JP5493359B2 - Method, kit, use of polynucleotide for fertility estimation or identification of infertility, use of polypeptide for fertility estimation or identification of infertility, and antibody - Google Patents

Description

  The present invention relates to a technique that enables estimation of fertility and identification of the cause of infertility. Specifically, a method for diagnosing fertility by detecting a gene deficiency or mutation correlated with fertility, and a kit, polynucleotide, polypeptide, and Relates to antibodies.

  In recent years, the problem of declining birthrate has been frequently taken up, and various measures are being sought to solve this problem. Needless to say, it is important to rescue couples who have no children due to infertility as a countermeasure to the declining birthrate. In fact, Non-Patent Document 1 reports that about 15% of couples desiring to have children cannot get pregnant even after two years, and the development of an effective infertility treatment method is strongly desired.

  In recent years, the development of in vitro fertilization (IVF) technology has enabled pregnancy and childbirth even when sperm activity is low. However, the details of the molecular mechanisms behind infertility remain unclear, which can make it difficult to select an appropriate treatment.

  On the other hand, according to Non-Patent Document 2, studies on male infertility in mice have revealed the existence of many genes that affect fertility, and mutations in these genes cause male infertility in humans. It may be a factor. For example, Calmegin, which is a male germ cell-specific antigen, is a calcium-binding protein localized in the endoplasmic reticulum, and is a membrane protein Calnexin that is a universally expressed endoplasmic reticulum molecular chaperone (see, for example, Non-Patent Document 3). Is a homologue. It has been reported that male knockout mice (− / −) of the Calmegin gene are almost infertile, and their sperm cannot adhere to the egg extracellular matrix (transparent body) (see Non-Patent Document 4). This is a chaperone molecule of a protein in which Calmegin is involved in adhesion of sperm to an ovum, and it hinders folding of ADAMs and Izumo molecules involved in fusion and adhesion (Non-Patent Document 5) due to abnormal or decreased expression of the molecule. As a result, it is thought that it may become infertile.

On the other hand, there is Calreticulin (Calr3) as a protein specifically expressed in the testis as in Calmegin. Calr3 is mainly localized in the endoplasmic reticulum as a calcium-binding chaperone, and suggests that it functions in concert with Calmegin in the folding of newly biosynthesized glycoproteins (Non-patent Document 6). ). Calr3 gene, has not yet become clear function in the infertility process, by the inventors of the present ^application, 39 th Annual in the Meeting of the Society of Reproduction (2006 years), reported that Calr3 knockout mouse is infertility It was done.

De Kretser, 1 other, Journal of Clinical Endocrinology & Metabolism, 1999, 84, 3443-3450.

Mazak, 1 other, Nature Cell Biology, 2002, Volume 4 (Supp1), S41-S49.

Bergeron, 3 others, Trends In Biochemical Sciences, 1994, Vol. 19, pages 124-128.

Ikawa, 6 others, Nature, 1997, 387, 607-611.

Rubinstein, 4 others, Seminars in cell and developmental biology, 2006, Vol. 17, No. 2, pages 254-263.

Person, 2 outside, Gene, 2002, Vol. 297, No. 1-2, pages 151-158.

An effective genetic diagnosis method for diagnosing male infertility has not yet been established. The causes of male infertility range from deletion of germ cells themselves to infertility of sperm. Therefore, in order to establish an effective treatment method according to a disease state, a diagnostic method capable of identifying the cause of various disease states is desired.
The present invention has been made in view of the above problems, and provides a fertility diagnostic method that enables estimation of fertility possibility based on the ability of sperm to fuse with an egg and identification of the cause of infertility. The purpose is to do.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that male mice lacking a Calmegin gene functionally similar to the Calr3 gene become infertile, and Calmegin molecule antibodies are fused between sperm and ovum. The Calr3 molecule is expected to be a chaperone molecule of a protein involved in the adhesion from the expression site to the sperm egg, and the Calr3 knockout mouse becomes infertile. In order to complete the present invention, it is considered that a mutation of Calr3 protein causes (or is likely to cause) male infertility, a human Calr3 gene sequence is analyzed, and a mutation associated with male infertility is found in the human Calr3 gene. It came.

That is, the present invention has a detection step of detecting the presence or absence of a deletion or mutation of the Calr3 gene in the human using a biological sample collected from a human. In the detection step, the first adenine of the translation initiation codon is detected. The base at position 128, base at position 129, base at position 233, base at position 251, base at position 313, base at position 328 in the translation region of the human Calr3 gene. Base, 346th base, 370th base, 385th base, 584th base, 586th base, 616th base, 644th base, 647th base, 742nd base, 782nd base, 783th base, 820th base, 945th base, 946th base, 947th base, 970th base, 976th base Base at position 98, Base, 1022 base, 1023 base, 1034 base, 1046 base, 1056 base, 1058 base, 1063 base, 1074 base Detecting the presence or absence of a mutation in one or more bases selected from the group consisting of a base, a base at position 1083, a base at position 1086, and a base at position 1111,
When a mutation in which adenine of the base at position 128 is replaced with thymine is detected,
When a mutation in which cytosine at the position 129 is replaced with adenine is detected,
When a mutation in which cytosine of the base at position 233 is replaced with adenine is detected,
When a mutation in which the thymine at position 251 is replaced with adenine is detected,
When a mutation in which the thymine at position 313 is substituted with adenine is detected,
When a mutation in which the adenine of the 328th base is replaced with thymine is detected,
When a mutation is detected in which the guanine of the 346th base is replaced with thymine,
When a mutation in which guanine at the 370th base is replaced with thymine is detected,
When a mutation in which the thymine at position 385 is replaced with adenine is detected,
When a mutation in which the guanine of the base at position 584 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 586 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 616 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 644 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 647 is replaced with thymine is detected,
When a mutation in which the guanine at the 742nd base is replaced with thymine is detected,
When a mutation in which adenine of the base at position 782 is replaced with thymine is detected,
When a mutation in which cytosine at position 783 is substituted with adenine is detected,
When a mutation in which guanine at the base at position 820 is substituted with adenine is detected,
When a mutation in which cytosine of the base at position 945 replaces adenine is detected,
When a mutation in which the thymine at position 946 is replaced with adenine is detected,
When a mutation in which the thymine at position 947 is replaced with adenine is detected,
When a mutation in which the thymine at position 970 is replaced with adenine is detected,
When a mutation in which the guanine at the 976th base is replaced with adenine is detected,
When a mutation in which the thymine at position 981 is replaced with adenine is detected,
When a mutation in which the guanine of the base at position 1022 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1023 is replaced with thymine is detected,
When a mutation in which cytosine of the base at position 1034 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 1046 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1056 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 1058 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1063 is replaced with thymine is detected,
When a mutation in which adenine of the base at position 1074 is replaced with thymine is detected,
When a mutation in which the thymine at position 1083 is substituted with adenine is detected,
When a mutation in which the guanine at the 1086th base is replaced with thymine is detected, or when a mutation in which the cytosine at the 1111th base is replaced with thymine is detected,
It is an object of the present invention to provide a method characterized in that it is estimated that the human fertility is low, or that the human infertility is highly likely due to a functional defect of the Calr3 molecule.
The present invention also relates to the base sequence of human Calr3 gene or the base sequence represented by SEQ ID NO: 1, the base at position 233, the base at position 586, the position 616, the position 644 in the translation region of the human Calr3 gene. , The 647th base, the 742nd base, the 820th base, the 976th base, the 1056th base, or the 1058th base, and the substituted base The present invention provides a use of a polynucleotide for estimating fertility or identifying the cause of infertility, characterized by comprising 10 to 100 consecutive nucleotide sequences containing or a nucleotide sequence complementary to the nucleotide sequence. For the purpose.
The present invention also relates to a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, wherein the base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, and the base at position 644 Is thymine, the base at position 647 is thymine, the base at position 742 is thymine, the base at position 820 is adenine, the base at position 976 is adenine, the base at position 1056 is thymine, or the base at position 1058 is A polynucleotide comprising a nucleotide sequence substituted with thymine, and an estimation of fertility or identification of infertility of the polypeptide comprising an amino acid sequence encoded by the polynucleotide The purpose is to provide use for.
Further, the present invention provides the polynucleotide represented by SEQ ID NO: 1, wherein the base at position 128 is thymine, the base at position 129 is adenine, the base at position 251 is adenine, the base at position 313 is adenine, The base at position 328 is thymine, the base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, the base at position 584 is thymine, the base at position 782 is thymine, and position 783 The base at position 945 is adenine, the base at position 946 is adenine, the base at position 947 is adenine, the base at position 970 is adenine, the base at position 981 is adenine, the base at position 1022 Is thymine, the base at position 1023 is thymine, the base at position 1034 is thymine, the base at position 1046 is thymine, the base at position 1063 is thymine, the base at position 1074 is thymine, A polynucleotide comprising a base sequence in which the base at position 1083 is substituted with adenine, the base at position 1086 is thymine, or the base at position 1111 is replaced with thymine, and an amino acid sequence encoded by the polynucleotide It is an object of the present invention to provide a use of a polypeptide characterized in that it is used to estimate fertility or identify the cause of infertility .
Furthermore, the present invention provides a kit for carrying out the above-described method , comprising a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation, wherein the mutation comprises a translation initiation codon adenine. When the first base is used, the 128th base, the 129th base, the 233th base, the 251st base, the 313th base, the 328th position of the translation region of the human Calr3 gene Base, position 346, base position 370, position base 385, position base 584, position base 586, position base 616, position base 644, position base 647 , 742th base, 782nd base, 783th base, 820th base, 945th base, 946th base, 947th base, 970th base, A base at position 976, a base at position 981; Base at position 1022, base at position 1023, base at position 1034, base at position 1046, base at position 1056, base at position 1058, base at position 1063, base at position 1074, position 1083 A mutation of one or more bases selected from the group consisting of base No. 1086, base No. 1111, and base sequence of the human wild-type Calr3 gene or a translation region thereof in was labeled by the polynucleotide, or pre-fluorescent dye the polynucleotide consisting of a nucleotide sequence complementary to the contiguous nucleotide sequence or the nucleotide sequence of 10 to 100, including a base portion which is subject to detect the presence or absence of a mutation high It is an object to provide a kit characterized by being a hybridization probe.

  By using the fertility diagnostic method and fertility diagnostic kit of the present invention, estimation of human fertility and the cause of infertility can be easily performed with high accuracy.

It is a physical map which shows the amplicon of PCR used for human Calr3 gene structure and sequence analysis. It is a schematic diagram explaining the principle of the invader method.

  The fertility diagnosis method according to the present invention is characterized by having a detection step of detecting the presence or absence of a deletion or mutation of the Calr3 gene in a human using a biological sample collected from the human. Human Calr3 gene deficiency and specific base mutations are highly correlated with male infertility. Therefore, it is possible to estimate fertility and identify the cause of infertility by collecting a biological sample from a subject and examining whether the subject's Calr3 gene is defective or mutated using this biological sample. Become.

  SEQ ID NO: 1 is obtained by extracting only the translation region from the base sequence of the transcription product of the wild-type Calr3 gene registered in GeneBank with the accession number NC — 001009.8. As shown in SEQ ID NO: 1, the wild type human Calr3 gene has a translation region consisting of 1155 bp. SEQ ID NO: 2 shows the deduced amino acid sequence of the wild type human Calr3 protein.

  FIG. 1 is a schematic diagram showing the structure of the wild type human Calr3 gene. Of the wild-type human Calr3 gene, the thin line portion is an intron and the thick line portion is an exon. “MET” is a start codon and “TER” stop codon. Furthermore, each numerical value under the wild type human Calr3 gene indicates the number of bases when the transcription start point is 1. According to the human genome resource constructed by the human genome project, the human Calr3 gene is located on the long arm of human chromosome 19 (19p13.11).

  As a result of the investigation shown in the Examples described later, the present inventors have found that there is a patient whose base is substituted in the translation region of the human Calr3 gene shown in SEQ ID NO: 1 in the population consisting of male infertility patients. I found it. On the other hand, these base substitutions were not found in a population of men with confirmed fertility.

  When the above substitution mutation occurs in the human Calr3 gene, a normal protein is not expressed. Therefore, even if the substitution mutation of the human Calr3 gene occurs only in one allele, the translation amount of the normal Calr3 protein decreases or the mutant Calr3 protein acts on a dominant negative. By doing so, it is estimated that there is a high possibility of causing infertility.

  In the present invention, mutation means that a base is deleted, substituted, or inserted in the base sequence of a gene. The mutation of the Calr3 gene detected in the detection step of the fertility diagnostic method according to the present invention is a mutation having a high correlation with male infertility. Specifically, by detecting XX mutations described below, it is diagnosed whether a human who has collected a biological sample used in the detection step is easily fertile or is infertile. In some cases, it can be determined whether the cause of infertility is due to a mutation in the Calr3 gene. “The X-position base of the translation region of the Calr3 gene” means “the X-position base of the translation region of the human Calr3 gene when the translation start codon adenine is the first base”. To do.

  As a mutation highly correlated with male infertility, there is a mutation in which adenine at position 128 in the translation region of the Calr3 gene is substituted with thymine. By this base substitution, aspartic acid at position 43 of the deduced amino acid sequence is encoded by valine.

  There is also a mutation in which cytosine at position 129 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, aspartic acid at position 43 in the deduced amino acid sequence is encoded by glutamic acid.

  There is also a mutation in which cytosine at position 233 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, serine at position 78 of the deduced amino acid sequence is encoded by tyrosine.

  There is also a mutation in which the thymine at position 251 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, phenylalanine at position 84 in the deduced amino acid sequence is encoded by tyrosine.

  There is also a mutation in which the thymine at position 313 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, the cysteine at position 105 of the deduced amino acid sequence is encoded by serine.

  There is also a mutation in which the adenine at position 328 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, isoleucine at position 110 of the deduced amino acid sequence is encoded by phenylalanine.

  There is also a mutation in which the guanine at position 346 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, aspartic acid at position 116 of the deduced amino acid sequence is encoded by tyrosine.

  There is also a mutation in which guanine at position 370 in the translation region of the Calr3 gene is replaced with thymine. This base substitution prevents the glycine at position 124 of the deduced amino acid sequence from being encoded (stop codon).

  There is also a mutation in which the 385th thymine in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, tyrosine at position 129 of the deduced amino acid sequence is encoded by asparagine.

  There is also a mutation in which guanine at position 584 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, serine at position 195 of the deduced amino acid sequence is encoded by isoleucine.

  There is also a mutation in which the adenine at position 586 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, isoleucine at position 196 of the deduced amino acid sequence is encoded by leucine.

  There is also a mutation in which adenine at position 616 in the translation region of the Calr3 gene is replaced with thymine. This base substitution prevents the lysine at position 206 of the deduced amino acid sequence from being encoded (stop codon).

  There is also a mutation in which the adenine at position 644 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, lysine at position 215 of the deduced amino acid sequence is encoded by methionine.

  There is also a mutation in which the adenine at position 647 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, aspartic acid at position 216 of the deduced amino acid sequence is encoded by valine.

  There is also a mutation in which the guanine at position 742 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, aspartic acid at position 248 of the deduced amino acid sequence is encoded by tyrosine.

  There is also a mutation in which adenine at position 782 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, the tyrosine at position 261 in the deduced amino acid sequence is encoded by phenylalanine.

  There is also a mutation in which cytosine at position 783 in the translation region of the Calr3 gene is substituted with adenine. By this base substitution, the tyrosine at position 261 of the deduced amino acid sequence is not encoded (stop codon).

  There is also a mutation in which the guanine at position 820 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, valine at position 274 of the deduced amino acid sequence is encoded by isoleucine.

  There is also a mutation in which cytosine at position 945 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, asparagine at position 315 of the deduced amino acid sequence is encoded by lysine.

  There is also a mutation in which thymine at position 946 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, phenylalanine at position 316 of the deduced amino acid sequence is encoded by isoleucine.

  In addition, there is a mutation in which thymine at position 947 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, phenylalanine at position 316 of the deduced amino acid sequence is encoded by tyrosine.

  There is also a mutation in which the thymine at position 970 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, tyrosine at position 324 of the deduced amino acid sequence is encoded by asparagine.

  There is also a mutation in which guanine at position 976 in the translation region of the Calr3 gene is replaced with adenine. As a result of this base substitution, aspartic acid at position 326 of the deduced amino acid sequence is encoded by asparagine.

  There is also a mutation in which the thymine at position 981 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, asparagine at position 327 of the deduced amino acid sequence is encoded by lysine.

  There is also a mutation in which guanine at position 1022 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, arginine at position 341 in the deduced amino acid sequence is encoded by methionine.

  There is also a mutation in which guanine at position 1023 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, arginine at position 341 in the deduced amino acid sequence is encoded by serine.

  There is also a mutation in which cytosine at position 1034 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, alanine at position 345 of the deduced amino acid sequence is encoded by valine.

  There is also a mutation in which the adenine at position 1046 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, lysine at position 349 of the deduced amino acid sequence is encoded by methionine.

  There is also a mutation in which the guanine at position 1056 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, methionine at position 352 of the deduced amino acid sequence is encoded by isoleucine.

  There is also a mutation in which the adenine at position 1058 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, lysine at position 353 of the deduced amino acid sequence is encoded by methionine.

  There is also a mutation in which the guanine at position 1063 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, alanine at position 355 of the deduced amino acid sequence is encoded by serine.

  There is also a mutation in which the adenine at position 1074 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, glutamic acid at position 358 in the deduced amino acid sequence is encoded by aspartic acid.

  There is also a mutation in which the thymine at position 1083 in the translation region of the Calr3 gene is replaced with adenine. By this base substitution, glutamic acid at position 361 of the deduced amino acid sequence is encoded by aspartic acid.

  There is also a mutation in which the guanine at position 1086 in the translation region of the Calr3 gene is replaced with thymine. By this base substitution, glutamic acid at position 362 of the deduced amino acid sequence is encoded by aspartic acid.

  In addition, there is a mutation in which cytosine at position 1111 in the translation region of the Calr3 gene is substituted with thymine. By this base substitution, the histidine at position 371 of the deduced amino acid sequence is encoded by tyrosine.

  In the fertility diagnostic method according to the present invention, in particular, the base at position 233, the base at position 586, the base at position 616, the base at position 644, the base at position 647 in the translation region of the Calr3 gene, It is preferable to detect the base at position 742, the base at position 820, the base at position 976, the base at position 1056, or the base at position 1058. This is because the correlation with fertility is higher. Moreover, in the fertility diagnosis method according to the present invention, one of the above mutations may be detected, or a plurality of mutations may be detected.

  The fertility diagnosis method according to the present invention uses these single nucleotide mutations as fertility genetic markers. For example, when these base mutations are not detected, it can be presumed that the subject from whom the biological sample used in the detection process has been collected is likely to have no problem with fertility. Moreover, when a test subject is an infertility patient, it can be determined that there is a high possibility that the cause of infertility is due to something other than a functional defect of the Calr3 gene. On the other hand, when a mutation is detected, it is estimated that the subject has a low possibility of fertility. Further, if the subject is an infertile patient, it can be determined that the cause is highly likely due to a defective function of the Calr3 molecule due to a deletion or mutation of the Calr3 gene. Therefore, the disease can be cured at an early stage by giving appropriate treatment such as TSE-ICSI (Testicular biopsy-intracytoplasmic super injection) to such a subject. That is, the fertility diagnostic method according to the present invention can be said to be a diagnostic method for the purpose of estimating fertility and identifying the cause of infertility. With this method, infertility is diagnosed. Because important information is provided, subjects can be presented with a qualified selection from existing treatments. For this reason, the fertility diagnostic method according to the present invention can be preferably used in the diagnosis of low fertility or infertility.

  The biological sample used in the fertility diagnostic method according to the present invention is particularly limited as long as it is a sample collected from a human and can be expected to contain a nucleic acid or protein derived from the Calr3 gene. Instead, for example, blood, serum, plasma, mucous membranes such as the inside of the cheek, semen, testicular tissue (eg, seminiferous tubules) collected from the subject by testicular biopsy, sperm cells, seminal plasma and the like can be mentioned. Here, the nucleic acid derived from the Calr3 gene means genomic DNA of the Calr3 gene, mRNA that is a transcription product of the gene, or the like. The protein derived from the Calr3 gene means a Calr3 molecule that is a protein expressed from the Calr3 gene, a degradation product thereof, or the like. In particular, when a protein derived from the Calr3 gene is to be detected, biological samples such as semen, testicular tissue (eg, seminiferous tubules) collected from subjects by testicular biopsy, sperm cells, seminal plasma, etc. may be used. preferable. The seminal plasma is considered to contain a protein expressed in the sperm cell, which is released when the sperm cell is destroyed even if it does not contain the sperm cell.

  It should be noted that the biological sample may be used in the detection step as it is by appropriately diluting it, or DNA such as genomic DNA previously extracted from the biological sample may be used. Alternatively, after extracting RNA such as mRNA from the biological sample, cDNA synthesized from the RNA by reverse transcription reaction may be used in the detection step.

  In the detection step of the fertility diagnostic method according to the present invention, the method for detecting a deficiency or mutation of the human Calr3 gene is not particularly limited, and various known techniques used in detection of gene mutation or polymorphism, for example, , Nucleotide sequencing method based on Sanger method, invader method, Taqman probe method, SMMD method (Simultaneous Multiple Mutation Detection System), PCR-RFLP (polymerase chain reaction-restriction fragment length method, elongation method) The method, the method which modified them, etc. can be used. Since detection sensitivity and accuracy are excellent, it is preferable to use a probe or primer having a base sequence that specifically recognizes a mutation site, such as the invader method or the Taqman probe method.

  Probes and primers for detecting a deletion or mutation of the human Calr3 gene can be designed and synthesized by a conventional method based on the human Calr3 gene and its surrounding base sequences. The human Calr3 gene and the surrounding nucleotide sequences are obtained from publicly known databases such as NCBI Human Genome Resources (http://www.ncbi.nlm.nih.gov/genome/guide/human/). Those skilled in the art can easily obtain them.

For example, as a primer for detecting the presence or absence of a mutation at position 233 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the adenine of the translation initiation codon is the first position. In the polynucleotide comprising the base sequence of the translation region of the human wild-type Calr3 gene, that is, the base sequence represented by SEQ ID NO: 1, the base at position 233 is substituted from cytosine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 233 and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that there is no mutation in the base at position 233, the base sequence of the translation region of the human wild-type Calr3 gene (base represented by SEQ ID NO: 1) is also used. A polynucleotide having a base sequence at position 233 is cytosine, and comprising a base sequence homologous or complementary to 10 to 100 consecutive base sequences including the base at position 233. Can be used.
On the other hand, as a primer or the like for detecting the presence or absence of a mutation at the position 233 of the translation region of the Calr3 gene using genomic DNA, the primer containing the base at the position 233 in the base sequence of the Calr3 gene is used. A polynucleotide comprising 100 consecutive base sequences and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 233rd base of the translation region of the Calr3 gene. That is, as a primer for detecting the mutation at position 233 of the translation region of the Calr3 gene, in the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4, the 101st nucleotide is replaced from cytosine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that there is no mutation in the base at position 233, a polynucleotide comprising the base sequence also represented by SEQ ID NO: 4, A polynucleotide comprising 10 to 100 consecutive base sequences containing bases and a homologous or complementary base sequence can be used.

Similarly, as a primer for detecting the presence or absence of a mutation at position 586 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 586 is substituted from adenine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 586 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 586 and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 586 of the translation region of the Calr3 gene using genomic DNA, the 103rd base in the base sequence represented by SEQ ID NO: 5 is changed from adenine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 103rd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 586, 10 to 100 containing the 103rd base in the base sequence similarly represented by SEQ ID NO: 5 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 5 is a partial base sequence of the Calr3 gene, and the 103rd base corresponds to the 586th base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 616 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as the base sequence represented by SEQ ID NO: 1. In which a base at position 616 is substituted from adenine to thymine, and a polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 616 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 616th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 616 of the translation region of the Calr3 gene using genomic DNA, the base at position 133 in the base sequence represented by SEQ ID NO: 5 is from adenine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 133rd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at the 616th position is a wild type that has not been mutated, the base sequence represented by SEQ ID NO: 5 contains 10th to 100th nucleotides containing the 133rd base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 5 is a partial base sequence of the Calr3 gene, and the 133rd base corresponds to the 616th base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 644 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction from the nucleotide sequence represented by SEQ ID NO: 1 , The nucleotide at position 644 is substituted from adenine to thymine, and a polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 644 and a homologous or complementary base sequence, SEQ ID NO: In the base sequence represented by 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 644 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 644 of the translation region of the Calr3 gene using genomic DNA, the 161st base in the nucleotide sequence represented by SEQ ID NO: 5 is changed from adenine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences including the 161st base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that the base at the position 644 is a wild type without mutation, the base sequence represented by SEQ ID NO: 5 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 5 is a partial base sequence of the Calr3 gene, and the 161st base corresponds to the 644th base in the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 647 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which a base at position 647 is substituted from adenine to thymine, and a polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 647 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 647 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 647 of the translation region of the Calr3 gene using genomic DNA, the 164th base in the nucleotide sequence represented by SEQ ID NO: 5 is changed from adenine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 164th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at the 647th position is a wild type that has not been mutated, the base sequence represented by SEQ ID NO: 5 contains 10 to 100 including the 164th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 5 is a partial base sequence of the Calr3 gene, and the 164th base corresponds to the 647th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 742 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 , The nucleotide at position 742 is substituted from guanine to thymine, and a polynucleotide or sequence comprising 10 to 100 consecutive base sequences containing the base at position 742 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 742nd base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 742 of the translation region of the Calr3 gene using genomic DNA, the 101st base in the base sequence represented by SEQ ID NO: 6 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that there is no mutation in the 742nd base, the base sequence represented by SEQ ID NO: 6 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 6 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 742nd base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 820 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as the base sequence represented by SEQ ID NO: 1. , The nucleotide at position 820 is substituted from guanine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 820 and a homologous or complementary base sequence, In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 820th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 820 of the translation region of the Calr3 gene using genomic DNA, the 101st base in the nucleotide sequence represented by SEQ ID NO: 7 is changed from guanine to adenine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting a wild type in which no mutation has occurred in the base at position 820, the base sequence represented by SEQ ID NO: 7 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 7 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 820th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 976 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 976 is substituted from guanine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 976 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 976th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 976 of the translation region of the Calr3 gene using genomic DNA, the base at position 132 in the base sequence represented by SEQ ID NO: 8 is changed from guanine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 132nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 976, 10 to 100 containing the 132nd base in the base sequence similarly represented by SEQ ID NO: 8 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 132nd base corresponds to the 976th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 1056 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 1056 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1056 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 1056th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 1056 in the translation region of the Calr3 gene using genomic DNA, the 135th base in the base sequence represented by SEQ ID NO: 9 is changed from guanine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 135th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the 1056th base is a wild type without mutation, the base sequence represented by SEQ ID NO: 9 contains 10th to 100th nucleotides containing the 135th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 135th base corresponds to the 1056th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 1058 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 1058 is substituted from adenine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1058 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 1058th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 1058 of the translation region of the Calr3 gene using genomic DNA, the base at position 137 in the base sequence represented by SEQ ID NO: 9 is from adenine to thymine. And a polynucleotide comprising 10 to 100 consecutive base sequences containing the 137th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at position 1058 is a wild type in which no mutation has occurred, 10 to 100 containing the 137th base in the base sequence similarly represented by SEQ ID NO: 9 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 137th base corresponds to the 1058th base of the translation region of the Calr3 gene.

Similarly, as a primer or the like for detecting the presence or absence of a mutation at position 128 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which a base at position 128 is substituted from adenine to thymine, a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 128 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 128th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 128 of the translation region of the Calr3 gene using genomic DNA, the 101st base in the base sequence represented by SEQ ID NO: 3 is from adenine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that there is no mutation at the 128th base, the base sequence represented by SEQ ID NO: 3 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 3 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 128th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 129 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which the base at position 129 is replaced by cytosine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 129 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 129th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 129 of the translation region of the Calr3 gene using genomic DNA, the base at position 102 in the nucleotide sequence represented by SEQ ID NO: 3 is from cytosine to adenine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 102nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting a wild type in which no mutation has occurred in the base at position 129, 10 to 100 containing the 102nd base in the base sequence also represented by SEQ ID NO: 3 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 3 is a partial base sequence of the Calr3 gene, and the 102nd base corresponds to the 129th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 251 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 251 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 251 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the base at position 251 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 251 of the translation region of Calr3 gene using genomic DNA, the 119th base is changed from thymine to adenine in the base sequence represented by SEQ ID NO: 4. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 119th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that there is no mutation in the 251st base, the base sequence represented by SEQ ID NO: 4 contains 10th to 100th nucleotides containing the 119th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 119th base corresponds to the 251st base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 313 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which the base at position 313 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 313 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 313 and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 313 of the translation region of the Calr3 gene using genomic DNA, the 181st base in the nucleotide sequence represented by SEQ ID NO: 4 is changed from thymine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 181st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at position 313 is a wild type without mutation, 10 to 100 containing the 181st base in the base sequence similarly represented by SEQ ID NO: 4 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 181st base corresponds to the 313rd base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 328 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 328 is substituted from adenine to thymine, and a polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 328 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 328 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 328 of the translation region of the Calr3 gene using genomic DNA, the 196th base in the base sequence represented by SEQ ID NO: 4 is changed from adenine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences including the 196th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at the position 328 is a wild type in which no mutation has occurred, 10 to 100 containing the 196th base in the base sequence also represented by SEQ ID NO: 4 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 196th base corresponds to the 328th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 346 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 346 is substituted from guanine to thymine, and a polynucleotide comprising 10 to 100 consecutive base sequences containing the base at position 346 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 346th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 346 of the translation region of the Calr3 gene using genomic DNA, the base at position 214 in the base sequence represented by SEQ ID NO: 4 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 214th base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that there is no mutation at the 346th base, the base sequence represented by SEQ ID NO: 4 contains 10th to 100th bases containing the 214th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 214th base corresponds to the 346th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 370 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. , The nucleotide at position 370 is substituted from guanine to thymine, and a polynucleotide or sequence comprising 10 to 100 consecutive base sequences containing the base at position 370 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 370th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 370 of the translation region of the Calr3 gene using genomic DNA, the 238th base in the nucleotide sequence represented by SEQ ID NO: 4 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences including the 238th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the 370th base is a wild type in which no mutation has occurred, 10 to 100 containing the 238th base in the base sequence similarly represented by SEQ ID NO: 4 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 238th base corresponds to the 370th base of the translation region of the Calr3 gene.

Similarly, as a primer or the like for detecting the presence or absence of a mutation at position 385 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which a base at position 385 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 385 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 385th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 385 of the translation region of the Calr3 gene using genomic DNA, the nucleotide at position 253 in the nucleotide sequence represented by SEQ ID NO: 4 is changed from thymine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 253rd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 385, the base sequence represented by SEQ ID NO: 4 contains 1053 to 100 including the 253rd base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 4 is a partial base sequence of the Calr3 gene, and the 253rd base corresponds to the 385th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at the position 584 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 584 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 584 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the 584th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting mutation of the 584th position in the translation region of the Calr3 gene using genomic DNA, the 101st base in the base sequence represented by SEQ ID NO: 5 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred at the 584th base, 10 to 100 containing the 101st base in the base sequence also represented by SEQ ID NO: 5 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 5 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 584th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 782 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 782 is substituted from adenine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 782 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the 782nd base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 782 of the translation region of the Calr3 gene using genomic DNA, in the base sequence represented by SEQ ID NO: 6, the 141st base is from adenine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 141st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 782, the base sequence represented by SEQ ID NO: 6 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 6 is a partial base sequence of the Calr3 gene, and the 141st base corresponds to the 782nd base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 783 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which a base at position 783 is substituted from cytosine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 783 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 783rd base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 783 of the translation region of the Calr3 gene using genomic DNA, in the base sequence represented by SEQ ID NO: 6, the 142nd base is from cytosine to adenine. And a polynucleotide comprising 10 to 100 consecutive base sequences containing the 142nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 783, the base sequence represented by SEQ ID NO: 6 contains 10 to 100 including the 142nd base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 6 is a partial base sequence of the Calr3 gene, and the 142nd base corresponds to the 783rd base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 945 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as the base sequence represented by SEQ ID NO: 1. In which the base at position 945 is substituted from cytosine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 945 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 945th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 945 of the translation region of the Calr3 gene using genomic DNA, the 101st base in the nucleotide sequence represented by SEQ ID NO: 8 is from cytosine to adenine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at position 945 is a wild type without mutation, 10 to 100 containing the 101st base in the base sequence similarly represented by SEQ ID NO: 8 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 945th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 946 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which the base at position 946 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 946 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 946 and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 946 of the translation region of Calr3 gene using genomic DNA, the base at position 102 in the base sequence represented by SEQ ID NO: 8 is changed from thymine to adenine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 102nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at the 946th position is a wild type without mutation, 10 to 100 containing the 102nd base in the base sequence similarly represented by SEQ ID NO: 8 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 102nd base corresponds to the 946th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 947 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which the base at position 947 is replaced by thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 947 and a homologous or complementary base sequence or SEQ ID NO: In the base sequence represented by 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 947 and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 947 of the translation region of the Calr3 gene using genomic DNA, the 103rd base in the base sequence represented by SEQ ID NO: 8 is changed from thymine to adenine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 103rd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 947, 10 to 100 containing the 103rd base in the base sequence similarly represented by SEQ ID NO: 8 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 103rd base corresponds to the 947th base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 970 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 is used. In which the base at position 970 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 970 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 970th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 970 of the translation region of the Calr3 gene using genomic DNA, the 126th base in the base sequence represented by SEQ ID NO: 8 is changed from thymine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 126th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that there is no mutation at the 970th base, the base sequence represented by SEQ ID NO: 8 contains 10th to 100th bases containing the 126th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 126th base corresponds to the 970th base in the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 981 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the base sequence represented by SEQ ID NO: 1 In which the base at position 981 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 981 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences containing the 981st base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 981 of the translation region of the Calr3 gene using genomic DNA, the 137th base is changed from thymine to adenine in the base sequence represented by SEQ ID NO: 8. And a polynucleotide comprising 10 to 100 consecutive base sequences containing the 137th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the base at position 981, the base sequence represented by SEQ ID NO: 8 contains 10 to 100 including the 137th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 8 is a partial base sequence of the Calr3 gene, and the 137th base corresponds to the 981st base in the translation region of the Calr3 gene.

Similarly, as a primer or the like for detecting the presence or absence of a mutation at position 1022 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 1022 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1022 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 1022 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation in the position 1022 of the translation region of the Calr3 gene using genomic DNA, the 101st base in the base sequence represented by SEQ ID NO: 9 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 101st base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that the base at position 1022 is a wild type without mutation, 10 to 100 containing the 101st base in the base sequence similarly represented by SEQ ID NO: 9 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 101st base corresponds to the 1022nd base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 1023 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction from this, the base sequence represented by SEQ ID NO: 1 In which the base at position 1023 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1023 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the 1023rd base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 1023 of the translation region of the Calr3 gene using genomic DNA, the base at position 102 in the base sequence represented by SEQ ID NO: 9 is from guanine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 102nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting a wild type in which no mutation has occurred in the base at position 1023, the base sequence represented by SEQ ID NO: 9 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 102nd base corresponds to the 1023rd base of the translation region of the Calr3 gene.

Similarly, as a primer for detecting the presence or absence of a mutation at position 1034 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 1034 is substituted from cytosine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences including the base at position 1034 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the 1034th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 1034 of the translation region of the Calr3 gene using genomic DNA, the base at position 113 in the base sequence represented by SEQ ID NO: 9 is from cytosine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 113th base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that there is no mutation in the 1034th base, the base sequence represented by SEQ ID NO: 9 contains 10th to 100th nucleotides containing the 113th base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 113th base corresponds to the 1034th base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 1046 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as a base sequence represented by SEQ ID NO: 1. In which a base at position 1046 is substituted from adenine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1046 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide consisting of 10 to 100 consecutive base sequences including the 1046th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 1046 of the translation region of the Calr3 gene using genomic DNA, the 125th base in the base sequence represented by SEQ ID NO: 9 is changed from adenine to thymine. A polynucleotide consisting of 10 to 100 consecutive base sequences containing the 125th base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred in the 1046th base, 10 to 100 containing the 125th base in the base sequence also represented by SEQ ID NO: 9 A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 125th base corresponds to the 1046th base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 1063 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as the base sequence represented by SEQ ID NO: 1. In which the base at position 1063 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1063 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the base at position 1063 and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at the 1063rd position in the translation region of the Calr3 gene using genomic DNA, the 142nd base in the base sequence represented by SEQ ID NO: 9 is from guanine to thymine. And a polynucleotide comprising 10 to 100 consecutive base sequences containing the 142nd base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that the base at position 1063 is a wild type in which no mutation has occurred, 10 to 100 containing the 142nd base in the base sequence similarly represented by SEQ ID NO: 9. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 142nd base corresponds to the 1063rd base of the translation region of the Calr3 gene.

Similarly, as a primer or the like for detecting the presence or absence of a mutation at position 1074 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which a base at position 1074 is substituted from adenine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1074 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 1074th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation in the position 1074 of the translation region of the Calr3 gene using genomic DNA, the 153rd base in the base sequence represented by SEQ ID NO: 9 is changed from adenine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 153rd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting that the base at the 1074th position is a wild type that has not been mutated, the base sequence represented by SEQ ID NO: 9 also contains the 153rd base. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 153rd base corresponds to the 1074th base of the translation region of the Calr3 gene.

Similarly, as a primer or the like for detecting the presence or absence of a mutation at position 1083 in the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction, the nucleotide sequence represented by SEQ ID NO: 1 In which the base at position 1083 is substituted from thymine to adenine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1083 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 1083th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer or the like for detecting a mutation at position 1083 of the translation region of the Calr3 gene using genomic DNA, the 162nd base in the nucleotide sequence represented by SEQ ID NO: 9 is changed from thymine to adenine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 162nd base and a homologous or complementary base sequence can be used. In addition, as a primer for detecting the wild type in which no mutation has occurred at the base at position 1083, the base sequence represented by SEQ ID NO: 9 contains 10th to 100th bases. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 162nd base corresponds to the 1083rd base of the translation region of the Calr3 gene.

Similarly, a primer for detecting the presence or absence of a mutation at position 1086 of the translation region of the Calr3 gene using mRNA or cDNA obtained from the reverse transcription reaction is used as the base sequence represented by SEQ ID NO: 1. In which the base at position 1086 is substituted from guanine to thymine, and a polynucleotide consisting of 10 to 100 consecutive base sequences containing the base at position 1086 and a homologous or complementary base sequence; In the base sequence represented by No. 1, a polynucleotide comprising 10 to 100 consecutive base sequences including the 1086th base and a homologous or complementary base sequence can be used.
On the other hand, as a primer for detecting a mutation at position 1086 in the translation region of the Calr3 gene using genomic DNA, the 165th base in the base sequence represented by SEQ ID NO: 9 is changed from guanine to thymine. A polynucleotide comprising 10 to 100 consecutive base sequences containing the 165th base and a homologous or complementary base sequence can be used. Further, as a primer or the like for detecting that there is no mutation in the base at position 1086, 10 to 100 containing the 165th base in the base sequence similarly represented by SEQ ID NO: 9. A polynucleotide comprising a continuous base sequence and a homologous or complementary base sequence can be used. The base sequence represented by SEQ ID NO: 9 is a partial base sequence of the Calr3 gene, and the 165th base corresponds to the 1086th base of the translation region of the Calr3 gene.

  Similarly, a primer for detecting the presence or absence of a mutation at position 1111 of the translation region of the Calr3 gene using mRNA, cDNA obtained by reverse transcription reaction from this, or genomic DNA is represented by SEQ ID NO: 1. A base sequence at position 1111 is replaced by cytosine to thymine, and a polymorphism consisting of 10 to 100 consecutive base sequences containing the base at position 1111 and a homologous or complementary base sequence. In the nucleotide or the nucleotide sequence represented by SEQ ID NO: 1, a polynucleotide consisting of a homologous or complementary nucleotide sequence with 10 to 100 consecutive nucleotide sequences including the base at position 1111 can be used.

  Thus, as a primer for detecting the presence or absence of each mutation described above, the base sequence of the translation region of the human wild-type Calr3 gene (SEQ ID NO: 4) when the translation start codon adenine is the first base. In the base sequence represented by 1), a polynucleotide comprising 10 to 100 consecutive base sequences including the mutation site and a homologous or complementary base sequence can be used. In addition, in the genomic DNA of the human wild-type Calr3 gene, a polynucleotide comprising 10 to 100 consecutive base sequences including the mutation site and a homologous or complementary base sequence can also be used. The length of these polynucleotides is preferably 10 to 70 bases, more preferably 10 to 50 bases, and even more preferably 10 to 30 bases. In addition, since the detection accuracy of the presence or absence of mutation is improved, the position of the base that can pair with the base of the mutation site is not particularly limited in the polynucleotide, but 1 to 5 bases from the 3 ′ end It is preferably within the range, and more preferably at the 3 ′ end.

  A kit for diagnosing human fertility and using a fertility diagnostic kit containing a reagent for detecting the presence or absence of a deletion or mutation of the human Calr3 gene according to the present invention. The fertility diagnosis method can be performed more simply. As a fertility diagnostic kit according to the present invention, when the translation initiation codon adenine is the first base, the 128th base, the 129th base, the 233rd position of the translation region of the human Calr3 gene Base, position 251, position 313, position 328, position 346, position 370, position 385, position 584, position 586 , 616th base, 644th base, 647th base, 742nd base, 782nd base, 783th base, 820th base, 945th base, Base at position 946, base at position 947, base at position 970, base at position 976, base at position 981, base at position 1022, base at position 1023, base at position 1034, position 1046 Base, position 1056, position 1058 It preferably contains a reagent for detecting a mutation in the base, the 1063 position base, the 1074 position base, the 1083 position base, the 1086 position base, or the 1111 position base. Position base, position 586 base, position 616 base, position 644 base, position 647 base, position 742 base, position 820 base, position 976 base, position 1056 More preferably, it contains a base or a reagent for detecting a mutation at the 1058th position. Moreover, the kit containing the reagent for detecting one type of mutation among these mutations may be sufficient, and the kit containing the reagent for detecting multiple types of mutation may be sufficient.

  Reagents for detecting these base mutations include, for example, a primer pair for amplifying a nucleic acid region containing the base by PCR and the like, and a base sequence for determining the base sequence of the obtained amplification product. And a determination reagent. Examples of the base sequence determination reagent include a homologous or complementary sequence of 10 to 100 consecutive base sequences including the mutation site in the base sequence of the human wild-type Calr3 gene described above or the base sequence of the translation region thereof. Examples thereof include a hybridization probe in which a polynucleotide comprising a base sequence is previously labeled with a fluorescent dye or the like. In addition, when the method for detecting the deletion or mutation of the human Calr3 gene is the PCR-RFLP method, a restriction enzyme used in the RFLP method may be used.

Moreover, the fertility diagnosis method and fertility diagnosis kit according to the present invention are not particularly limited as long as they detect the presence or absence of a mutation that causes a functional defect in the human Calr3 gene. For example, the fertility diagnostic method and fertility diagnostic kit may detect that a human Calr3 gene of a subject contains a mutation that causes a functional defect in the gene. You may detect that the Calr3 gene does not contain the mutation which causes the functional defect | deletion of this gene.
For example, in the case of detecting the presence of the mutation described above, the fertility diagnosis method and fertility diagnosis kit need only be able to detect that these mutations occur in at least one allele. On the other hand, when detecting the absence of these mutations, the fertility diagnostic method and fertility diagnostic kit preferably detect that these mutations have not occurred in both alleles.

  More specifically, in one embodiment, the fertility diagnosis method and the fertility diagnosis kit according to the present invention include at least one of the above mutation sites (hereinafter, referred to as “therespective of human Calr3 gene”). The presence or absence of the above-described substitution mutation may be detected by determining the base sequence of the region containing the base of “mutation detection site”. For the determination of the base sequence, the Sanger method or various known techniques applying the Sanger method can be used.

In this case, the fertility diagnostic kit uses a mutation detection site as a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation in order to amplify the region containing the mutation detection site of the human Calr3 gene. It is preferred that a set of primers designed to be sandwiched is included. More specifically, the primer set includes a primer having a part or all of the base sequence upstream of the mutation detection site of the human Calr3 gene and a base sequence downstream of the mutation detection site of the human Calr3 gene. And a primer having a base sequence complementary to part or all.
By performing PCR using the above-described primer set using genomic DNA contained in a biological sample collected from a subject as a template, the region containing the base of the mutation detection site of the human Calr3 gene is amplified as a DNA fragment. By determining the base sequence using one or both of the above primer sets, the presence or absence of a substitution mutation can be reliably and easily detected. The base sequence can be easily determined by using, for example, ABI-PRISM 310 Genetic Analyzer (Applied Biosystems Inc.) according to the instruction manual.

  In addition, a primer different from a primer used for PCR for amplification of a DNA fragment may be used as a primer used for determination of a base sequence. That is, the fertility diagnostic kit includes a primer set for amplification of a DNA fragment and a primer used for determination of a base sequence as a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation. You may go out.

  In the primer set for amplification of the DNA fragment, the length of the region sandwiched between the primers is preferably 5 kb or less, more preferably 3 kb or less, and even more preferably 2 kb or less. , 1.5 kb or less is particularly preferable. By shortening the length of the region sandwiched between the primers, the region including the base of the mutation detection site can be effectively amplified.

The primer used for determining the base sequence is preferably set at a position within 2 kb from the base of the mutation detection site, preferably set at a position within 1.5 kb, and within 1.0 kb. Is more preferably set to a position within 0.7 kb. With the above configuration, the base sequence can be accurately determined.
Furthermore, the primer used for determining the base sequence is preferably set at a position separated by 20 bp or more from the base of the mutation detection site, more preferably set at a position separated by 30 bp or more, and separated by 40 bp or more. It is more preferable that the position is set, and it is particularly preferable that the position is set at a position separated by 50 bp or more. Moreover, it is preferable that the said primer group is what amplifies the area | region 20 bp or more containing the base of the mutation detection site | part of a human Calr3 gene. In the vicinity of the primer, the determination result of the base sequence tends to be inaccurate, but the base sequence can be accurately determined by the above configuration.

  Each primer preferably has at least 10 bases, more preferably 15 bases or more, and more preferably 18 bases or more, the same base sequence as that of the Calr3 gene or its periphery, or a complementary base sequence. It is preferable to have 20 bases or more. By lengthening the length of the same (or complementary) base sequence of the Calr3 gene or its surroundings, it is possible to reliably amplify only the target region including at least one of the mutation sites of the Calr3 gene. .

Examples of the above primer pairs include primers consisting of base sequences represented by SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19, respectively (1, 2r, 3 in FIG. 4r, 5.1, 6.1r, 7, 8r, 9 and 10R) can be used, but it goes without saying that the base sequences of the primers that can be used in the present invention are not limited to this sequence. A person skilled in the art can easily obtain the base sequence of the human Calr3 gene and the surrounding base sequence from the above-mentioned human genome resources and GeneBank, and therefore, Oligo (registered) based on the obtained base sequence. Primers can be designed using trademarks such as National Bioscience Inc. or GENETYX (software development).
Each primer can be synthesized by a known method such as a phosphoramidite method, and can be easily synthesized by using, for example, Applied Biosystems Inc. 392 synthesizer according to the instruction manual.

Each primer described above may have a fluorescent label. When a fluorescent label is attached to the primer, the base sequence of the amplification region can be determined by the die primer method without using a radioisotope.
On the other hand, when the fluorescent label is not attached to each primer, the base sequence can be determined by the dye terminator method. In this case, the fertility diagnostic kit includes deoxyribonucleic acid composed of monomers A, T, G, and C, fluorescently labeled A, T, G, and C, in addition to the primer set. A dideoxyribonucleic acid (so-called dye terminator) comprising a monomer, a polymerase, and the like may further be included.

  In one embodiment, the fertility diagnostic method and the fertility diagnostic kit according to the present invention may detect the presence or absence of a base substitution mutation at the mutation detection site of the human Calr3 gene by an invader method. Good. Details of the invader method are described in a paper by Lyamichev et al. (Lyamichev V et al., Nat biotechnol. 17, 292, 1999), which is incorporated herein by reference.

FIG. 2 is a schematic diagram illustrating the principle of the invader method. In the invader method, an invader oligo 2 and an allele oligo 3 which are non-fluorescently labeled probes and a fret probe 5 which is a fluorescently labeled probe are used.
The invader probe 2 has a base sequence complementary to the target gene 1 and is designed so that the base at the 3 ′ end corresponds to the mutation (or single nucleotide polymorphism) detection site 1a of the target gene 1 ( It is not a complementary base to any base). On the other hand, the allele oligo 3 has a base sequence complementary to the flap 3b, which is a base sequence irrelevant to the target gene 1, and a target gene 1, and the 5 ′ terminal base is a mutation (or one) of the target gene 1. Nucleotide polymorphism) A nucleotide 3a designed to complement the detection site 1a is linked. Here, the flap 3b is linked to the 5 ′ end of the nucleotide 3a.
When the invader probe 2 and the allele oligo 3 designed in this way are hybridized with the target gene 1, they hybridize with the target gene 1 while taking the structure in which the allele oligo 3 and the invader probe 2 overlap each other, and at the mutation detection site 1a. The invader probe 2 enters only one base under the allele oligo 3. Then, the flap endonuclease (also called “clear base”) 4 recognizes this invasion structure and cleaves the allele oligo 3 at the 3 ′ side of the overlapped base. As a result, a flap free body 3c in which the flap 3b and the mutation detection site are linked is generated.
The fret probe 5 is composed of a nucleotide 5a capable of hybridizing with itself at the 5 'end side and hybridizing with the flap 3b at the 3' end side, a fluorescent dye 5b, and a quencher (luminescence inhibitor) 5c. Here, the nucleotide 5a has a base sequence complementary to the flap educt 3c at the 3 'end. The fluorescent dye 5b is bound to the 5 ′ end of the nucleotide 5a. However, in the fret probe 5, the fluorescence of the fluorescent dye 5b is suppressed by the quencher 5c.
When the flap free body 3c hybridizes to such a fret probe 5, a 3 entry structure is formed again at the mutation detection site of the flap free body 3c. As a result, the above-described flap endonuclease 4 recognizes this invasion structure again, and the 5 ′ end portion of the fret probe 5 is cleaved. As a result, the fluorescent dye 5b bonded to the 5 ′ end of the fret probe 5 is released and fluorescence is generated.

  Therefore, the fertility diagnostic method and fertility diagnostic kit according to the present invention may detect the presence or absence of a human Calr3 gene deletion or mutation by an invader method. In this case, the fertility diagnostic kit preferably includes invader probe 2, allele oligo 3, fret probe 5 and the like as reagents for detecting the presence or absence of a human Calr3 gene deletion or mutation.

When fluorescence is generated when there is a substitution mutation, the invader probe 2 has a base sequence complementary to the human Calr3 gene, and the 3 ′ terminal base is each mutant type of the mutant Calr3 gene. Designed to correspond to a base (but not complementary). In addition, nucleotide 3a of allele oligo 3 has a base sequence complementary to the human Calr3 gene, and is designed so that the base at the 5 ′ end is complementary to the above mutant base of the mutant Calr3 gene. The fret probe 5 is a flap free body 3c that is cleaved by the flap endonuclease 4 when the invader probe 2 and the allele oligo 3 are hybridized with the mutant Calr3 gene to form an invasion structure by the invader probe 2. Designed to be detectable.
With the above configuration, when the above-mentioned base is mutated in the subject Calr3 gene, the 3 ′ end of the invader probe 2 invades the 5 ′ end of the nucleotide 3a hybridized to the target gene. 3c is cut out and fluorescence is generated. On the other hand, when the subject's Calr3 gene is a wild type, no invasion structure is formed by the terminal portion of the invader probe 2, so that nucleotide 3c is not cut out and thus fluorescence does not occur.

In the above example, the fluorescence is generated when the Calr3 gene of the subject is a substitution mutation type. However, the present invention is not limited to this, and the fluorescence is generated when the Calr3 gene of the subject is a wild type. In the case of the substitution mutant type, the fluorescence may be suppressed, or various modifications can be made such as using different fluorescent dyes for the wild type and the mutant type.
In each of the above methods, the configuration for determining the presence or absence of substitution mutations by examining the sense strand of the Calr3 gene (codon-encoding side strand) has been described in detail, but the same applies by examining the antisense strand. Needless to say, it is possible to make a judgment.

  The fertility diagnostic method and fertility diagnostic kit according to the present invention may detect the presence or absence of a mutation causing a functional defect in the human Calr3 gene at the protein level. That is, the detection method and fertility diagnostic kit according to the present invention may determine whether or not normal human Calr3 protein is expressed in human, and abnormal human Calr3 protein is expressed. It may be determined whether or not there is. Here, the normal protein is a human wild-type Calr3 polypeptide and means a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. On the other hand, an abnormal protein is, for example, a polynucleotide consisting of an amino acid sequence encoded by a polynucleotide in which any one of the above-mentioned single-base substitution mutations occurs in a polynucleotide consisting of the base sequence of the translation region of the human wild-type Calr3 gene. Means peptide. Specifically, in the polynucleotide consisting of the base sequence of the translation region of the human wild-type Calr3 gene (base sequence represented by SEQ ID NO: 1) when the translation start codon adenine is the first base, The base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, the base at position 644 is thymine, the base at position 647 is thymine, the base at position 742 is thymine, and position 820 Wherein the base at position 976 is adenine, the base at position 1056 is thymine, or the base at position 1058 is substituted with thymine. In addition, when the translation start codon adenine is the first base, in the polynucleotide comprising the base sequence of the translation region of the human wild-type Calr3 gene, the base at position 128 is thymine and the base at position 129 is adenine. The base at position 251 is adenine, the base at position 313 is adenine, the base at position 328 is thymine, the base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, The base at position 584 is thymine, the base at position 782 is thymine, the base at position 783 is adenine, the base at position 945 is adenine, the base at position 946 is adenine, the base at position 947 is adenine, and position 970 The base at position 981 is adenine, the base at position 1022 is thymine, the base at position 1023 is thymine, the base at position 1034 is thymine, the position 104 The base in position is thymine, the base in position 1063 is thymine, the base in position 1074 is thymine, the base in position 1083 is adenine, the base in position 1086 is thymine, or the base in position 1111 is replaced with thymine It may be a polynucleotide characterized by comprising a base sequence.

  The method for detecting whether the human Calr3 protein contained in the biological sample is a normal protein or an abnormal protein is not particularly limited. For example, when the normal protein and the abnormal protein have clearly different molecular weights, such as mutations at positions 370 and 616, the size of the human Calr3 protein contained in the biological sample is determined by electrophoresis or the like. By examining, it is possible to detect whether or not an abnormal protein is present. Further, by using an antibody, it can be detected with higher accuracy whether the human Calr3 protein contained in the biological sample is a normal protein or an abnormal protein.

  Specifically, for example, the detection step of the fertility diagnostic method according to the present invention includes a human mutant Calr3 protein encoded by a human mutant Calr3 gene containing at least one mutation among the above-described mutations, and It may be one that detects whether or not an abnormal protein is present in a biological sample by using an antibody that binds and does not bind to human wild-type Calr3 protein (normal protein). . Further, as a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation contained in the fertility diagnostic kit according to the present invention, the reagent binds to an abnormal protein of human Calr3 and does not bind to a normal protein. The antibody may be used. Such an antibody can be obtained by selecting an antibody that does not cross the normal protein of human Calr3 from polyclonal antibodies and monoclonal antibodies prepared by a conventional method using the abnormal protein of human Calr3 as an antigen. .

More specifically, the antibody according to the present invention can be produced by immunizing an experimental animal with the above-described human mutant Calr3 polypeptide containing the amino acid substitution mutation or a fragment thereof containing the mutation part as an antigen. (See, for example, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354n (1985). )
In general, animals can be immunized with free peptides. However, it may be possible to efficiently immunize by coupling the free peptide to a macromolecular carrier (eg, heal limpet hemocyanin (KLH) or tetanus toxide). For example, a peptide containing cysteine can be coupled to a carrier by a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be coupled to the carrier using more common linking agents such as glutaraldehyde.
Animals such as rabbits, rats and mice can be immunized by intraperitoneal and / or intradermal injection of an emulsion containing free peptide or carrier-coupled peptide and Freund's adjuvant. In order to obtain an anti-peptide antibody with a useful titer that can be detected by ELISA, booster injections are preferably performed at intervals of about 2 weeks.
The antibody can be obtained by collecting blood from the immunized animal and extracting the IgG fraction from the serum. Further, the antibody titer is improved by purifying the antibody by a known technique.
The antibody according to the present invention binds to the above-described human mutant Calr3 polypeptide, but does not bind to human wild-type Calr3 polypeptide. Therefore, the antibody according to the present invention can be obtained by selecting antibodies that do not bind to the human wild-type Calr3 protein from the antibodies obtained by the above procedure. In order to facilitate the above selection, it is desirable to select an oligopeptide having about 10 residues consisting of the mutant portion of the human mutant Calr3 polypeptide and the surrounding amino acid sequence as an antigen peptide to be injected into the animal.

Thus, in one embodiment, the fertility diagnostic method and fertility diagnostic kit according to the present invention include a human mutation encoded by a human mutant Calr3 gene containing at least one base substitution among the above-described mutations. It may be used to determine whether a human mutant Calr3 protein is expressed in a biological sample collected from a human using an antibody that binds to a type Calr3 protein and does not bind to a human wild type Calr3 protein .
Whether or not the human mutant Calr3 protein is expressed using the above antibody is determined, and if it is determined that the human mutant Calr3 protein is expressed in the subject, the substitution mutation described above occurs in the human Calr3 gene. At the same time, it can be estimated that the subject is less likely to be fertile or likely to be infertile. Moreover, when a test subject is infertile, it can be estimated that the cause of infertility originates in abnormality of human Calr3 gene.

In another embodiment, the detection method and fertility diagnostic kit according to the present invention may determine whether or not normal human Calr3 protein is expressed in humans. More specifically, the detection step of the fertility diagnostic method according to the present invention uses an antibody characterized in that it binds to human wild-type Calr3 protein and does not bind to abnormal human Calr3 protein. It may be one that detects whether or not human wild-type Calr3 protein is present in the sample. Further, as a reagent for detecting the presence or absence of a human Calr3 gene deficiency or mutation contained in the fertility diagnostic kit according to the present invention, the reagent binds to human wild-type Calr3 protein and does not bind to an abnormal protein. (Anti-Carr3 antibody) may be used.
Whether or not the human Calr3 protein is expressed using an anti-Carr3 antibody is determined, and if it is determined that the human Calr3 protein is not expressed in the subject, it is suggested that the human Calr3 gene is deleted or mutated At the same time, it can be estimated that the subject has a low possibility of fertility or a high possibility of infertility. Moreover, when a test subject is infertile, it can be estimated that the cause of infertility is due to the fact that normal human Calr3 protein is not expressed.

The technique for detecting human wild-type Calr3 protein or human mutant Calr3 protein in a biological sample using an antibody is not particularly limited, and any known technique may be used. Examples thereof include Western blotting and ELISA (Enzyme-linked Immunosorbent Assay).
When the fertility diagnostic kit is a kit suitable for a fertility diagnostic method using Western blotting or ELISA, the fertility diagnostic kit includes a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation. In addition to the anti-human wild type Calr3 protein antibody or the anti-Calr3 antibody, an anti-globulin antibody labeled with a specific enzyme such as peroxidase necessary for the color reaction may be contained.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example, A various change is possible in the range shown to the claim. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

[Example 1]
In this example, the human Calr3 gene sequence was analyzed, and mutations related to male infertility were detected in the human Calr3 gene.
[Person to analyze]
Non-obstructive infertility Japanese male subjects (N = 1000 or more) were divided into subgroups based on the degree of spermatogenesis deficiency. Of these infertile subjects, 13% have non-obstructive azoospermia, 31% have severe hypospermia (<5 × 10 ⁶ cells / mL), 33% have ataxia, the rest 23% were morphologically normal. When examined for genetic factors, all of these subjects had primary idiopathic infertility (Birmingham A et al. Fertil Steril 2004; 9: 2313-2317). The control group (N = 200 or more) was a father of a child born to a pregnant woman at the obstetrics and gynecology clinic, and was a male with confirmed fertility. Informed consent was obtained from all donors that the samples were used for genomic DNA analysis (Osaka University Hospital Urology).

[Mutation screening]
In each of the following steps, each treatment was performed according to a conventional method or an instruction manual attached to the kit unless otherwise specified.
Genomic DNA was isolated and prepared from blood samples or semen samples by protease treatment and phenol extraction (Sambrook J et al., Isolation of DNA from Mammalian Cells. New York: Cold Spring Harbor Press. 1989: pp. 9.16-9.21) . As blood-derived genomic DNA, DNA amplified using GenomiPhi (manufactured by GE Healthcare Bioscience) was used.
As shown in FIG. 1, the Calr3 gene has an amino acid coding region formed from 9 exons. To amplify each exon, the Calr3 gene is divided into 5 replication units (amplicons) as shown in FIG. Primer was designed. Table 1 shows the respective primers, and Table 2 shows the PCR reaction conditions.

  Using these primers, PCR reaction was performed under the reaction conditions described in Table 2. The obtained PCR amplified fragment was purified by AMPure (Agencourt Bioscience). Using this purified PCR amplified fragment as a template, sequencing reaction was performed using the primers used when each amplicon was amplified. Specifically, the reaction product purified by CleanSEQ (manufactured by Agencourt Bioscience) was performed using BigDye (registered trademark) Terminator V3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) and ABI-PRISM 3730 GeneticA (Genetic Bioscience). (Applied Biosystems). The base sequence was determined by comparing the determination results in both directions.

[Sequence analysis results]
For each infertile subject and fertility confirmed subject, the amplicon amplified with primers 1 and 2r, the amplicon amplified with primers 3 and 4r, the amplicon amplified with primers 5.1 and 6.1r, When the base sequences of the amplicons amplified with primers 7 and 9.5r and the amplicons amplified with primers 9 and 10r were analyzed, as shown in Tables 3 to 5, known SNPs (5 A total of 44 substitution mutations including) were detected. Of these, 35 substitution mutations with amino acid changes were detected in infertile subjects, and these substitution mutations were not detected at all from fertility confirmed subjects, confirming that there was a correlation with fertility It was done. Hereinafter, each of these 35 substitution mutations will be described.

One subject out of 634 infertile subjects was found in which the adenine at position 128 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, aspartic acid at position 43 of the deduced amino acid sequence is encoded by valine.
Moreover, one test subject in which cytosine at position 129 in the translation region of the Calr3 gene was substituted with adenine in one allele was found among 634 infertile subjects. By this base substitution, aspartic acid at position 43 in the deduced amino acid sequence is encoded by glutamic acid.

Two of 784 infertile subjects were found in which the cytosine at position 233 of the translated region of the Calr3 gene was substituted with adenine in one allele. By this base substitution, serine at position 78 of the deduced amino acid sequence is encoded by tyrosine.
In addition, one subject out of 784 infertile subjects was found in which the thymine at position 251 in the translation region of the Calr3 gene was substituted with adenine in one allele. By this base substitution, phenylalanine at position 84 in the deduced amino acid sequence is encoded by tyrosine.
Moreover, one test subject in which thymine at position 313 in the translation region of the Calr3 gene was substituted with adenine in one allele was found among 784 infertile subjects. By this base substitution, the cysteine at position 105 of the deduced amino acid sequence is encoded by serine.
In addition, one subject out of 784 infertile subjects was found in which the adenine at position 328 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, isoleucine at position 110 of the deduced amino acid sequence is encoded by phenylalanine.
Moreover, one test subject in which the guanine at position 346 in the translated region of the Calr3 gene was replaced with thymine in one allele was found among 784 infertile subjects. By this base substitution, aspartic acid at position 116 of the deduced amino acid sequence is encoded by tyrosine.
Moreover, one test subject in which guanine at position 370 in the translation region of the Calr3 gene was replaced with thymine in one allele was found among 784 infertile subjects. This base substitution prevents the glycine at position 124 of the deduced amino acid sequence from being encoded (stop codon).
In addition, one subject out of 784 infertile subjects was found in which the thymine at position 385 in the translation region of the Calr3 gene was substituted with adenine in one allele. By this base substitution, tyrosine at position 129 of the deduced amino acid sequence is encoded by asparagine.

One of 300 infertile subjects was found in which the guanine at position 584 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, serine at position 195 of the deduced amino acid sequence is encoded by isoleucine.
In addition, three subjects in 300 infertile subjects were found in which the adenine at position 586 in the translation region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, isoleucine at position 196 of the deduced amino acid sequence is encoded by leucine.
In addition, 4 subjects were found among 300 infertile subjects in which the adenine at position 616 in the translated region of the Calr3 gene was replaced with thymine in one allele. This base substitution prevents the lysine at position 206 of the deduced amino acid sequence from being encoded (stop codon).
In addition, two subjects in 300 infertile subjects were found in which the adenine at position 644 in the translation region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, lysine at position 215 of the deduced amino acid sequence is encoded by methionine.
In addition, two subjects in 300 infertile subjects were found in which the adenine at position 647 in the translation region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, aspartic acid at position 216 of the deduced amino acid sequence is encoded by valine.

Fifteen subjects were found among 671 infertile subjects who had guanine at position 742 in the translated region of the Calr3 gene replaced with thymine in one allele. By this base substitution, aspartic acid at position 248 of the deduced amino acid sequence is encoded by tyrosine.
In addition, one subject out of 671 infertile subjects was found in which the adenine at position 782 in the translation region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, the tyrosine at position 261 in the deduced amino acid sequence is encoded by phenylalanine.
In addition, one test subject was found among 671 infertile subjects who had cytosine at position 783 in the translation region of the Calr3 gene replaced with adenine in one allele. By this base substitution, the tyrosine at position 261 of the deduced amino acid sequence is not encoded (stop codon).
In addition, 19 subjects out of 671 infertility subjects were replaced with adenine in the allelic guanine at position 820 in the translation region of the Calr3 gene, and subjects who substituted adenine in both alleles. One of 671 infertile subjects was found. By this base substitution, valine at position 274 of the deduced amino acid sequence is encoded by isoleucine.

One of 464 infertile subjects was found in which the cytosine at position 945 in the translated region of the Calr3 gene was substituted with adenine in one allele. By this base substitution, asparagine at position 315 of the deduced amino acid sequence is encoded by lysine.
Moreover, one test subject in which thymine at position 946 in the translation region of the Calr3 gene was substituted with adenine in one allele was found among 464 infertile subjects. By this base substitution, phenylalanine at position 316 of the deduced amino acid sequence is encoded by isoleucine.
In addition, one subject out of 464 infertile subjects was found in which the thymine at position 947 in the translated region of the Calr3 gene was replaced with adenine in one allele. By this base substitution, phenylalanine at position 316 of the deduced amino acid sequence is encoded by tyrosine.
In addition, one subject out of 464 infertile subjects was found in which the thymine at position 970 in the translated region of the Calr3 gene was replaced with adenine in one allele. By this base substitution, tyrosine at position 324 of the deduced amino acid sequence is encoded by asparagine.
In addition, six subjects among 464 infertile subjects were found in which guanine at position 976 in the translation region of the Calr3 gene was substituted with adenine in one allele. As a result of this base substitution, aspartic acid at position 326 of the deduced amino acid sequence is encoded by asparagine.
Further, among 464 infertile subjects, a subject was found in which the thymine at position 981 in the translated region of the Calr3 gene was substituted with adenine in one allele. By this base substitution, asparagine at position 327 of the deduced amino acid sequence is encoded by lysine.
Moreover, one test subject in which guanine at position 1022 in the translated region of the Calr3 gene was replaced with thymine in one allele was found among 464 infertile subjects. By this base substitution, arginine at position 341 in the deduced amino acid sequence is encoded by methionine.
Moreover, one test subject in which the guanine at position 1023 in the translation region of the Calr3 gene was replaced with thymine in one allele was found among 464 infertile subjects. By this base substitution, arginine at position 341 in the deduced amino acid sequence is encoded by serine.
Moreover, one test subject in which the cytosine at position 1034 in the translation region of the Calr3 gene was replaced with thymine in one allele was found among 464 infertile subjects. By this base substitution, alanine at position 345 of the deduced amino acid sequence is encoded by valine.
In addition, one subject out of 464 infertile subjects was found in which the adenine at position 1046 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, lysine at position 349 of the deduced amino acid sequence is encoded by methionine.
In addition, two subjects in whom guanine at position 1056 in the translated region of the Calr3 gene was replaced with thymine in one allele were found among 464 infertile subjects. By this base substitution, methionine at position 352 of the deduced amino acid sequence is encoded by isoleucine.
In addition, 27 subjects were found among 464 infertile subjects whose adenine at position 1058 in the translation region of the Calr3 gene had been replaced with thymine in one allele. By this base substitution, lysine at position 353 of the deduced amino acid sequence is encoded by methionine.
Moreover, one test subject in which the guanine at position 1063 in the translated region of the Calr3 gene was replaced with thymine in one allele was found among 464 infertile subjects. By this base substitution, alanine at position 355 of the deduced amino acid sequence is encoded by serine.
In addition, one subject out of 464 infertile subjects was found in which the adenine at position 1074 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, glutamic acid at position 358 in the deduced amino acid sequence is encoded by aspartic acid.
Moreover, one test subject in which thymine at position 1083 in the translation region of the Calr3 gene was substituted with adenine in one allele was found among 464 infertile subjects. By this base substitution, glutamic acid at position 361 of the deduced amino acid sequence is encoded by aspartic acid.
In addition, one subject out of 464 infertile subjects was found in which the guanine at position 1086 in the translated region of the Calr3 gene was replaced with thymine in one allele. By this base substitution, glutamic acid at position 362 of the deduced amino acid sequence is encoded by aspartic acid.
Moreover, one test subject in which the cytosine at position 1111, in the translation region of the Calr3 gene, was replaced with thymine in one allele was found among 464 infertile subjects. By this base substitution, the histidine at position 371 of the deduced amino acid sequence is encoded by tyrosine.

  Among these substitution mutations, in particular, the base at position 233, the base at position 586, the base at position 616, the base at position 644, the base at position 647, the base at position 742, and the base at position 820 , The appearance rate of the substitution mutation of the base at position 976, the base at position 1056, or the base at position 1058 was high, and the correlation with male infertility was high.

  In subjects who have found the above substitution mutation, the function of the Calr3 protein becomes insufficient because the translation amount of the normal Calr3 protein is reduced or the mutant Calr3 protein acts on a dominant negative. It was speculated that he became infertile.

  In this study, single nucleotide polymorphisms were found in the Calr3 gene in addition to the substitution mutations described above. However, these single nucleotide polymorphisms have patterns of infertility and fertility confirmed subjects. There was no significant difference between the two, and it was not found to affect fertility in particular.

  By using the fertility diagnostic method and the like according to the present invention, human fertility can be estimated and the cause of infertility can be identified. Therefore, the present invention can be suitably used in the pharmaceutical field and the medical field.

  1 ... target gene, 1a ... mutation (or single nucleotide polymorphism) detection site, 2 ... invader oligo, 3 ... allele oligo, 3a ... nucleotide, 3b ... flap, 3c ... flap free body, 4 ... flap endonuclease, 5 ... fret Probe, 5a ... nucleotide, 5b ... fluorescent dye, 5c ... quencher (luminescence inhibitor).

Claims (20)

Using a biological sample collected from a human, having a detection step of detecting the presence or absence of a deletion or mutation of the Calr3 gene in the human,
In the detection step, when the translation initiation codon adenine is the first base, the 128th base, the 129th base, the 233rd base, the 251st position of the translation region of the human Calr3 gene , 313th base, 328th base, 346th base, 370th base, 385th base, 584th base, 586th base, 616th base , 644th base, 647th base, 742nd base, 782nd base, 783th base, 820th base, 945th base, 946th base, Base at position 947, base at position 970, base at position 976, base at position 981, base at position 1022, base at position 1023, base at position 1034, base at position 1046, position 1056 Base, position 1058, position 10 3-position of the base, the 1074-positional base, to detect the presence or absence of a mutation of the 1083 position of the base, one or more bases selected from the group consisting of the 1086 position of the base, and a 1111 of the base,
When a mutation in which adenine of the base at position 128 is replaced with thymine is detected,
When a mutation in which cytosine at the position 129 is replaced with adenine is detected,
When a mutation in which cytosine of the base at position 233 is replaced with adenine is detected,
When a mutation in which the thymine at position 251 is replaced with adenine is detected,
When a mutation in which the thymine at position 313 is substituted with adenine is detected,
When a mutation in which the adenine of the 328th base is replaced with thymine is detected,
When a mutation is detected in which the guanine of the 346th base is replaced with thymine,
When a mutation in which guanine at the 370th base is replaced with thymine is detected,
When a mutation in which the thymine at position 385 is replaced with adenine is detected,
When a mutation in which the guanine of the base at position 584 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 586 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 616 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 644 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 647 is replaced with thymine is detected,
When a mutation in which the guanine at the 742nd base is replaced with thymine is detected,
When a mutation in which adenine of the base at position 782 is replaced with thymine is detected,
When a mutation in which cytosine at position 783 is substituted with adenine is detected,
When a mutation in which guanine at the base at position 820 is substituted with adenine is detected,
When a mutation in which cytosine of the base at position 945 replaces adenine is detected,
When a mutation in which the thymine at position 946 is replaced with adenine is detected,
When a mutation in which the thymine at position 947 is replaced with adenine is detected,
When a mutation in which the thymine at position 970 is replaced with adenine is detected,
When a mutation in which the guanine at the 976th base is replaced with adenine is detected,
When a mutation in which the thymine at position 981 is replaced with adenine is detected,
When a mutation in which the guanine of the base at position 1022 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1023 is replaced with thymine is detected,
When a mutation in which cytosine of the base at position 1034 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 1046 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1056 is replaced with thymine is detected,
When a mutation in which the adenine of the base at position 1058 is replaced with thymine is detected,
When a mutation in which the guanine of the base at position 1063 is replaced with thymine is detected,
When a mutation in which adenine of the base at position 1074 is replaced with thymine is detected,
When a mutation in which the thymine at position 1083 is substituted with adenine is detected,
When a mutation in which the guanine at the 1086th base is replaced with thymine is detected, or when a mutation in which the cytosine at the 1111th base is replaced with thymine is detected,
The fertility potential of human estimated low, or infertility cause of the human and judging that there is a high possibility due to loss of function of Calr3 molecules, methods. In the detection step, when the adenine of the translation initiation codon is the first position, the position of the position 233, the position 586, the position 616, the position 644 of the translation region of the human Calr3 gene One or more mutations selected from the group consisting of: base No. 647, base No. 647, base No. 742, base No. 820, base No. 976, base No. 1056 and base No. 1058 The method according to claim 1, wherein the presence or absence of the data is detected. Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 233 is substituted from cytosine to adenine, and 10 to 100 consecutive base sequences including the base at position 233.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 4, the 101st base is substituted from cytosine to adenine, and 10 to 100 consecutive base sequences containing the 101st base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, a base at position 586 is substituted with adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 586.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 5, the 103rd base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences including the 103rd base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) A base sequence represented by SEQ ID NO: 1, wherein the base at position 616 is substituted with adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 616.
(B) A base sequence complementary to the base sequence of (a).
(C) A base sequence represented by SEQ ID NO: 5, wherein the 133rd base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences including the 133rd base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) A base sequence represented by SEQ ID NO: 1, wherein the base at the 644th position is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the base at the 644th position.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 5, the 161st base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 161st base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 647 is substituted from adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 647.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 5, the 164th base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 164th base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 742 is substituted from guanine to thymine, and 10 to 100 consecutive base sequences containing the base at position 742.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 6, the 101st base is substituted from guanine to thymine, and 10 to 100 consecutive base sequences containing the 101st base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 820 is substituted from guanine to adenine, and a continuous base sequence of 10 to 100 containing the base at position 820.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 7, the 101st base is substituted from guanine to adenine, and 10 to 100 consecutive base sequences containing the 101st base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, a base sequence at position 976 is substituted from guanine to adenine, and 10 to 100 consecutive base sequences including the base position at position 976.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 8, the 132nd base is substituted from guanine to adenine, and 10 to 100 consecutive base sequences containing the 132nd base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 1056 is substituted from guanine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 1056.
(B) A base sequence complementary to the base sequence of (a).
(C) In the base sequence represented by SEQ ID NO: 9, the 135th base is substituted from guanine to thymine, and 10 to 100 consecutive base sequences containing the 135th base.
(D) A base sequence complementary to the base sequence of (c). Use of a polynucleotide comprising any one of the following base sequences (a) to (d) for estimating fertility or identifying the cause of infertility:
(A) In the base sequence represented by SEQ ID NO: 1, the base at position 1058 is substituted from adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 1058.
(B) A base sequence complementary to the base sequence of (a).
(C) A base sequence represented by SEQ ID NO: 9, wherein the 137th base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 137th base.
(D) A base sequence complementary to the base sequence of (c). In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, the base at position 644 is thymine, and position 647 Base thymine, base 742 is thymine, base 820 is adenine, base 976 is adenine, base 1056 is thymine, or base 1058 is thymine Use of a polynucleotide characterized by comprising a sequence for estimating fertility or identifying the cause of infertility . In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the 128th base is thymine, the 129th base is adenine, the 251st base is adenine, the 313th base is adenine, and 328th position The base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, the base at position 584 is thymine, the base at position 782 is thymine, the base at position 783 Is adenine, the base at position 945 is adenine, the base at position 946 is adenine, the base at position 947 is adenine, the base at position 970 is adenine, the base at position 981 is adenine, and the base at position 1022 is thymine The base at position 1023 is thymine, the base at position 1034 is thymine, the base at position 1046 is thymine, the base at position 1063 is thymine, the base at position 1074 is thymine, 1083 position of the base is adenine, the 1086 position base is thymine, or the 1111 position of the base is estimated or cause of infertility fertility potential of a polynucleotide, comprising the base sequence substituted thymine Use for identification . In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, the base at position 644 is thymine, and position 647 Base thymine, 742th base is thymine, 820th base is adenine, 976th base is adenine, 1056th base is thymine, or 1058th base is substituted with thymine A polynucleotide comprising a sequence, or
In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the 128th base is thymine, the 129th base is adenine, the 251st base is adenine, the 313th base is adenine, and 328th position The base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, the base at position 584 is thymine, the base at position 782 is thymine, the base at position 783 Is adenine, the base at position 945 is adenine, the base at position 946 is adenine, the base at position 947 is adenine, the base at position 970 is adenine, the base at position 981 is adenine, and the base at position 1022 is thymine The base at position 1023 is thymine, the base at position 1034 is thymine, the base at position 1046 is thymine, the base at position 1063 is thymine, the base at position 1074 is thymine, 1083 position of the base is adenine, the 1086 position base is thymine, or the 1111 position of the base the estimation of fertility potential of a polypeptide consisting of the amino acid sequence encoded by a polynucleotide consisting of the base sequence substituted thymine Or use to identify the cause of infertility . In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, the base at position 644 is thymine, and position 647 Base thymine, 742th base is thymine, 820th base is adenine, 976th base is adenine, 1056th base is thymine, or 1058th base is substituted with thymine A polynucleotide comprising a sequence, or
In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the 128th base is thymine, the 129th base is adenine, the 251st base is adenine, the 313th base is adenine, and 328th position The base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, the base at position 584 is thymine, the base at position 782 is thymine, the base at position 783 Is adenine, the base at position 945 is adenine, the base at position 946 is adenine, the base at position 947 is adenine, the base at position 970 is adenine, the base at position 981 is adenine, and the base at position 1022 is thymine The base at position 1023 is thymine, the base at position 1034 is thymine, the base at position 1046 is thymine, the base at position 1063 is thymine, the base at position 1074 is thymine, A sequence that binds to a polypeptide comprising an amino acid sequence encoded by a polynucleotide comprising a base sequence in which the base at position 1083 is adenine, the base at position 1086 is thymine, or the base at position 1111 is substituted with thymine; An antibody that does not bind to a polypeptide consisting of the amino acid sequence represented by No. 2. Using a biological sample collected from a human, the detection step of detecting the presence or absence of mutation of the Calr3 polypeptide expressed in the human by the antibody of claim 16,
When a mutation in the Calr3 polypeptide is detected, the human fertility is estimated to be low, or the human infertility is determined to be highly likely due to a functional defect in the Calr3 molecule. The way . A kit for carrying out the method according to claim 1, comprising a reagent for detecting the presence or absence of a deletion or mutation of the human Calr3 gene,
When the mutation is adenine of the translation initiation codon as the first base, the 128th base, the 129th base, the 233rd base, the 251st base of the translation region of the human Calr3 gene , 313th base, 328th base, 346th base, 370th base, 385th base, 584th base, 586th base, 616th base, Base at position 644, base at position 647, base at position 742, base at position 782, base at position 783, base at position 820, base at position 945, base at position 946, position 947 Base, 970th base, 976th base, 981st base, 1022nd base, 1023rd base, 1034th base, 1046th base, 1056th base , Base at position 1058, base at position 1063 The 1074 base at nucleotide position, the 1083 position of a base, is one or more base mutations selected from the group consisting of the 1086 position of the base, and a 1111 of the base,
Wherein the reagents in the nucleotide sequence or nucleotide sequence of the translation region of the human wild-type Calr3 gene, complementary to the contiguous nucleotide sequence or the nucleotide sequence of 10 to 100, including a base portion which is subject to detect the presence or absence of a mutation A kit comprising a polynucleotide comprising a base sequence, or a hybridization probe in which the polynucleotide is previously labeled with a fluorescent dye. The kit according to claim 18, wherein the polynucleotide comprises any one of the following base sequences.
(1a) In the base sequence represented by SEQ ID NO: 1, the base at position 233 is substituted from cytosine to adenine, and a continuous base sequence of 10 to 100 containing the base at position 233.
(1b) A base sequence complementary to the base sequence of (1a).
(1c) In the base sequence represented by SEQ ID NO: 4, the 101st base is substituted from cytosine to adenine, and 10 to 100 consecutive base sequences containing the 101st base.
(1d) A base sequence complementary to the base sequence of (1c).
(2a) A base sequence represented by SEQ ID NO: 1, wherein the base at position 586 is substituted from adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 586.
(2b) A base sequence complementary to the base sequence of (2a).
(2c) A base sequence represented by SEQ ID NO: 5, wherein the 103rd base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 103rd base.
(2d) A base sequence complementary to the base sequence of (2c).
(3a) A base sequence represented by SEQ ID NO: 1, wherein the base at position 616 is substituted with adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 616.
(3b) A base sequence complementary to the base sequence of (3a).
(3c) A base sequence represented by SEQ ID NO: 5, wherein the 133rd base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 133rd base.
(3d) A base sequence complementary to the base sequence of (3c).
(4a) A base sequence represented by SEQ ID NO: 1, wherein the base at position 644 is substituted from adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 644.
(4b) A base sequence complementary to the base sequence of (4a).
(4c) A base sequence represented by SEQ ID NO: 5, wherein the 161st base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 161st base.
(4d) A base sequence complementary to the base sequence of (4c).
(5a) A base sequence represented by SEQ ID NO: 1, wherein the base at position 647 is substituted with adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 647.
(5b) A base sequence complementary to the base sequence of (5a).
(5c) A base sequence represented by SEQ ID NO: 5, wherein the 164th base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 164th base.
(5d) A base sequence complementary to the base sequence of (5c) above.
(6a) A base sequence represented by SEQ ID NO: 1, wherein the base at position 742 is substituted from guanine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 742.
(6b) A base sequence complementary to the base sequence of (6a).
(6c) In the base sequence represented by SEQ ID NO: 6, the 101st base is substituted from guanine to thymine, and 10 to 100 consecutive base sequences containing the 101st base.
(6d) A base sequence complementary to the base sequence of (6c) above.
(7a) In the base sequence represented by SEQ ID NO: 1, the base at position 820 is substituted from guanine to adenine, and 10 to 100 consecutive base sequences including the base at position 820.
(7b) A base sequence complementary to the base sequence of (7a).
(7c) In the base sequence represented by SEQ ID NO: 7, the 101st base is substituted from guanine to adenine, and 10 to 100 consecutive base sequences containing the 101st base.
(7d) A base sequence complementary to the base sequence of (7c).
(8a) A base sequence represented by SEQ ID NO: 1, wherein the 976th base is substituted from guanine to adenine, and 10 to 100 consecutive base sequences containing the 976th base.
(8b) A base sequence complementary to the base sequence of (8a).
(8c) In the base sequence represented by SEQ ID NO: 8, the 132nd base is substituted from guanine to adenine, and 10 to 100 consecutive base sequences containing the 132nd base.
(8d) A base sequence complementary to the base sequence of (8c).
(9a) In the base sequence represented by SEQ ID NO: 1, the base at position 1056 is substituted from guanine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 1056.
(9b) A base sequence complementary to the base sequence of (9a).
(9c) A base sequence represented by SEQ ID NO: 9, wherein the 135th base is substituted from guanine to thymine, and 10 to 100 consecutive base sequences containing the 135th base.
(9d) A base sequence complementary to the base sequence of (9c).
(10a) In the base sequence represented by SEQ ID NO: 1, the base at position 1058 is substituted from adenine to thymine, and a continuous base sequence of 10 to 100 containing the base at position 1058.
(10b) A base sequence complementary to the base sequence of (10a).
(10c) A base sequence represented by SEQ ID NO: 9, wherein the 137th base is substituted from adenine to thymine, and 10 to 100 consecutive base sequences containing the 137th base.
(10d) A base sequence complementary to the base sequence of (10c).
(11) In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the base at position 233 is adenine, the base at position 586 is thymine, the base at position 616 is thymine, the base at position 644 is thymine, The base at position 647 is thymine, the base at position 742 is thymine, the base at position 820 is adenine, the base at position 976 is adenine, the base at position 1056 is thymine, or the base at position 1058 is replaced by thymine Base sequence.
(12) In the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, the 128th base is thymine, the 129th base is adenine, the 251st base is adenine, the 313th base is adenine, The base at position 328 is thymine, the base at position 346 is thymine, the base at position 370 is thymine, the base at position 385 is adenine, the base at position 584 is thymine, the base at position 782 is thymine, 783 Position base is adenine, position 945 is adenine, position 946 is adenine, position 947 is adenine, position 970 is adenine, position 981 is adenine, position 1022 The base is thymine, the base at position 1023 is thymine, the base at position 1034 is thymine, the base at position 1046 is thymine, the base at position 1063 is thymine, and the base at position 1074 is Min, the 1083 position base is adenine, the base sequence No. 1086 of the base is thymine, or the 1111 position of the base is replaced by thymine. A kit for estimating human fertility or identifying the cause of infertility , comprising a reagent for detecting the presence or absence of a human Calr3 gene deletion or mutation,
When the mutation is adenine of the translation initiation codon as the first base, the 128th base, the 129th base, the 233rd base, the 251st base of the translation region of the human Calr3 gene , 313th base, 328th base, 346th base, 370th base, 385th base, 584th base, 586th base, 616th base, Base at position 644, base at position 647, base at position 742, base at position 782, base at position 783, base at position 820, base at position 945, base at position 946, position 947 Base, 970th base, 976th base, 981st base, 1022nd base, 1023rd base, 1034th base, 1046th base, 1056th base , Base at position 1058, base at position 1063 The 1074 base at nucleotide position, the 1083 position of a base, is one or more base mutations selected from the group consisting of the 1086 position of the base, and a 1111 of the base,
Kit wherein said reagent binds to the abnormal protein of human Calr3, and characterized in that it is an antibody that does not bind to normal human proteins Calr3.

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