JP2002153299A - GENOPOLYMORPHISM OF CONSTITUENT MASP-1 OF HUMAN COMPLEMENT-ACTIVATED LECTIN RaRF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a genopolymorphism involved in the onset of infectible diseases, particularly those in the babyhood. SOLUTION: This method for detecting a genopolymorphism involved in the onset of infectible diseases comprises amplifying a given region including the 148th base of the 6th intron of the constituent MASP-1 gene of human complement-activated lectin RaRF and detecting a mutation at the 148th base of the 6th intron. This method is useful for predicting the onset of infectible diseases, particularly those in the babyhood.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting a genetic polymorphism useful for a clinical laboratory diagnosis for predicting the onset of susceptibility to infection, particularly to infants. Methods and methods for detecting polymorphisms in the complement-activating component MASP-1 of the human complement-activating lectin Ra-reactive factor (RaRF) associated with the development of susceptibility to infection, especially in childhood Primers to be used.

[0002]

2. Description of the Related Art With regard to protection against infection with pathogenic microorganisms, attention has been paid to protection against infection by lectins, which is one of the defense defense responses by mechanisms other than antibody production. Antibodies are specific for each foreign substance, but their production takes a certain period of time, whereas the complement-activating lectin RaRF, which is always produced and increases in a short time when needed, is By binding to polysaccharides present on the surface and activating complement, it plays an important role in host defense in the early stage of infection before antibody production. It has also been found that mutations in the genes of RaRF component proteins increase the susceptibility to infection.

[0003] The complement-activating lectin RaRF present in the body fluids of vertebrates such as humans binds to polysaccharides present on the surface of a wide range of microorganisms such as viruses, bacteria, fungi and protozoa.
It induces defense reactions such as phagocytosis, inflammation and bactericidal activity by inducing the “MB lectin pathway” different from the conventional complement activation pathway.

[0004] RaRF is composed of a polysaccharide binding component that recognizes and binds to a polysaccharide, and a complement activating component for activating complement. The polysaccharide binding component is a mannan binding lectin (man
nan-binding lectin (MBL), and as a complement activating component, a mannan-binding lectin-related serine protease
(mannan-binding lectin associated serine protease,
In addition to MASP-1 and MASP-2, which are called MASPs, a truncated MASP-2 protein (small MBL-associated
protein, sMAP) has been found.

RaRF is an acute phase protein that increases in a short time during inflammation, whereas MBL also increases during bacterial or viral infection, reaching several tens times the normal average.
(Aittoniemi, J. et al., APMIS, 105: 617-622, 1997)
. For MASP-1, it has been reported that the expression level of MASP-1 mRNA increases several times during inflammation (Knittel, T. et.
al., Lab. Inv., 77: 221-230, 1997).

In recent years, it has been reported that mutation of the gene of MBL, which is a component of RaRF, causes various diseases including susceptibility to infection. The mild recurrent infections often seen in infants and infants have been considered frail, but abnormalities in the complement-activating lectin are attracting attention as one of the leading factors causing such susceptibility. . For example, it has been reported that many children with chronic gastrointestinal disorders and otitis media have low serum MBL levels [Super, M. et al., Lancet (ii), 1236-1339, 198.
9]. Later, it was reported that when the MBL gene was mutated, the serum concentration of the MBL protein decreased (Lipscombe, RJ e
t al., Immunol. Lett., 32: 253-257, 1992; Sumiya,
M. et al., Lancet, 337: 1569-1570, 1991; Super, M. e.
tal., Nature Genet., 2: 50-55, 1992). This is the MBL
Gly54 in the collagen region by codon mutation in the gene
Becomes Asp, and the structural change of the protein region occurs. Mutation Gly57 → Glu and Arg5 at another site
It has been reported that serum MBL concentration decreases even with 2 → Cys.

[0007] When the MBL gene is mutated,
Numerous clinical observations have shown that not only decreases in MBL but also repeated infections during childhood.
This suggests that lectins play an important role in childhood until immunity against various pathogenic microorganisms is completed. In addition, it has been reported that when the MBL gene mutation is present on both homologous chromosomes, serious infections can occur in adults (Summerfield,
JA et al., Lancet, 345: 886-889, 1995). further,
HIV-infected individuals with MBL mutations have been reported to develop AIDS early and have a short survival time (Gerred,
P. et al., Lancet, 349: 236-240, 1997). Also abnormal
People with MBL are more susceptible to diseases other than infectious diseases. For example, in severe atherosclerosis, Gl
The mutations in y54 → Asp and Arg52 → Cys are (Madsen, HO et a
L., Lancet, 352: 959-960, 1998), and it has been reported that Gly54 → Asp mutations are significantly higher in systemic lupus erythematosus (Lau YL et al., Arthritis Rheum., 39: 706-70).
8, 1996).

[0008] Another RaRF component, the complement activating component MASP-1, is a serine protease component. RaRF MB
When L binds to the polysaccharide of the microorganism, the information is transmitted to the MSAP and activated, and the N-terminal side of the serine protease component of the MASP polypeptide is cleaved to an activated form, which is thought to activate complement. Therefore, it is considered that mutation of this gene reduces resistance to infection and causes diseases including susceptibility to infection. Therefore, MASP-1
The detection of the gene mutation is very important for predicting diseases such as communicable diseases.

The MASP-1 gene is a long gene of 50,000 base pairs or more present on the long arm of human chromosome 3 and is composed of 16 or more exons (Takada, F. et al., Ge
nomics, 25: 757-759, 1995; Takayama, Y. et al., Mo
l. Immunol., 36: 505-514, 1999). The nucleotide sequence of this gene was sequenced for the exon part, but the sequence of only a part of the intron was determined. In addition, no exon or intron polymorphism has been reported for this gene.

[0010] Usually, a mutation in a regulatory gene such as a promoter involved in the expression of an exon of a certain gene or a protein is often closely linked to a mutation (allotype) of an intron in the vicinity thereof. Since mutations in those genes are closely related to protein production or changes in function, detection of such allotypes makes it possible to diagnose diseases caused by protein production or function changes, and the severity of the disease. Is being done.

[0011]

SUMMARY OF THE INVENTION The present invention relates to a communicable disease,
In particular, it is an object of the present invention to provide a method for detecting a gene polymorphism of MASP-1, which is a component of RaRF, involved in the development of susceptibility to infectious diseases in childhood.

[0012]

Means for Solving the Problems The present inventors have recognized that the gene polymorphism of MASP-1, a complement activation component of RaRF, which is involved in the development of susceptibility to infectious diseases, in particular, infantile susceptibility, may be associated with this gene. The present invention was found to be present in the sixth intron portion of the present invention, and the present invention was completed.

[0013] That is, the gist of the present invention is a method for detecting a genetic polymorphism associated with the onset of susceptibility to infection, particularly the development of susceptibility to infection in childhood, comprising the following steps. (1) a step of collecting a sample containing genomic DNA; (2) the genomic DNA contains the 148th base of the 6th intron of the serine protease MASP-1 gene which is a component of the human complement-activating lectin RaRF. Amplifying the constant region; and (3) detecting a mutation at the 148th base of the sixth intron of the MASP-1 gene in the obtained amplification product.

In the above method, the amplification is preferably performed by a polymerase chain reaction method using an oligonucleotide primer set based on the nucleotide sequence of exon 6 and the nucleotide sequence of intron 6 of the MASP-1 gene. It is particularly preferable to use, as the oligonucleotide primer, a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4. In addition, mutation detection
Single-stranded DNA High-order structural polymorphism analysis can be performed.

Furthermore, the present invention relates to a gene for the serine protease MASP-1, which is a component of the human complement-activating lectin RaRF, based on the nucleotide sequence of exon 6 and the nucleotide sequence of intron 6 of the MASP-1 gene. It also relates to an oligonucleotide primer set so as to amplify a certain region including the 148th base site in the sixth intron.
Preferred examples of the oligonucleotide primer include:
A primer pair including a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4 is exemplified. The oligonucleotide primers of the present invention are useful for detecting gene polymorphisms associated with the development of susceptibility to infection, particularly in childhood.

The present invention relates to MASP-1 which is an important component of RaRF.
The detection of the allotype of this gene makes it possible to clinically predict the susceptibility to susceptibility, especially in childhood, as in the case of MBL. , Which allows the selection of an appropriate treatment. In addition, there is a possibility that diagnosis of diseases other than infectious diseases can be predicted.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present inventors have analyzed the upstream base sequence of the sixth intron of the MASP-1 gene by sequencing using the dideoxy method, and found that there is a 148th point mutation from cytosine to thymine. SEQ ID NO: 1 shows a cytosine nucleotide sequence, and SEQ ID NO: 2 shows a thymine nucleotide sequence.
When the genotype determined by this point mutation is determined for 200 Japanese as described in the Examples below, the appearance of a homozygous genotype having both thymine alleles is 6.5%. It is determined to be polymorphic. It is believed that the method of the present invention makes it possible to predict the onset of compromised disease.

As described above, the polymorphism in the sixth intron exists in the gene of MASP-1, which is a component of RaRF, which is involved in the development of susceptibility to infection, particularly in childhood. Type detection is performed as described in detail below.

The method for detecting a gene polymorphism according to the present invention comprises:
(1) collecting a sample containing genomic DNA, (2) genome
Amplifying a certain region containing the 148th base of the sixth intron of the serine protease MASP-1 gene, which is a component of the human complement-activating lectin RaRF, in the DNA; and (3) obtaining the amplified product In the MASP-
Detecting a mutation at the 148th base of the sixth intron of one gene.

In the method of the present invention, genomic DNA is measured, and a material for preparing the DNA can be used without any limitation as long as it is a human-derived sample. For example, genomic DNA may be extracted and purified from a sample such as cells of body fluids such as blood, bone marrow fluid, semen, and peritoneal fluid, tissue cells such as oral mucosa and liver, and hair root cells attached to hair according to a conventional method.

In order to detect the mutation of the sixth intron of the MASP-1 gene using the prepared genomic DNA, first, a DNA fragment is prepared by amplifying a DNA region containing the 148th mutation site of this intron. . Amplification of DNA can be performed, for example, according to the polymerase chain reaction method (PCR method). In this case, primers appropriately set so as to specifically amplify a certain region including the mutation site of the sixth intron are used. . Primers were prepared using the nucleotide sequence of exon 6 of the MASP-1 gene (SEQ ID NO: 5)
Based on the intron base sequence, the sixth of the MASP-1 gene
What is necessary is just to set so that an amplified DNA of several tens to several hundreds of base pairs including the mutation site in the intron can be obtained. When the obtained amplified DNA is analyzed by a single-stranded DNA higher-order structural polymorphism analysis method, it is preferable to obtain an amplified DNA of about 300 to 500 base pairs. The length of a primer used for DNA amplification is preferably about 18 to 25 bases.

The following primers are preferably used as primers used in such a PCR method. Forward primer: 5'-GGGCCTTTCTGTGGAGAGAA-3 '
(SEQ ID NO: 3) Reverse primer: 5'-AATCTCTCTATGCGATACTGAA-3 '
(SEQ ID NO: 4) After amplifying DNA by PCR, etc.,
Excess primers and nucleotides in the product are removed by a column or the like.

Next, the 148th mutation of the sixth intron of MASP-1 is detected by using the DNA fragment amplified as described above, and the method for detecting the mutation is not limited at all. Any method can be used. For example,
Methods for detecting mutations in DNA fragments that can be used in the present invention include:
Single-stranded conformation polymorphism (single-stranded conformati
on polymorphism, SSCP) analysis method, restriction fragment length polymorphism
(restriction fragmentlength polymorphism, RFLP) analysis, heteroduplex analysis, HE
T) method, denaturing gradient gel electrophoresis
ient gel electrophoresis (DGGE) method, direct sequencing (d
irect sequence, DS) method, carbodiimide modification (carbodi
imide modification, CDI) method [see Bio Manual Series 1, Basic Technology of Genetic Engineering, Masaru Yamamoto, Yodosha (1993)].

Various operations that can be employed in the mutation detection method of the present invention, for example, chemical synthesis of DNA, isolation of DNA,
Purification, amplification, selection, etc. may all be in accordance with conventional methods. For example, DNA can be isolated and purified by agarose gel electrophoresis or the like, and the sequence is determined by the dideoxy method (San
ger, F. et al., Proc. Natl. Acad. Sci., USA, 7
4: 5463-5467, 1977) and the Maxam-Gilbert method (Maxam,
AM et al., Method in Enzymology, 65: 499-560, 198
0) etc. can be used. The determination of the DNA base sequence may be performed using a commercially available sequence kit or the like. DNA
PCR method for amplifying a specific region of is also a conventional method (for example, Saiki, RK et al., Science, 230: 1350-1354, 19
85).

For detection of a DNA fragment, the DNA fragment can be labeled with the following substances. As a method for labeling a DNA fragment, there are a method of labeling a primer in advance, a method of using a labeled base component (for example, radioactive labeling of phosphorus) when performing PCR, and a method of labeling after performing PCR.

The substance that can be used for the label is not particularly limited.
For example, the 5 ′ end of DNA may be labeled with a radioactive substance, a fluorescent substance, a chemiluminescent substance, biotin (detected with an enzyme-labeled avidin), or the like. Particularly preferably, a primer (Amersham Pharmacia Biotech, Inc.) in which the 5 ′ end of the oligonucleotide primer for PCR is fluorescently labeled with Cy5 in advance is used. An application of this fluorescent labeling method to the SSCP method is called a fluorescent SSCP method.

In the present invention, to detect a mutation, a single-stranded DNA conformational polymorphism analysis method (SSCP method) or a restriction enzyme fragment length polymorphism analysis method (RFLP method) can be preferably used. In particular, a method in which the amplification method by the PCR method and the above-described fluorescent SSCP method are combined is preferable. This method uses the PCR method
Since this is a combination with the DNA amplification method, simple and easy detection using a small amount of a DNA sample and with high sensitivity and accuracy is possible. Also, the preferred conditions for performing the SSCP method are 6-
8% acrylamide gel, electrophoresis temperature 15-25 ° C,
In particular, it is advantageous to use a gel of about 6% acrylamide (acrylamide: bisacrylamide = 99: 1) at a migration temperature of about 20 ° C. in terms of accurate detection of polymorphism.

In the following examples, an example of detecting a mutation in the sixth intron of the MASP-1 gene in genomic DNA using fluorescent PCR-SSCP will be described. And any other detection method can be used.

[0029]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

The reagents and the like used in this example are as follows. <40% acrylamide stock solution> 79.2 g of acrylamide,
Dissolve 0.8 g of NN'-methylenebisacrylamide in purified water to make 200 ml, add 10 g of Amberlite, and slowly shake overnight in the dark. Store at 4 ° C in the dark. <20 × TME (6.2)> Tris 36.3 g, MES 115.2 g, EDTA
Dissolve 3.7 g with purified water to make 500 ml. <6% acrylamide gel> 40% acrylamide stock solution
60 ml of purified water was added to 11.25 ml and 3.75 ml of 20 × TME (6.2).
Add μl and TEMED 40 μl.

The experimental procedure used in this example will be described below. (1) Preparation of Human Peripheral Blood Genomic DNA Genomic DNA was purified from human peripheral blood using SepaGene (Sanko Junyaku Co., Ltd.) according to the kit. It is prepared according to the following method.

100 μl of human peripheral blood collected using the anticoagulant EDTA-2Na was placed in a 1.5 ml microtube,
100 μl of the reagent I (sample diluent) of the SepaGene kit is added, mixed with a mixer, and allowed to stand at room temperature for 10 minutes. Next, add 100 μl of the reagent II (pretreatment liquid) of the same kit and mix gently with a mixer.
Add 400 μl of Reagent IV (extractant) of the same kit and mix vigorously 10 times. This is centrifuged at 12,000 rpm for 15 minutes at 4 ° C., and 500 μl of the supernatant is placed in a new 1.5 ml microtube. Add 2 μl of coprecipitant, 50 μl of reagent V (precipitating aid) of the same kit, and 600 μl of isopropanol.
After centrifugation for minutes, remove the supernatant. 70% ethanol 500μ
l In addition, after gently mixing by inversion, centrifuge at 15,000 rpm at 4 ° C for 5 minutes to remove the supernatant. 500% of 100% ethanol
In addition, after gently mixing by inversion, centrifuge at 15,000 rpm at 4 ° C. for 5 minutes to remove the supernatant. After air-drying the precipitate, add 50 parts of sterile distilled water.
Add μl.

(2) DNA amplification operation 1 μm of the genomic DNA solution (50-100 ng / μl) prepared in (1) above
Add 37.5 μl of sterile distilled water and 5 μl of 10 × KOD Dash PCR solution
l, 2 mM dNTPs (4 kinds of deoxyribonucleoside triphosphates) 5 μl, 20 μM forward primer 0.5 μl, 20 μM
μM reverse primer 0.5 μl, 2.5 U / μl KOD Dash
Add 0.5 μl to make the total volume 50 μl, further overlay 2 drops of mineral oil, and perform 35 cycles of reaction at 94 ° C. for 30 seconds, 60 ° C. for 10 seconds and 74 ° C. for 1 minute. The primer for PCR has the following nucleotide sequence. These primers are Cy5 labeled at the 5 'end. <Oligonucleotide primer> Forward primer: 5'-GGGCCTTTCTGTGGAGAGAA-3 '
(SEQ ID NO: 3) Reverse primer: 5'-AATCTCTCTATGCGATACTGAA-3 '
(SEQ ID NO: 4) Confirmation of the PCR product was performed after electrophoresis on a 3% agarose gel.
Perform staining with ethidium bromide.

(3) Excessive primer removal operation To remove excess primers from the PCR product, use MicroS
Using pin S-300 HR Column (Amersham Pharmacia Biotech), perform the following operations according to the column instruction manual.

First, PCR in which mineral oil is layered
Add 50 μl of chloroform to the product, mix with a mixer,
Centrifuge to remove mineral oil. After mixing the resin packed in the column with a mixer, place it in a 2.0 ml microtube and centrifuge at 3,000 rpm for 1 minute to remove the extra buffer added to the column. Replace this column with a new
Place the PCR product in a 1.5 ml microtube, remove the mineral oil, and centrifuge at 3,000 rpm for 2 minutes.

(4) SSCP operation Fluorescent SSCP analysis was performed using an AL equipped with an external cooling circulation tank.
F. Using express DNA sequencer (Amersham Pharmacia Biotech), containing 1 × TME (6.2) buffer 6
% Acrylamide (acrylamide: bisacrylamide = 99: 1) gel (270 mm high x 308 mm wide x 0.5 m thick)
m) is used. Transfer 3 μl of the PCR product from which excess primer and nucleotides have been removed to 3 μl of loading solution for SSCP.
After heat denaturation at 95 ° C. for 5 minutes in addition to l, 2 μl of this was applied to a gel and electrophoresed. The composition of the loading solution is a deionized formamide solution containing 0.05% of blue dextran. The electrophoresis buffer used was 1 × TME (6.2). To analyze the measurement data, use the analysis software Fragme
Use nt Manager V1.2 (Amersham Pharmacia Biotech).

(5) SSCP electrophoresis conditions Electrophoresis temperature 20 ° C., voltage 1500 V, current 100 mA, power 30 W
Use 1 × TME (6.2) as electrophoresis buffer and perform electrophoresis for 500 minutes.

(6) Genotyping Judgment of the DNA fragment obtained by amplification treatment by PCR is performed by heat denaturation, and the peak separated by the difference in the mobility of the single-chain higher-order structure is determined.
Analysis is performed using the analysis software Fragment Manager V1.2 (Amersham Pharmacia Biotech) to determine the genotype. The genotype was determined as follows: when the pattern in Fig. 1.A was detected, the 148th base in intron 6 was homozygous for cytosine; when the pattern in Fig. 1.B was detected, cytosine and thymine were detected. If the heterozygote, the pattern in Figure 1.C, is detected, it is regarded as a thymine homozygote. For nucleotide sequence confirmation, use Thermo Sequenase Cy5 Dye Terminator
It is performed by nucleotide sequence analysis using Kit (Amersham Pharmacia Biotech). At this time, 5′-TTCCATAGTGACAACTCGGG-3 ′ is used as an oligonucleotide primer.

Example 1 This example was performed in accordance with each of the above experimental operations. 20 randomly selected Japanese adults
For 0 subjects, prepare genomic DNA, amplify, remove excess primer, remove SSCP according to the above experimental procedures (1) to (6).
Mutation was detected and the genotype was determined. Table 1 shows the frequency of appearance of each genotype. The MASP-1 gene 6
The genotype of the homozygote in which the 148th base of the intron is both cytosine is CC, the genotype of the heterozygote that is both cytosine and thymine is TC, and the genotype of the homozygote that is both thymine are TT It was written.

[Table 1] Number of occurrences of MASP-1 genotype in healthy subjects 200 CC TC TT people (frequency) 116 (58.0%) 71 (35.5%) 13 (6.5%)

The results show that the genotype CC of a homozygote whose alleles are cytosine is 58.0%, the genotype TC of a heterozygote which is cytosine and thymine is 35.5%, and the genotype of a homozygote whose both are thymine are 35.5%. The TT is 6.5%, and therefore, the mutation from cytosine to thymine at the 148th base of the intron 6 of the MASP-1 gene is determined to be a polymorphism.

[0042]

According to the present invention, gene polymorphisms associated with susceptibility to infection, particularly susceptibility to infection in childhood, that is, gene polymorphism of MASP-1, a component of the human complement-activating lectin RaRF, are analyzed. A method is provided for doing so. Detection of this polymorphism may lead to a clinical predictive diagnosis of susceptible diseases, particularly susceptible diseases in early childhood, and the diagnosis may allow for the selection of treatments for those diseases.

[Brief description of the drawings]

[Figure 1] Single-stranded DNA higher-order polymorphism analysis (PCR-SSCP method)
FIG. 6 is a diagram showing a pattern generated by the above.

[Sequence List] SEQUENCE LISTING <110> SUMIKIN BIO-SCIENCE, INC. <120> GENETIC POLYMORPHISM OF MASP-1 OF HUMAN COMPLEMENT-ACTIVATING LECT IN, RA-REACTIVE FACTOR <130> S15X12P <140> <141> <160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 259 <212> DNA <213> Homo sapiens <400> 1 gtaacctctt ccctcccagg tcacaccgag ctgctgtgct tagcccttgc agaggcagtg 60 gctgagggag gaggtg gcgtcgcc gaggtc gcctc gag gcgtc gcgtc gcgtc gcgtgg gggcaagggc aacaataatg ttgggtggac tagactgatg gttttcaaaa catgtacttc 240 agtatcgcat agagagatt 259 <210> 2 <211> 259 <212> DNA <213> Homo sapiens <400> 2 gtaacctctt ccctcccagg tcacaccgag ctgctgtgct tagcccttgc agaggcagtg 60 gctgagggag gaggtgtgca ggcttggatc tcctctggag acctgatggg actcccagtt 120 gagcagctgg ggaccaaggc acgtgctttc tcctgactca gttttccatc tataaaatgg 180 gggcaagggc aacaataatg ttgggtggac tagactgatg gttttcaaaa catgtacttc 240 agtatcgcat agagagatt 259 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: primer <400> 3 gggcctttct gtggagagaa 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 aatctctcta tgcgatactg aa 22 <210> 5 <211> 148 <212> DNA <213> Homo sapiens <400> 5 atcaaagttg gtccaaaagt tttggggcct ttctgtggag agaaagcccc agaacccatc 60 agcacccaga gccacagtgt cctgatcctg ttccatagtg acaactcggg agactag

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor, Tsuyoshi Ishida 10203-4, Tana, Sagamihara-shi, Kanagawa Prefecture Sumikin Bioscience Corporation (72) Inventor Hiromitsu Tazawa 10203-4, Tana, Sagamihara-shi, Kanagawa Prefecture Sumikin Bioscience Corporation (72) Inventor Masaya Kawakami 2-3-3 Kamizuruma, Sagamihara-shi, Kanagawa F-term (reference) 4B024 AA13 BA80 CA01 HA11 HA20 4B063 QA01 QA17 QA19 QQ03 QQ42 QR08 QR62 QR66 QS16 QS20 QS25 QS36 QX02

Claims (9)

[Claims] 1. A method for detecting a genetic polymorphism associated with the development of a susceptible disease, comprising the following steps: (1) a step of collecting a sample containing genomic DNA; (2) a human complement-activating lectin RaRF in genomic DNA;
Amplifying a certain region containing the 148th base of the 6th intron of the serine protease MASP-1 gene which is a component of (3);
Detecting the mutation at the 148th base of the intron. 2. The method according to claim 1, wherein the susceptible disease is a susceptible disease in childhood. 3. The amplification according to claim 1 or 2, wherein the amplification is performed by a polymerase chain reaction method using an oligonucleotide primer set based on the nucleotide sequence of exon 6 and the nucleotide sequence of intron 6 of the MASP-1 gene. The described method. 4. As an oligonucleotide primer,
The method according to claim 3, wherein a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4 are used. 5. The method according to claim 1, wherein the mutation is detected by single-stranded DNA conformational polymorphism analysis. 6. A serine protease MA which is a component of the human complement-activating lectin RaRF based on the nucleotide sequence of exon 6 and the nucleotide sequence of intron 6 of the MASP-1 gene.
An oligonucleotide primer set so as to amplify a certain region including the 148th base site in the sixth intron of the SP-1 gene. 7. The oligonucleotide primer according to claim 6, comprising a forward primer having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer having the nucleotide sequence of SEQ ID NO: 4. 8. The oligonucleotide primer according to claim 6, for detecting a genetic polymorphism associated with the development of a susceptible disease. 9. The oligonucleotide primer according to claim 8, wherein the susceptible disease is a susceptible disease in childhood.