JP5648948B2 - Genetic mutation screening method for hypophosphatasia - Google Patents

Description

  The present invention relates to detection based on genetic mutations in hypophosphatasia. Furthermore, the present invention relates to genetic mutation screening and genetic diagnosis for hypophosphatasia.

  Hypophosphatasia is a hereditary disease caused by a gene mutation of tissue non-specific alkaline phosphatase (TNSALP), and is divided into five types (pathology) depending on the degree of symptoms. Especially in the perinatal period, which has a high degree of severity, bone formation throughout the body is impaired before birth, and the ribs that support the respiratory muscles break, resulting in respiratory failure and early death after birth. There have been no effective treatments so far, but in recent years a case has been reported in which a patient was saved by transplanting bone marrow, mesenchymal cells, etc. of a healthy person. Also, in the project for the realization of regenerative medicine by the Ministry of Education, Culture, Sports, Science and Technology, iPS cells were established from the patient's own cells under the theme of “Development of genetically modified mesenchymal stem cell transplantation treatment for severe congenital bone metabolic disease”, and the ALP gene was introduced. After that, research aimed at radical treatment of hypophosphatasia patients by inducing to mesenchymal cells or differentiating into osteoblasts and transplanting was started.

  Diagnosis of hypophosphatasia is simple if the presence or absence of O-leg, physical characteristics such as bone pain due to fracture, blood and urine test results, serum ALP activity and ALP isozyme (ALP type) activity are reduced It depends on whether or not there are rickets-like changes such as enlargement of the epiphyseal line, cupping (claw-shaped depression), and fling (hair bristle) at the metaphysis by X-ray photographs. However, changes in the metaphysis alone are difficult to distinguish from other bone system diseases such as vitamin D deficiency rickets, hypophosphatemic rickets, metaphyseal dysplasia, etc. However, it was difficult to identify the clinical type of hypophosphatasia from serum ALP. Furthermore, since this hereditary disease has an autosomal recessive inheritance format, only 1 in every tens of thousands actually develops this rare disease. However, this genetic disease is often found because the bone formation of a pregnant child suddenly fails for a couple with no family history and no symptoms. In children with hypophosphatasia and benign perinatal forms, it is often pointed out that osteogenesis is suspected to be a failure of bone formation during pregnancy, and it is difficult to estimate the child's condition based on the ALP activity of the parents. From the genetic mutation analysis so far, the relationship between the gene mutation site and the disease type has become clear.

  Among the TNSALP gene abnormalities, it is known that the 1559T deletion mutation is homozygous and causes perinatal hypophosphatasia, which has been reported only to Japanese (see Non-Patent Document 1). Asymptomatic carriers with heterozygous 1559T deletion mutations are predicted to be particularly common in Japanese. The gene mutation site exists in each exon, and the relationship between the mutation site and the disease (pathology) is becoming clear (see Non-Patent Document 2). Conventionally, gene mutation sites require time because PCR was performed for each exon and the base sequence was analyzed and identified.

  On the other hand, conventionally, a melting temperature curve analysis method using a DNA-binding dye has been known as a method for analyzing gene mutation (see Patent Document 1).

Special table 2008-510698

Mornet E. Hypophosphatasia. Orphanet J Rare Dis. 2007 Oct 4; 2: 40. Orimo H et al., J Bone Miner Metab. 2002; 20 (1): 28-33.

  An object of the present invention is to provide a method for screening a gene mutation for hypophosphatasia, a method for detecting hypophosphatasia based on a gene mutation, and a kit for detection.

  The present inventor has intensively studied to develop a detection method based on a mutation of a gene related to hypophosphatasia present in peripheral blood.

  Methods for analyzing mutations in DNA sequences can be divided into two general categories. One is to identify the genotype of a known sequence variation, and the second is to scan for an unknown variation. Many methods are available to identify the genotype of a known sequence variation. In contrast, most methods for unknown mutations require gel electrophoresis or column separation after PCR. Specifically, small amplicons or unlabeled probes are used when genotyping known sequence mutations, and large amplicons are required when scanning unknown mutations.

  Genetic testing for many genetic diseases can be difficult because the causative mutations are often distributed throughout the gene. Simultaneous mutation scanning and genotyping can be used to detect these mutated portions. Specifically, for any particular gene, each exon is amplified using primers outside the general splice site. If sequence changes are seen frequently, this smaller locus can be amplified by first identifying the genotype of the site or using an additional set of primers. Mutation scanning and genotyping of a specific site can be performed in different regions, or when scanning is positive, a base sequence analysis can be performed using the product to identify a gene mutation site.

  We created 12 pairs of primers corresponding to TNSALP, the causative gene of hypophosphatasia. Using this primer, the target region of the causative gene can be amplified by PCR. High resolution melting curve (HRM) of samples after PCR is measured using a gene mutation analyzer called Light Scanner (Idaho Technology Inc .; http://www.jki.co.jp/product/LightScanner.htm) By doing so, it became possible to screen and diagnose mutation sites easily. This makes it possible to easily detect a TNSALP gene abnormality that is not currently known, and is expected to be useful for the diagnosis of hypophosphatasia.

  In addition, among the TNSALP gene abnormalities, the homozygous 1559T deletion mutation is known to cause perinatal hypophosphatasia, which can be an important diagnostic point for this disease. Asymptomatic carriers with heterozygous 1559T deletion mutations are predicted to be particularly common in Japanese. Therefore, examining the presence or absence of the 1559T deletion mutation is an important point for this disease. Therefore, a pair of primers was prepared for a method of rapidly screening for the presence or absence of 1559T deletion (ALP SAG).

That is, the present invention is as follows.
[1] A method of comprehensively detecting mutations in the tissue non-specific alkaline phosphatase (TNSALP) gene that is a causative gene of hypophosphatasia using the following 12 primer pairs:
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 2 comprising the nucleotide sequence shown in SEQ ID NO: 1;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 4 comprising the nucleotide sequence shown in SEQ ID NO: 3;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 6 comprising the nucleotide sequence shown in SEQ ID NO: 5;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 8 comprising the nucleotide sequence shown in SEQ ID NO: 7;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 10 comprising the nucleotide sequence shown in SEQ ID NO: 9;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 12 comprising the nucleotide sequence shown in SEQ ID NO: 11;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 14 comprising the nucleotide sequence shown in SEQ ID NO: 13;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 16 comprising the nucleotide sequence shown in SEQ ID NO: 15;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 18 comprising the nucleotide sequence shown in SEQ ID NO: 17;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 20 comprising the nucleotide sequence shown in SEQ ID NO: 19;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 22 comprising the nucleotide sequence shown in SEQ ID NO: 21;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 24 comprising the nucleotide sequence shown in SEQ ID NO: 23.

[2] Twelve pairs of tissue non-specific alkaline phosphatase (TNSALP) gene are amplified using the 12 primer pairs of [1], and mutations are detected by melting curve analysis using the obtained amplification product. A method for comprehensively detecting the mutation of [1].
[3] Using a primer consisting of the nucleotide sequence shown in SEQ ID NO: 25 and a primer pair consisting of the primer consisting of the nucleotide sequence shown in SEQ ID NO: 26, the tissue non-specific alkaline phosphatase (TNSALP) gene that is the causative gene of hypophosphatasia A method for detecting a deletion mutation of 1559T.

[4] A tissue non-specific alkaline phosphatase (TNSALP) gene fragment was amplified using a primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer pair consisting of the primer consisting of the base sequence shown in SEQ ID NO: 26. A method for detecting a deletion mutation of [3], wherein the mutation is detected by melting curve analysis using the amplified product.
[5] Using a sample obtained from the subject, detecting a mutation in the tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene of hypophosphatasia, by any of the methods [1] to [4] A test method for determining that a subject is suffering from hypophosphatasia or has a high risk of suffering from hypophosphatasia when a genetic mutation is present.

[6] A kit for comprehensively detecting mutations in a tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene for hypophosphatasia, including the following 12 primer pairs:
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 2 comprising the nucleotide sequence shown in SEQ ID NO: 1;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 4 comprising the nucleotide sequence shown in SEQ ID NO: 3;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 6 comprising the nucleotide sequence shown in SEQ ID NO: 5;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 8 comprising the nucleotide sequence shown in SEQ ID NO: 7;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 10 comprising the nucleotide sequence shown in SEQ ID NO: 9;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 12 comprising the nucleotide sequence shown in SEQ ID NO: 11;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 14 comprising the nucleotide sequence shown in SEQ ID NO: 13;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 16 comprising the nucleotide sequence shown in SEQ ID NO: 15;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 18 comprising the nucleotide sequence shown in SEQ ID NO: 17;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 20 comprising the nucleotide sequence shown in SEQ ID NO: 19;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 22 comprising the nucleotide sequence shown in SEQ ID NO: 21;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 24 comprising the nucleotide sequence shown in SEQ ID NO: 23.

[7] A tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene for hypophosphatasia, comprising a primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer pair consisting of the primer consisting of the base sequence shown in SEQ ID NO: 26 Kit for detecting mutations.

[8] Comprehensive detection of mutations in tissue non-specific alkaline phosphatase (TNSALP) gene, the causative gene of hypophosphatasia, including the 12 primer pairs of [6], to detect hypophosphatasia in subjects Or a kit for determining the risk of suffering from hypophosphatasia.
[9] A tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene for hypophosphatasia, comprising a primer consisting of the base sequence shown in SEQ ID NO: 25 and a primer pair consisting of the primer consisting of the base sequence shown in SEQ ID NO: 26 A kit for detecting a mutation, detecting hypophosphatasia in a subject, or determining a risk of suffering from hypophosphatasia.

  By using the primer of the present invention as described in Examples, it is possible to comprehensively screen and analyze a mutation of a tissue non-specific alkaline phosphatase (TNSALP) gene that is a causative gene of hypophosphatasia. The presence or absence of a deletion mutation can be examined. The presence of these gene mutations indicates that there is a high risk of morbidity or onset of hypophosphatasia, and by detecting the above mutations, the presence or absence of morbidity of hypophosphatasia and the risk of onset can be known.

It is a figure which shows the primer for detecting the variation | mutation of a tissue non-specific type alkaline phosphatase (TNSALP) gene. It is a figure which shows the position of the SNP of exon 7 of a tissue non-specific type alkaline phosphatase (TNSALP) gene, and the melting curve of the amplified fragment used for detection. It is a figure which shows the position of the SNP of exon 9 of a tissue non-specific type alkaline phosphatase (TNSALP) gene, and the melting curve of the amplified fragment used for detection. It is a figure which shows the position of the SNP of exon 12-2 of a tissue non-specific alkaline phosphatase (TNSALP) gene, and the melting curve of the amplified fragment used for detection. It is a figure which shows the result of a detection of 1559T deletion mutation of a tissue non-specific type alkaline phosphatase (TNSALP) gene.

Hereinafter, the present invention will be described in detail.
In the present invention, a mutation in a tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene of hypophosphatasia such as perinatal hypophosphatasia, is screened. The mutation referred to in the present invention includes all mutations that cause a mismatch between a wild-type normal gene and a mutant gene, and includes deletion, substitution, addition, insertion, etc. of one or more bases. SNP).

  In the present invention, 14 fragments of the TNSALP gene are amplified using 12 primer pairs corresponding to the TNSALP gene, amplification products are obtained, mutations are detected, and screening is performed.

  The primer pair set corresponding to the TNSALP gene is a primer consisting of the nucleotide sequences of SEQ ID NOs: 1 to 24 shown in FIG.

  The present invention comprises at least one pair of the above primer pairs, and further comprises at least one pair, preferably two pairs, preferably three pairs, preferably four pairs, preferably five pairs, preferably six pairs, preferably six of the above primer pairs. Includes a kit for detecting a gene mutation comprising a primer pair comprising 7 pairs, preferably 8 pairs, preferably 9 pairs, preferably 10 pairs, preferably 11 pairs, more preferably all 12 pairs.

  The kit may further contain a heat-resistant polymerase for performing gene amplification and a DNA-binding dye for performing melting curve analysis. The kit can be used as a genetic diagnosis kit for hypophosphatasia.

  Amplification of the gene using the above primer set can be performed by a known gene amplification method such as PCR. As gene amplification methods other than PCR, strand displacement amplification (SDA method), isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN method), NASBA method, cascade rolling circle amplification Examples include cascade rolling circle amplification (CRCA method), Q beta replicase mediated amplification method, LAMP method, transcription-mediated amplification (TMA method), and the like.

  It is desirable to detect mutations by a high resolution melting curve analysis method because it is necessary to detect mutations in a wide range at once. However, other mutation detection methods such as Direct sequencing method, Cel1 method, and TGCE method can also be used.

  In melting curve analysis, after the DNA probe is hybridized to the target DNA, the temperature is gradually raised, and during this temperature rise, the signal that changes due to the melting of the hybrid is measured. This is a method for analyzing a melting curve of a hybrid with DNA. The melting temperature obtained by analyzing the melting curve reflects the homology between the DNA probe and the target DNA. Therefore, by determining the melting temperature, the identity of the target DNA can be determined, and as a result, mutations can be screened. . The melting process can be followed by a fluorescent dye that specifically binds to double-stranded DNA. Since the fluorescent dye is dissociated as the double-stranded DNA melts, the fluorescence intensity decreases. Therefore, by plotting the fluorescence intensity against the temperature, a melting temperature curve showing the melting process of the double-stranded DNA is obtained. Analysis can be performed by the change in fluorescence intensity accompanying this temperature rise, and the melting temperature curve of DNA with mutations such as deletion, substitution, addition, insertion, etc. in DNA is different from the melting temperature curve of the wild type, Analytical instruments can be used to identify differences in melting temperature curves and screen for mutations.

Melting curve analysis can be performed according to the method described in, for example, Reed G et al., Clin Chem., 50 (2004) 1748-54 and Special Table 2008-51098, for example, high-resolution mutation analysis It can be carried out using LightScanner or LS32 / LightScanner (Idaho Technology) known as an apparatus. In the case of the latter apparatus, PCR to melting curve analysis can be performed consistently. Examples of fluorescent dyes used for melting curve analysis include dyes that saturate and bind to double-stranded DNA (saturated dsDNA binding dyes). Saturated and bound dyes are those that significantly increase PCR when present in a concentration that gives the maximum fluorescent signal for the amount of dsDNA normally produced by PCR in the absence of dye, specifically about 10 ng / μL. A pigment that does not inhibit. Suitable dyes for use in the present invention include cyanine dyes containing one or more divalent moieties -C (R) = disposed in a chain connecting two heterocycles containing nitrogen. Here, R is hydrogen or an arbitrary carbon substituent, and examples thereof include alkyl including C _1-6 alkyl which may be substituted. When two or more -C (R) = are present in the cyanine dye, each R may be independently selected. Such cyanine dyes may be monomers or dimers as defined in more detail than the representative general formulas described herein. An example of such a cyanine dye is LCGreenPlus (trademark) (Japanese Patent Publication No. 2008-510698). However, other dyes could be used in methods that do not require detection of SNPs and other minor changes. Examples of the dye other than the cyanine dye include an acridine-based dye, a phenanthridinium intercalator, a phenanthroline-based metallointercalator, and the like, for example, SYBR (registered trademark) Green.

  As the sample, any sample containing the subject's DNA, such as the subject's peripheral blood and skin biopsy sample, can be used, but in view of noninvasiveness, the use of peripheral blood is desirable. Distribute the sample obtained from the subject to a 96-well or 384-well microplate, amplify the DNA of the sample in each well using the primer set described above, and amplify the DNA (amplicon) amplified in the well. Data analysis may be performed by analyzing the melting curve using an analysis apparatus such as the above-described Light Scanner. The data analysis may be performed by analyzing the melting curve using software such as Call-IT attached to the apparatus used for the above melting curve analysis. Furthermore, the present invention includes a primer pair for detecting a deletion mutation at the 1559th base T (1559T) of the TNSALP gene. The primer consists of a primer consisting of the base sequence shown in SEQ ID NO: 25 in FIG. 1 and a primer consisting of the base sequence shown in SEQ ID NO: 26. Using this primer pair, a fragment containing the 1559th base of TNSALP was amplified, and by melting curve analysis, it was determined whether the TNSALP 1559T deletion mutation was homozygous, heterozygous, or nonmutant. Can be determined and detected. By the method of the present invention, it is possible to screen and identify a mutation in a tissue non-specific alkaline phosphatase (TNSALP) gene that can cause the onset of hypophosphatasia. By detecting a mutation in the TNSALP gene, a subject's hypophosphatasia can be detected, and the subject's risk of suffering from hypophosphatasia can be determined and evaluated.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Comprehensive Analysis of TNSALP Gene Mutations Genomic DNA extracted from peripheral blood collected from hypophosphatasia patients or subjects considered to be carriers of TNSALP gene mutations was used as a sample, and the sample was treated with 10 mM Tris- After diluting in HCl (pH 7.5) and 0.1 mM EDTA, a fluorescent dye was added to each exon of the TNSALP gene shown in FIG. 1 with a specific primer and subjected to polymerase chain reaction (PCR). High resolution melting curve (HRM) of the sample after PCR was measured using a gene mutation analyzer called Light Scanner (Idaho Technology Inc .; http://www.jki.co.jp/product/LightScanner.htm) did.

  PCR was performed in a total volume of 10 μl per well of each exon 96 well plate. The reaction mixture contained 10 × ExPCR buffer, 200 μM dATP, dGTP dCTP, and dTTP, 0.3 μM primers (Forward, Reverse), 1 μl ExTaq polymerase, and dye (1/10 of 10 × LC Green Plus), respectively. Depending on the exon, PCR was performed by adding 10% DMSO or changing the enzyme to Takara Primer Star-CFX96. The polymerase chain reaction is performed in a PCR machine by first denaturing at 95 ° C for 2 minutes, followed by cooling to 94 ° C for 30 seconds, exon (annealing) temperature at 20 ° C / second (60 ° C to 72 ° C). A cycle of heating for 30 seconds was performed by 45 cycles. The size of the amplicon varies from exon to exon, but is 400 bp or less. After PCR, the sample was heated to 94 ° C. for 30 seconds and then cooled to 25 ° C.

  Samples were then transferred to a high resolution DNA melting analyzer, Light Scanner (http://www.jki.co.jp/product/LightScanner.htm) (Idaho Technology, Salt Lake City, UT), and 96 well melting curves were analyzed simultaneously. . Melting curve analysis was performed as described in the examples.

  The results are shown in FIGS. 2-1 to 2-3. FIGS. 2-1 to 2-3 show the positions of the SNPs of exons 7, 9 and 12-2, respectively, and the melting curves of the amplified fragments used for detection. In all the primer pairs, it was confirmed that the PCR product showed a single band, and the melting curve also showed the fluorescence intensity. Here, homozygous SNP (melting curve in FIG. 2-1), heterozygous SNP (melting curve in FIG. 2-2), and heterozygous deletion (melting curve in FIG. 2-3) are high resolution melting. Detected from analysis.

Example 2 Detection of 1559T deletion mutation of TNSALP gene Small amplicon genotyping (SAG) method (Gundry) is a technique for identifying genotypes of known sequence mutations of the 1559T deletion mutation of TNSALP gene, which is common in Japanese. CN et al. Nucleic Acids Res. 2008, Vol. 36, 3401-3408). Primers were designed at positions immediately adjacent to the 1559T deletion mutation so that the amplified fragment was short (50-100 bp). PCR was performed in a total volume of 10 μl per well of a 96 well plate. The reaction mixture is 2.5x High Sensitivity Genotyping master mix, 200 μM dATP, dGTP dCTP, and dTTP, 0.3 μM primer (Forward, Reverse), 1 μl ExTaq polymerase, and dye (1/10 of 10 × LC Green Plus) Was included. The polymerase chain reaction is performed in a PCR machine by first denaturing at 95 ° C for 2 minutes, followed by cooling to 94 ° C for 30 seconds, exon (annealing) temperature at 20 ° C / second (60 ° C to 72 ° C). A cycle of heating for 30 seconds was performed by 45 cycles. The size of the amplicon varies from exon to exon, but is 400 bp or less. After PCR, the sample was heated to 94 ° C. for 30 seconds and then cooled to 25 ° C. Samples were then transferred to a high resolution DNA melting analyzer, Light Scanner (http://www.jki.co.jp/product/LightScanner.htm) (Idaho Technology, Salt Lake City, UT), and 96 well melting curves were analyzed simultaneously. . Melting analysis was performed as described in the examples.

  Specific results are shown in FIG. Homozygous and heterozygous deletions were detected from high resolution melting analysis.

  According to the method of the present invention, it is possible to screen a tissue non-specific alkaline phosphatase (TNSALP) gene mutation associated with hypophosphatasia in a subject, and determine the presence and risk of developing a low phosphatase disease based on the presence of the mutation. Can be evaluated.

SEQ ID NO: 1-26 Primer

Claims (4)

A method for comprehensive screening of tissue non-specific alkaline phosphatase (TNSALP) gene mutations that cause hypophosphatasia using the following 12 primer pairs by melting temperature curve analysis :
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 2 comprising the nucleotide sequence shown in SEQ ID NO: 1;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 4 comprising the nucleotide sequence shown in SEQ ID NO: 3;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 6 comprising the nucleotide sequence shown in SEQ ID NO: 5;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 8 comprising the nucleotide sequence shown in SEQ ID NO: 7;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 10 comprising the nucleotide sequence shown in SEQ ID NO: 9;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 12 comprising the nucleotide sequence shown in SEQ ID NO: 11;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 14 comprising the nucleotide sequence shown in SEQ ID NO: 13;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 16 comprising the nucleotide sequence shown in SEQ ID NO: 15;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 18 comprising the nucleotide sequence shown in SEQ ID NO: 17;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 20 comprising the nucleotide sequence shown in SEQ ID NO: 19;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 22 comprising the nucleotide sequence shown in SEQ ID NO: 21;
A pair of plug i Mar consisting of the nucleotide sequence shown in primers and SEQ ID NO: 24 comprising the nucleotide sequence shown in SEQ ID NO: 23. Using a sample obtained from a subject and using the 12 primer pairs according to claim 1 , the tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene for hypophosphatasia, was analyzed by melting temperature curve analysis . A test method for detecting a mutation and determining that a subject is suffering from hypophosphatasia or has a high risk of suffering from hypophosphatase when a genetic mutation is present. Using a sample obtained from a Japanese subject , a low phosphatase by a melting temperature curve analysis method using a primer consisting of a base sequence shown in SEQ ID NO: 25 and a primer pair consisting of a primer consisting of a base sequence shown in SEQ ID NO: 26 To determine whether the deletion mutation of 1559T, which is a deletion of one base of the tissue non-specific alkaline phosphatase (TNSALP) gene that is the causative gene of the disease , is homozygous, heterozygous, or not mutated A test method for determining that the patient is suffering from perinatal hypophosphatasia or has a high risk of suffering from perinatal hypophosphatasia . A mutation of the tissue non-specific alkaline phosphatase (TNSALP) gene, which is a causative gene for hypophosphatasia, comprising the primer pair according to claim 1 or 3 is comprehensively detected by a melting temperature curve analysis method. A kit for detecting phosphatasia or determining the risk of suffering from hypophosphatasia.

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