JPH1099099A - Diagnosis of abnormity of human ldl receptor gene and probe used therefor and oligonucleotide primer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To diagnose the subject genetic abnormity causing familial hypercholesterolemia, etc., by probing whether the base sequence of the codon 109 of the exon of human LDL receptor gene is TCA or TCCA. SOLUTION: This method for diagnosing the abnormity of human LDL receptor gene by probing whether the base sequence of the codon 109 of the exon 4 of the human LDL receptor gene is TCA or TCCA comprises annealing a normal probe and an abnormal probe with a gene region containing the codon 109 of the exon 4 of the human LDL receptor gene and subsequently detecting the hybridized probe. The normal probe has a base sequence complementary to the codon 109-containing region of a normal gene in which the codon 109 of the exon 4 of the human LDL(low density lipoprotein) receptor gene is TCA, and an abnormal probe has a base sequence complementary to the codon 109- containing region of an abnormal gene in which the codon 109 is TCCA produced by the insertion of C into the codon 109.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for diagnosing a human LDL receptor gene abnormality, and a probe and an oligonucleotide primer used therein.

[0002]

BACKGROUND OF THE INVENTION Familial hypercholesterolemia (FH)
Is a disease in which blood cholesterol levels are genetically high,
For this reason, it is a disease in which the risk of causing ischemic heart disease such as myocardial infarction increases from the young age to the middle age. It has been found that the cause of this disease is mainly an abnormality of the LDL (low-density lipoprotein) receptor gene. LDL
Receptor abnormalities are extremely diverse, with more than 200 being discovered by 1994.

[0003]

[0005] In some cases, despite the fact that FH is diagnosed clinically, none of the conventionally known abnormalities in the LDL receptor gene is found. Therefore, it is considered that an unknown abnormality which has not been reported so far exists in the LDL receptor gene.

Accordingly, an object of the present invention is to provide a means for diagnosing an unknown abnormality of the human LDL receptor gene which has not been reported so far.

[0005]

SUMMARY OF THE INVENTION As a result of intensive studies, the present inventors have found that exon 4 of the human LDL receptor gene is present.
The present inventors have found a genetic abnormality that has not been known before, and have completed the present invention.

That is, according to the present invention, the nucleotide sequence of codon 109 of exon 4 of the human LDL receptor gene is TC
It is intended to provide a method for diagnosing a human LDL receptor gene abnormality, which comprises examining whether it has become A or whether C has been inserted into it to form TCCA. The present invention also provides oligonucleotide primers and probes used in the method of the present invention.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found that 109 codons in exon 4 of the human LDL receptor gene (hereinafter simply referred to as "codon 109") become TCCA by inserting C into normal TCA. Found an anomaly. Therefore, by examining whether the base sequence of the codon is TCA or TCCA, one type of human LDL receptor gene abnormality can be diagnosed.

Exon 4 of the human LDL receptor gene
Whether the nucleotide sequence of codon 109 in the middle is TCA or TCCA can be determined by various methods.

First, as a first method, a so-called PCR
-A method called RFLP method. In this method, first, a gene fragment containing codon 109 is amplified by a nucleic acid amplification method such as PCR. The gene used as a template may be any human genomic DNA collected from any cell. Genomic DNA can be obtained from peripheral blood or the like by a known method. In addition, PCR is a typical method of nucleic acid amplification, but amplification by other nucleic acid amplification methods is also possible. The nucleotide sequence of exon 4 of the human LDL receptor gene and its surrounding region is already known (Hobbs, HH, et al., (1989) J. Clin. In
vest. 84, 656-664), so that the nucleotide sequences of primers necessary for nucleic acid amplification can be easily known.

Next, the amplified fragment is digested with a restriction enzyme DdeI. As shown in FIG. 1, the normal gene has a DdeI restriction enzyme site composed of CTCAG. However, since the abnormal gene has C inserted into CTCCAG, the DdeI restriction enzyme site disappears. Therefore, when the amplified fragment is digested with DdeI, the gene is cut at codon 109 if the gene is normal, and not cut if it is abnormal. Therefore, whether the gene is normal or abnormal can be determined by examining the size of the amplified fragment after DdeI digestion.

For example, as described in the following example,
When amplification is carried out by PCR using a pair of 25 bp oligonucleotide primers that hybridize to introns adjacent to both sides of exon 4 of the human LDL receptor gene, 431 bp containing exon 4 (432 bp in the case of an abnormal type) is obtained. Are amplified.
When this is digested with DdeI, a fragment having the size shown in FIG. 2 is obtained. Therefore, when this is subjected to gel electrophoresis, a band pattern as shown in FIG. 3 is observed. Therefore, it is possible to diagnose whether the codon 109 is normal or abnormal by this method. Since a pair of human genomic DNAs originating from both parents are present, when the actual human-derived material is examined by the above-described method, it is shown in FIG. 3 in the case of normal or abnormal homozygous. Such a pattern is seen, but in the case of a heterojunction, a pattern in which these are mixed is seen.

As a second method, a method using a probe can be mentioned. That is, normal codon 109
A normal type probe having a nucleotide sequence complementary to the region containing codon 109, which has a nucleotide sequence complementary to the gene region containing, and a codon of an abnormal gene in which C has been inserted into codon 109 to form TCCA. An abnormal probe having a base sequence complementary to the region including the region 109 is prepared. As described above, since the nucleotide sequence of human LDL receptor gene exon 4 is known, the nucleotide sequence of the probe can be easily set. It is also possible to react a probe obtained by digesting a genomic gene with an appropriate restriction enzyme,
To increase sensitivity, first, codon 109
It is preferable to amplify a gene fragment containing the above by a nucleic acid amplification method and react the amplified fragment with a probe. By examining which probe the sample hybridizes with, it is possible to know whether codon 109 is normal or abnormal. When reacting with the normal probe, a mixture of the normal probe and the unlabeled abnormal probe is used. Similarly, when reacting with the abnormal probe, a mixture of the abnormal probe and the unlabeled normal probe is used. By using the competing probe (compet
By performing annealing in the presence of a negative probe, a more accurate determination can be made.

The method of the present invention is not limited to the above two methods, and the base sequence of codon 109 may be TCA or TCA.
Any method that can check for CCA is included in the scope of the present invention. For example, codon 109
May be amplified by a nucleic acid amplification method, and the nucleotide sequence of the amplified fragment may be directly examined.

The present invention also provides an oligonucleotide primer used in the above-mentioned first method. As described above, exon 4 of the human LDL receptor gene
And the nucleotide sequence of its surroundings are known. Therefore, the primer of the present invention has a base sequence complementary to the end of the region sandwiching codon 109 among the known base sequences.
There is a limit to the size of nucleic acid that can be amplified by the nucleic acid amplification method,
Also, amplifying too large a fragment does not make sense,
The distance between the region where the primer hybridizes and the codon 109 is 500 bp or less, more preferably 300 bp or less. The primer can be set in a region including codon 109, but when performing RFLP after amplification, if the amplified fragment is too small, the band will not be clearly detected by electrophoresis. The distance between the codon and the primer is preferably 20 bp or more. Further, the length of the primer is not particularly limited, but is preferably about 15 bp to 50 bp, particularly preferably about 15 bp to 30 bp.

The present invention further provides a normal probe and an abnormal probe used in the second method.
The length of the probe is not particularly limited.
bp, particularly preferably about 15 bp to 30 bp. As is well known, the probe is labeled with a radiolabel, a fluorescent label, a biotin label, an enzyme label, or the like. These labels and their methods of attachment are well known.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Example 1 Diagnosis by PCR-RFLP method (1) Materials The following three materials were used. Patient 1: Peripheral blood of an FH patient in which a single base pair insertion of cytosine exists at codon 109 of exon 4 of the LDL receptor gene in a heterozygous manner, and no other genetic abnormality is found. Patient 2: Exon 4 of the LDL receptor gene FH patients with no abnormalities in codon 109 (codon 1
Peripheral blood with abnormalities in regions other than 09) Healthy subjects: placenta of a woman with normal cholesterol level

(2) Amplification of exon 4 of LDL receptor gene by PCR method Human genomic DNA extracted from each of the above materials by a conventional method
From 0.1 μg, a gene region containing the LDL receptor gene exon 4 (431 bp, found according to the invention) using primers that hybridize outside of the LDL receptor gene exon 4 reported by Hobbset et al. In the case of the abnormal sequence, 432 bp) was amplified. The sequences of the oligonucleotide primers used are shown in SEQ ID NOs: 1 and 2 in the Sequence Listing. PCR reaction solution (25μ
The composition of l) is 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl ₂ , 0.01% gelatin,
0.25 units Taq polymerase. Also, PC
The conditions of R were as follows: denaturation at 94 ° C for 3 minutes, denaturation at 94 ° C,
1 minute, annealing 65 ° C, 1 minute, extension 72 ° C, 2
A 30 minute cycle was performed. Then 4 ℃
Incubated.

(3) Detection of abnormal site by RFLP method (i) 5 μl of the reaction solution after the above PCR was completed, 1 μl of Boehringer's restriction enzyme incubation buffer H, 4 μl of sterilized distilled water, and 1 unit of restriction enzyme DdeI Mix and incubate at 37 ° C. for 2 hours.

(Ii) Polyacrylamide gel electrophoresis After 2 μl of the gel loading solution was mixed with the reaction solution after the restriction enzyme treatment, the whole was applied to a 12.5% polyacrylamide gel, and electrophoresed at 100 V for 2 hours. The gel after electrophoresis was immersed in a 0.5 μg / ml ethidium bromide solution, and the DNA band was photographed under ultraviolet irradiation.

(Iii) Result The obtained electrophoresis pattern is shown in FIG. 202, 106, 99b when the DNA of Patient 2 and a healthy subject were used
Three bands of p were detected (it is considered that the band of 24 bp was not confirmed as a band because it was too small). On the other hand, in the DNA of patient 1 in which a single base pair insertion of cytosine (C) was found at codon 109, 202, 1
Four bands of 24, 106 and 99 bp were detected.

From the above results, it was confirmed by the PCR-RFLP method that the abnormality at this site exists in the heterozygous state. Therefore, it was confirmed that the present site can be diagnosed by the method of the present invention.

Example 2 Diagnosis Using Probes 5 μl of the reaction solution after completion of PCR in Example 1 was taken.
Using a blotting apparatus, spotting was performed on a nylon filter, and hybridization with allele-specific oligonucleotides was performed using normal or abnormal oligonucleotide probes labeled with ³² P in the presence of competitive probes. The sequence of the oligonucleotide probe used was such that the normal type was shown in SEQ ID NO: 3 in the sequence listing and the abnormal type was shown in SEQ ID NO: 4. After hybridization, the nylon filter was
The plate was washed with a 2 × SSPE solution containing% SDS at 45 ° C. for 30 minutes, exposed to an X-ray film, developed, and detected.

FIG. 5 shows the results. In the case of DNA of a healthy subject, a reaction was observed only with a normal-type probe, whereas in the DNA of a patient in which a single base pair insertion of cytosine (C) at codon 109 was detected in a heterologous form, normal A reaction was observed with both the type probe and the abnormal type probe. On the other hand, the DNA of Patient 2 in which no abnormality was observed at this site
Reacted only with the normal probe. Therefore, according to this method, it was confirmed that it specifically reacted only with the DNA of a patient having an abnormality at this site, and that it was possible to discriminate between homo and hetero genotypes.

[0025]

According to the present invention, there has been provided a method for diagnosing an unknown abnormality of the human LDL receptor gene which has not been reported so far, and a primer and a probe therefor.

[0026]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Sequence TGGTCTCGGC CATCCATCCC TGCAG 25

SEQ ID NO: 2 Sequence length: 25 Sequence type: nucleic acid sequence ACGCCCCGCC CCCACCCTGC CCCGC 25

SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid sequence TCTGTGACTC AGACCGGGAC 20

SEQ ID NO: 4 Sequence length: 21 Sequence type: nucleic acid sequence TCTGTGACTC CAGACCGGGA C 21

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the normal and abnormal genes near exon 4 codon 109 of human LDL receptor gene as DdeI
It is a figure shown with a restriction | limiting part.

FIG. 2 is a diagram showing the sizes and positions of fragments detected by the PCR-RFLP method in Example 1 for normal and abnormal genes.

FIG. 3 is a diagram schematically showing a band pattern obtained when a mixture of fragments shown in FIG. 2 is subjected to gel electrophoresis.

FIG. 4 is a diagram schematically showing a gel electrophoresis pattern obtained by the method of Example 1.

FIG. 5 is a view schematically showing an image of an X-ray photosensitive film obtained by the method of Example 2.

Claims (8)

[Claims] 1. Diagnosis of a human LDL receptor gene abnormality, comprising examining whether the nucleotide sequence of codon 109 of exon 4 of the human LDL receptor gene is TCA or whether C is inserted into TCCA to form TCCA. Method. 2. A method comprising amplifying a gene fragment containing codon 109 of exon 4 of the human LDL receptor gene by a nucleic acid amplification method, digesting the amplified fragment with a restriction enzyme DdeI, and examining the size of the digested gene fragment. The method of claim 1. 3. A normal probe having a nucleotide sequence complementary to a region containing codon 109 in a normal gene in which codon 109 of exon 4 of the human LDL receptor gene is TCA, and C is inserted into codon 109. Been TCC
An aberrant probe having a nucleotide sequence complementary to the region containing codon 109 of the abnormal gene in A was ligated to codon 109 of exon 4 of the human LDL receptor gene.
2. The method according to claim 1, comprising detecting a probe that has annealed with the gene region containing and hybridized. 4. The method according to claim 3, wherein the normal and abnormal probes are hybridized after a gene fragment containing codon 109 of exon 4 of the human LDL receptor gene is amplified by a nucleic acid amplification method. 5. An oligonucleotide primer capable of hybridizing with a part of a human LDL receptor gene and used in the method according to claim 2. 6. The primer according to claim 5, which has the nucleotide sequence shown in SEQ ID NO: 1 or 2 in the sequence listing. 7. The normal type probe used in the method according to claim 3, wherein codon 109 of exon 4 of the human LDL receptor gene has a nucleotide sequence complementary to a normal gene which is TCA and is labeled. . 8. The human LDL receptor gene according to claim 3, which has a nucleotide sequence complementary to an abnormal gene in which C is inserted into codon 109 of exon 4 of exon 4 to form TCCA, and is labeled. Abnormal probe used in the method.