification containing rs4538475:
5'-TGATCTCTCTGCTTCTGCTTGGTTTCTCCTTCAGC-3 '(SEQ ID NO: 18)
T type minor recognition probe:
5'-CCGTGTCCACTCTAGAAAAACCTTCTTGAAGAGCAGATTTTAATATT-3 '(SEQ ID NO: 43)
C type major recognition probe:
5'-ACCACCGCTTGAATACAAAACATCTTGAAGAGCAGATTTTAATATC-3 '(SEQ ID NO: 44)
Common probe:
5'-TGAAATTTGTCTTGCCAAGGGTAGGAAACCACGCGGATTTACATTTAACCAGCC-3 '(SEQ ID NO: 45)

(Autism spectrum test kit)
The test kit for an autism spectrum of the present invention includes any one or more of the following single-nucleotide polymorphism minor type recognition probes. Specific sequences are those described above, but are not particularly limited. Furthermore, if necessary, a major recognition probe, a common probe, a region amplification forward primer containing a single nucleotide polymorphism, and / or a region amplification reverse primer containing a single nucleotide polymorphism may be included.
(1) rs4301112 minor type recognition probe (2) rs28532698 minor type recognition probe (3) rs10001565 minor type recognition probe (4) rs11931532 minor type recognition probe (5) rs12645693 minor type recognition probe (6) rs4698412 Minor type recognition probe (7) Minor type recognition probe of rs4538475 (8) Minor type recognition probe of rs11724635 (9) Minor type recognition probe of rs12502586 (10) Minor type recognition probe of rs4273468

  The test method of the present invention includes a step of determining the type of single nucleotide polymorphism based on a polynucleotide such as DNA or mRNA obtained from a sample or a polynucleotide fragment obtained by the amplification. As a method for determining the type of single nucleotide polymorphism, a method known per se can be used. For example, in addition to the direct sequence method (dideoxy method) for directly determining the nucleotide sequence using a sequencer or the like, RELP (restriction fragment) PCR-RELP method, TaqMan method, DigiTag2 method, single nucleotide primer extension method, SnaPshot method (ABI), ASP-PCR method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP ( sequence specific primer) method, PCR-SSCP (single-stranded conformational polymorphism analysis) method, confirmation of the length of nucleic acid by electrophoresis, and a method using a genetic polymorphism testing instrument such as a DNA chip.

  The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. All the examples were approved by the Kanazawa University School of Medicine Ethics Committee, followed the Declaration of Helsinki, and were done after informed consent to the participants.

  In this example, it was confirmed whether the base substitution of the protein coding region on CD157 / BST-1 is involved in the risk of autism. Details are as follows.

(participant)
A total of 147 subjects (113 males, 34 females, age 15.6 ± 0.6 years) with autism spectrum disorder were recruited from Kanazawa University Hospital. All subjects with autism spectrum disorder met DSM-IV criteria for pervasive developmental disorder. The diagnosis was made by two experienced child psychiatrists based on interviews and medical chart reviews. The subject had no obvious physical malformations. Two experienced child psychiatrists independently diagnosed the majority of patients with autism spectrum disorder through semi-structural behavioral observations and interviews between the subject and their parents. Interviews with the parents were used to evaluate autism-specific behaviors and symptoms. The child psychiatrist was interviewed by Asperger Syndrome Diagnostic Interview (Autism 2001, 5: 57-66.), Revised Autism Diagnostic Interview-Revised (J Autism Dev Disord 1994, 24: 659 -685.), Pervasive Developmental Disorders Autism Society Japan Rating Scale (Autism Society Japan; 2006.) and diagnostic interviews for social and communication disorders (Diagnostic Interview for Social and Communication) Disorders: J Child Psychol Psychiat 2002, 43: 307-325.) And Tokyo Autistic Behavior Scale. A control group of 150 (115 males, 35 females, age 23.8 ± 0.3 years) is healthy volunteers who are not relatives of each other. All patients and controls are Japanese without non-Japanese parents or grandparents.

{Genotyping (determination of genotype)}
Genomic DNA was extracted from venous blood using the Wizard Genomic DNA Purification kit (Promega, Madison, Wisconsin, USA) or from the nail using the ISOHAIR DNA Extraction kit (Nippon Gene, Tokyo, Japan). According to the yield, the obtained genomic DNA was increased by the whole genome amplification method. Detection of SNP is based on the DigiTag2 method (Nishida et al., Analyt. Biochem. 364: 78-85, 2007) using a combination of sequence-specific primer (SSP) -PCR and fluorescence correlation spectroscopy. I did. First, the region around the target SNP is amplified by PCR, then two types of allele-specific probes (query probes) and a common probe based on the opposite sequence across the target SNP are placed on the PCR product. After annealing, the ligation reaction was performed, and further, two kinds of each were detected with Alexa555 and Alexa647 and detected. The SNPs studied were the International HapMap Project (http://www.hapmap.org) and the National Center for Biotechnology Information (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP) ) Information. SNPs with a minor allele frequency (MAF) greater than 0.1 were selected.
In addition, Linkage disequilibrium (LD) blocks were analyzed using HaploView 4.2.

(Statistical analysis)
The genotype and allele frequency data of this Example 1 were analyzed using a contingency table and Fisher's exact test (GraphPad Prism 6; GraphPad Software Inc., San Diego, CA, USA) with a P value of 0.05. If smaller, it was considered to be statistically significant. Also, Nyholt's method (http://genepi.qimr.edu.au/general/daleN/SNPSpD) is used, the effective number of independent SNPs is estimated to be 23, and the P value of each SNP analysis is If less than 0.002, it was judged that there was a statistically significant difference.

(Gene analysis results)
Of the 121 SNPs examined this time (see: Fig. 1), 93 SNPs with a success rate higher than 95% were further statistically analyzed. Of these, three types of SNPs detected significantly higher allele frequencies in the ASD group compared to the control group (rs4301112, odds ratio = 6.4, 95% confidence interval = 1.9 − 22, P = 0.0007; rs28532698, Odds ratio = 6.2, 95% confidence interval = 1.8-21, P = 0.0012 and rs10001565, odds ratio = 5.5, 95% confidence interval = 1.6-19, P = 0.0038; Fisher's exact test; see: Table 1 below) . rs4301112, rs28532698, and rs10001565 are located in the fourth, sixth, and seventh introns, respectively (see: FIG. 2). When calibrating the multiple test using the effective total SNP count, significantly higher allele frequencies were observed for rs4301112 and rs28532698 (see: Table 1). For rs10001565, only genotype C / T was significantly higher in the ASD group compared to the control group (odds ratio = 15, 95% confidence interval = 2.0–117, P = 0.0007; Fisher's exact test; Table 1 ).
Analyzed SNPs included those reported to be associated with Parkinson's disease. These are rs11931532, rs12645693, rs4698412, rs4538475, rs11724635, rs12502586 and rs4273468 (FIG. 2).
Linkage disequilibrium (LD) analysis of these SNPs revealed the existence of two haplotype blocks. One spans 5 kilobases (kb), including rs4301112, rs28532698 and rs10001565 associated with ASD (Block 1; Figure 3), and the other SNP associated with Parkinson's disease spanning 12 kb. It contains groups (Block 2; FIG. 3).
From the above, it was concluded that the base substitutions of rs4301112, rs28532698, and rs10001565 are significantly involved in the risk of autism. Furthermore, single nucleotide polymorphisms in linkage disequilibrium, rs11931532, rs12645693, rs4698412, rs4538475, rs11724635, rs12502586 and rs4273468, are considered to be involved in the risk of autism.

  To provide a new method for testing the autism spectrum.

Claims (4)

A method for examining an autism spectrum, comprising detecting a single nucleotide polymorphism of a base present on a BST-1 gene in a sample obtained from a subject or a single nucleotide polymorphism of a base in linkage disequilibrium.
The single nucleotide polymorphism of the base is any one or more from rs4301112, rs28532698 and rs10001565, and the single nucleotide polymorphism of the base in linkage disequilibrium is rs11931532, rs12645693, rs4698412, rs4538475, rs11724635, rs12502586 and It is any one or more from rs4273468, The inspection method of Claim 1 characterized by the above-mentioned.
The method according to claim 1 or 2, wherein it is determined whether the single nucleotide polymorphism of the base and the single nucleotide polymorphism of the base in linkage disequilibrium are major or minor.
An autism spectrum test kit comprising one or more of the following probes.
(1) rs4301112 minor type recognition probe (2) rs28532698 minor type recognition probe (3) rs10001565 minor type recognition probe (4) rs11931532 minor type recognition probe (5) rs12645693 minor type recognition probe (6) rs4698412 Minor type recognition probe (7) Minor type recognition probe of rs4538475 (8) Minor type recognition probe of rs11724635 (9) Minor type recognition probe of rs12502586 (10) Minor type recognition probe of rs4273468