JP2008237114A - Fertility diagnosis kit, mutation detection method, polynucleotide, polypeptide, expression vector, and expression kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fertility diagnosis kit enabling the estimation of fertility possibility and the identification of an infertility cause, and to provide a mutation detection method. <P>SOLUTION: This fertility diagnosis kit contains a reagent for detecting the presence or absence of a substitution mutation of cytosine base at the 283 position of human Centrin 1 gene. When the substitution of thymine base at cytosine base at the 283 position of a genome DNA obtained from a test subject is detected with the fertility diagnosis kit, the test subject is judged that the possibility of fertility is low. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fertility diagnostic kit and a mutation detection method capable of estimating fertility and identifying the cause of infertility, and a mutant polypeptide used for research on a method for treating infertility and the same The present invention relates to a polynucleotide encoding.

  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.

  Non-Patent Document 1 reports that about 15% of couples who want children can not get pregnant even after two years. In recent years, the development of in vitro fertilization (IVF) technology has enabled pregnancy and birth even when sperm activity is low, but the molecular mechanism behind infertility remains unclear. is the current situation.

  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 contribute to male infertility in humans. It may have become.

  Maturation of mammalian male germ cells occurs through a number of structural and functional changes. The process is called spermatogenesis, and is mainly involved in (1) proliferation and differentiation of spermatogonia, (2) meiosis in the early stage of spermatocytes, and (3) differentiation from haploid round sperm cells to sperm. It includes three stages of rapid shape change.

  Centrin1 protein is known as a mouse protein that is particularly highly expressed in the testis where such male germ cells are present (Non-patent Document 3). Centrin protein is found in a wide variety of organisms from yeast to mammals, and functions as one of the components constituting the centrosome. From experiments in yeast and the like, the Centrin protein is known to play an important role in the replication and separation of centrosomes.

  The mouse has three genes encoding Centrin protein, Centrin1, Centrin2, and Centrin3. Of these, the Centrin1 protein is particularly strongly expressed in mature male testis and is localized in the tail in sperm. From these facts, the Centrin1 protein is considered to be involved in the function of the centrosome during meiosis or the structure of the sperm flagella.

The gene encoding this Centrin protein also exists in humans (Non-patent Document 4). It is known that there are three genes encoding the Centrin protein even in humans. Especially, human Centrin1 protein has high homology with mouse Centrin1 protein, and is considered to be derived from the same origin (nonpatent literature 3).
de Kretser DM et al., J Clin Endocrinol Metab. 1999; 84: 3443-3450. Matzuk MM et al., Nat Cell Biol. 2002; 4 (Supp1): S41-S49 / Nat Med. 2002; 8 (Supp1): S33-S40. Hart EP et al., Genomics. 1999; 60: 111-120. Errabolu R et al., J Cell Sci. 1994; 107: 9-16.

  As mentioned above, since the Centrin1 gene is conserved across species in mammals and encodes a protein that is particularly strongly expressed in haploid germ cells, mutations in these genes can cause infertility. Conceivable. However, to date, no case has been reported that suggests a relationship between a mutation in the Centrin1 gene and infertility.

  If the relationship between the human Centrin1 gene mutation and fertility becomes clear, fertility can be diagnosed with a simple genetic diagnosis. Specifically, it is possible to easily determine whether or not a subject is highly likely to suffer from infertility, or whether or not the cause of the infertility patient is due to these genes. .

  Furthermore, if there is a mutant human Centrin1 protein found in patients with infertility, it will be an extremely useful tool in research on drugs and treatment methods for the treatment of infertility caused by the mutant protein.

  The present invention has been made in view of the above problems, and an object thereof is to provide a fertility diagnostic kit and a mutation detection method that enable estimation of fertility and identification of the cause of infertility. is there.

  Another object of the present invention is to provide a tool useful for studying a method for treating infertility.

  As a result of careful analysis of the base sequence of the human Centrin1 gene in a group consisting of male infertility patients and healthy males, as shown in the Examples described later, the present inventors have found mutations that cause functional defects in the human Centrin1 gene. And infertility were found to be related, and the present invention was completed.

  That is, in order to solve the above-described problems, the fertility diagnostic kit according to the present invention includes a reagent for detecting the presence or absence of a mutation that causes a functional defect of the human Centrin1 gene.

  Moreover, it is preferable that the said reagent detects the presence or absence of the variation | mutation of the 283rd base of a human Centrin1 gene, when the adenine base which codes a translation start codon is made into the 1st base.

  Further, the reagent is preferably one that detects that the base at position 283 of the human Centrin1 gene is substituted with thymine.

  In addition, the reagent includes a primer set for amplifying the region containing the base at position 283 of the human Centrin1 gene, and a base sequence determination reagent for determining the base sequence of the region. There may be.

  Alternatively, the reagent may detect the presence or absence of the mutation by an invader method.

  Alternatively, the reagent may detect the presence or absence of the mutation by the SMMD method.

  Moreover, in order to solve the said subject, the mutation detection method which concerns on this invention is characterized by including the process of detecting the presence or absence of the mutation which causes the functional defect | deletion of a human Centrin1 gene.

  In the above step, it is preferable to detect the presence or absence of a mutation in the position 283 of the human Centrin1 gene when the adenine base encoding the translation initiation codon is the position 1 base.

  In the polynucleotide of the present invention, when the adenine base encoding the translation initiation codon is the first base, the base at position 283 is substituted with thymine in the base sequence of the translation region of the human wild-type Centrin1 gene. It consists of the base sequence which was made.

  The polypeptide according to the present invention is characterized by comprising an amino acid sequence encoded by the polynucleotide.

  Moreover, the expression vector according to the present invention is characterized by having the above polynucleotide. And the expression kit which concerns on this invention is characterized by including the said expression vector.

  Since the fertility diagnostic kit and mutation detection method according to the present invention include a reagent (step) for detecting the presence or absence of a mutation that causes a functional defect in the human Centrin1 gene, estimation of fertility and infertility The cause of the can be identified.

  The polynucleotide according to the present invention provides a human Centrin1 gene containing a substitution mutation. Moreover, the polypeptide, the expression vector, and the expression kit according to the present invention provide a human substitution mutant Centrin1 protein found in infertile patients. Therefore, it is a useful tool for studying infertility caused by a deletion mutation of the Centrin1 gene.

(1. Fertility diagnostic kit and mutation detection method)
The fertility diagnostic kit and mutation detection method according to the present invention will be described. The mutation detection method according to the present invention is a method for detecting a gene mutation in a subject using a biological sample collected from the subject (human), and diagnoses whether the subject is hypofertile or infertile. It provides useful information for doing so. The fertility diagnostic kit is for carrying out the mutation detection method. In the mutation detection method, the presence or absence of a mutation in the human Centrin1 gene is detected. In addition, Centrin1 may be simply called Centrin or may be abbreviated as Cetn1.

  SEQ ID NO: 1 is obtained by extracting only the translation region from the base sequence of the transcription product of the human Centrin1 gene registered under the accession number gi3337234 in GeneBank. As shown in SEQ ID NO: 1, the Centrin1 gene has a translation region consisting of 519 bp. SEQ ID NO: 2 shows the deduced amino acid sequence of human Centrin1 protein.

  As described above, the Centrin1 protein is particularly strongly expressed in a mature male testis and has been reported to be localized in the tail in sperm. FIG. 1 is a schematic diagram showing the structure of the human Centrin1 gene. According to the Human Genome Resources (http://www.ncbi.nlm.nih.gov/genome/guide/human/) constructed by the Human Genome Project, the human Centrin1 gene is a retroposon-like gene without an intron. And is located on the short arm of human chromosome 18. The transcript of this gene is about 1.2 kb.

  As a result of the investigation shown in the Examples described later, the inventors of the present application have found that in the population of male infertility patients, cytosine at position 283 (hereinafter simply referred to as “C” in the translation region of the human Centrin1 gene shown in SEQ ID NO: 1). ")" Was found to be replaced by thymine (hereinafter simply "T") in one allele. On the other hand, the substitution of C at position 283 was not observed at all in the group consisting of males with confirmed fertility.

  When the above substitution mutation occurs in the human Centrin1 gene, the substitution site that originally encoded glutamine will encode a stop codon, and thus normal protein will not be expressed. Therefore, even if the substitution mutation from C to T at position 283 in the human Centrin1 gene occurs in only one allele, infertility is caused by a decrease in the translation amount of normal Centrin1 protein. It is estimated that there is a high possibility of causing.

  Thus, the subject's Centrin1 gene is examined for the presence of the substitution mutation at position C at position 283. If a substitution mutation for T is found, the subject is infertile or fertile (or possible). It is considered that A subject who is determined to be infertile or hypofertile can be presented with a suitable selection of an existing therapy such as ICSI (Intracytoplasmic Sperm Injection).

  In addition to the substitution mutation at position 283, it is presumed that a mutation causing a functional defect in the human Centrin1 gene is likely to cause infertility.

  From the above, the mutation detection method according to the present invention is isolated from a genomic DNA contained in a biological sample (blood, mucous membrane inside cheek, etc.) collected from a subject (human) or from a biological sample of the subject. And detecting the presence or absence of a mutation causing a functional defect in the human Centrin1 gene.

  In addition, the fertility diagnostic kit according to the present invention is a mutation that causes a functional defect of the human Centrin1 gene in genomic DNA contained in a biological sample collected from a subject or in genomic DNA isolated from a subject's biological sample. Reagents for detecting the presence or absence of

  The embodiment of the mutation detection method and fertility diagnostic kit is not particularly limited as long as it detects the presence or absence of a mutation that causes a functional defect in the human Centrin1 gene. For example, the mutation detection method and the fertility diagnostic kit may detect that the human Centrin1 gene of the subject contains a mutation that causes a functional defect in the gene, or the human Centrin1 gene of the subject Further, it may be detected that a mutation causing a functional defect of the gene is not included.

  For example, in the case of detecting the presence of the mutation, the mutation detection method and fertility diagnostic kit only need to be able to detect that the mutation causing the functional defect occurs in at least one allele. On the other hand, when detecting the absence of the mutation, the mutation detection method and the fertility diagnostic kit preferably detect that the mutation causing the functional defect does not occur in both alleles.

  The presence or absence of the mutation is examined by the mutation detection method or the fertility diagnostic kit. As a result, if no mutation is detected, it can be determined that there is a high possibility that there is 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 function defect of the Centrin1 gene.

  On the other hand, when a mutation is detected, it can be determined that there is a high possibility that it is not low fertility or infertility. Moreover, when a test subject is an infertility patient, it can be determined that the cause of infertility is highly likely due to a functional defect of the Centrin1 gene.

  Thus, according to the mutation detection method and fertility diagnostic kit, it is possible to estimate the possibility of fertility and identify the cause of infertility. That is, according to the mutation detection method or fertility diagnostic kit, information useful for diagnosing infertility is provided. Therefore, it can be said that the step of detecting the presence or absence of mutation in the mutation detection method is preferably used in a method for diagnosing hypofertility or infertility.

  The mutation detection method and fertility diagnostic kit detect the presence or absence of a substitution mutation at the position 283 of the human Centrin1 gene when the adenine encoding the translation initiation codon is the position 1 base. Is preferred.

  Furthermore, the mutation detection method and the fertility diagnostic kit preferably detect that the base at position 283 of the human Centrin1 gene is substituted with thymine. In this case, if the mutation detection method and fertility diagnostic kit detect that the base at position 283 of the Centrin1 gene is substituted with thymine, the subject is infertile or fertile. It can be determined that the possibility is high. Further, when the subject is an infertility patient, it can be determined that the cause of infertility is due to a mutation in the Centrin1 gene. Accordingly, it is possible to promptly propose an appropriate infertility treatment.

  By the way, as the mutation detection method and fertility diagnostic kit, various known methods, for example, base sequence determination method, invader method, or SMMD method (simultaneous multiple mutation detection system), PCR-RFLP method, MASA method, base Those based on the extension method, Taqman probe method and the like can be used. The fertility diagnostic kit may include an instruction manual describing how to use it.

  In one embodiment, the mutation detection method and the fertility diagnostic kit detect the presence or absence of the substitution mutation by determining the base sequence of the region containing the base at position 283 of the human Centrin1 gene. There may be. For the determination of the base sequence, the Sanger method or various known techniques applying the Sanger method can be used.

  In this case, it is preferable that the fertility diagnostic kit includes a set of primers designed so as to sandwich position 283 in order to amplify a region containing the base at position 283 of the human Centrin1 gene. More specifically, the primer set includes a primer having a part or all of the base sequence upstream of position 283 of the human Centrin1 gene and a base sequence downstream of position 283 of the human Centrin1 gene. And a primer having a base sequence complementary to part or all.

  When PCR (polymerase chain reaction) is performed using genomic DNA contained in a biological sample collected from a subject as a template and the above primer set, a region containing the base at position 283 of the human Centrin1 gene is amplified as a DNA fragment. Furthermore, if the nucleotide sequence is determined using one or both primers of the above primer set, 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. The primer used for determining the base sequence may be different from the primer used for PCR for amplification of the DNA fragment.

  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 the Centrin1 gene or its periphery, or a complementary base sequence. It is preferable to have 20 bases or more. By increasing the length of the same (or complementary) base sequence as the Centrin1 gene or its periphery, only the target region including the 283rd position of the Centrin1 gene can be reliably amplified.

  Further, in the above primer pair, the length of the region sandwiched between the primers is preferably 5 kb or less, more preferably 3 kb or less, further preferably 2 kb or less, and 1.5 kb or less. It is particularly preferred that By shortening the length of the region sandwiched between the primers, the region containing the 283rd base can be effectively amplified.

  Further, the primer used for determining the base sequence is preferably set at a position within 2 kb from the base at position 283, preferably set at a position within 1.5 kb, and within 1.0 kb. More preferably, the position is 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 that is 20 bp or more away from the base at position 283, more preferably 30 bp or more, and 40 bp or more. Is more preferable, and it is particularly preferable to set it at a position separated by 50 bp or more. Moreover, it is preferable that the said primer group amplifies 20 bp or more area | region containing the said 283rd base of the human Centrin1 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.

  As the above primer set, for example, primers (PrimerF1 and PrimerR1 in FIG. 1) having the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 can be used, respectively, but the primer bases that can be used in the present invention Needless to say, the sequence is not limited to this sequence. A person skilled in the art can easily obtain the base sequence of the human Centrin1 gene and the surrounding base sequence from the above-described 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, an Applied Biosystems Inc. type 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 above 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 mutation detection method and the fertility diagnostic kit may detect the presence or absence of a substitution mutation at the position 283 of the human Centrin1 gene by an invader method. 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 probe 2 and a signal probe 3 that are non-fluorescently labeled probes and a fret probe 5 that 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. On the other hand, the signal probe 3 has a flap 3b which is a base sequence unrelated to the target gene 1 and a base sequence complementary to the target gene 1, and the base at the 5 'end is a mutation (or a target gene 1). Single nucleotide polymorphism) A nucleotide 3a designed to correspond to the detection site 1a is linked. Here, the flap 3b is linked to the 5 'end of the nucleotide 3a.

  When the probes 2 and 3 designed in this way are hybridized with the target gene 1, the target gene 1, the invader probe 2 and the signal probe 3 form a triple chain at the mutation detection site 1a. Then, the flap endonuclease (also called “cleavase”) 4 recognizes this triple chain and cleaves the signal probe 3 on the 3 ′ side of the triple chain. 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 on the 5 'end side and hybridizing with the flap 3b on the 3' end side, a fluorescent dye 5b, and a quencher (luminescence inhibitor) 5c. Here, nucleotide 5a has a base sequence complementary to the mutation detection site of flap educt 3c at the 5 'end. The fluorescent dye 5b is bonded 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 triple chain 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 triple chain 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, when the fertility diagnostic kit detects the presence or absence of the above substitution mutation by the invader method, the fertility diagnostic kit may include the invader probe 2, the signal probe 3, the fret probe 5, and the like. preferable.

  When fluorescence is generated when there is a substitution mutation, the invader probe 2 has a base sequence complementary to the human Centrin1 gene consisting of the base sequence shown in SEQ ID NO: 1, and the base at the 3 ′ end is mutated. It is designed to correspond to the base (T) at position 283 of the type Centrin1 gene. The nucleotide 3a of the signal probe 3 has a base sequence complementary to the human Centrin1 gene, and is designed so that the base at the 5 'end corresponds to the base at position 283 of the mutant Centrin1 gene. The fret probe 5 detects the flap free body 3c that is cleaved by the flap endonuclease 4 when the invader probe 2 and the signal probe 3 are hybridized with the mutant Centrin1 gene to form a triple chain structure. Designed to be able to.

  When the base at position 283 of the subject's Centrin1 gene is substituted with T due to the above configuration, the 3 ′ end of nucleotide 3a overlaps with the 5 ′ end of invader probe 2 to form a triple chain. Forms fluorescence. On the other hand, when the subject's Centrin1 gene is a wild type, no triple chain is formed at the terminal portion of the invader probe 2, and thus fluorescence does not occur.

  In the above example, the fluorescence is generated when the subject's Centrin1 gene is a substitution mutation type. However, the present invention is not limited to this, and the subject's Centrin1 gene has a wild-type fluorescence. In the case of the substitution mutant type, the fluorescence may be suppressed, and various modifications can be made.

  In one embodiment, the mutation detection method and the fertility diagnostic kit may detect the presence or absence of a substitution mutation at the position 283 of the human Centrin1 gene by the SMMD method. Details of the SMMD method are described in a paper by Wakai et al. (Wakai J et al., Nucleic Acids Res., 2004. 32 (18), e141.), Which is incorporated herein by reference.

  FIG. 3 is a schematic diagram for explaining the principle of the SMMD method. The SMMD method detects single nucleotide polymorphisms, substitution mutations, deletion mutations, or insertion mutations using an electrochemical reaction. First, as shown in FIG. 3, in order to amplify a region including a mutation detection site using genomic DNA 11 of a subject as a template by PCR, asymmetric PCR is performed using counter primer 13 and special primer 12 in a ratio of 1: 4. (Process A). Here, the special primer 12 includes an oligomer 12a having a base sequence complementary to a region downstream of the mutation detection site 11a of the gene 11 whose mutation is to be detected, and a tag 12b. The tag 12b has a base sequence complementary to the region from the base one base downstream of the mutation detection site 11a of the gene 11 to the base one base upstream of the base to which the oligomer 12a is hybridized. It is linked to the 5 'end of 12a.

  As a result of the asymmetric PCR, the PCR product 14 shown in Step B is generated (Step B). Among the PCR products 14, the single-stranded asymmetric PCR product 14 including the special primer 12 includes the tag 12b, and thus forms a self-loop to form a special target (steps C and D).

  Next, the mutant type probe 20 and the wild type probe 30 are prepared. The probes 20 and 30 both have the same base sequence as the gene 11 whose mutation is to be detected, and an oligomer having the mutation detection sites 11a and 11b as the 3 ′ end is fixed to the gold electrode 25 on the 5 ′ end side. It is a thing. Here, the mutant probe 20 has a mutant base (T) at the 3 ′ end of the oligomer, whereas the wild type probe 30 has a wild type base (C) at the 3 ′ end of the oligomer. ing. These mutant probe 20 and wild type probe 30 can hybridize with the special target 15 because the oligomer has a base sequence complementary to the 3 ′ end of the special target 15 described above. A detailed method for immobilizing the oligomer on the gold electrode 25 is described in a paper by Takenaka et al. (Takenaka S et al., Anal Chem., 2000, 72, 1334-1341). This is used.

Before the probes 20 and 30 are hybridized with the special target 15, a background response is made in advance in a solution containing ferrocenylnaphthalene diimide (hereinafter abbreviated as “FND”), and the current I ₀ is measured. (Step E).

  Then, hybridization and ligation of the special target 15 with the mutant probe 20 and the wild type probe 30 are performed. Here, when the special target 15 is a mutant type (that is, when the subject has a mutant gene), the special target 15 is ligated because the nucleotide sequence is completely identical to the mutant probe 20. On the other hand, since the base sequence is different from that of the wild-type probe 30, the ligation is not performed (step F).

  Thereafter, when a denaturation treatment is performed, the mutant special target 15 is dissociated from the wild type probe 30 in the combination of the wild type probe 30 and the mutant special target 15. On the other hand, in the set of the mutant probe 20 and the mutant special target 15, since ligation is performed, dissociation does not occur (step G).

Then, each sample is washed to remove excess DNA, FND is added, and the electrochemical current I ₁ of the FND molecule is measured (step H). Here, in the electrode having the double-stranded DNA, the current I ₁ is larger than that of the electrode having the single-stranded DNA (Step I).

  Thereby, it can determine based on the electric current value which measured whether a test subject's genomic DNA was a mutant type or a wild type. In the SMMD method, a large number of mutations can be detected simultaneously by forming the mutant probe 20 and the wild-type probe 30 as an ECA (Electrochemial array) chip in an array.

  Therefore, when the fertility diagnostic kit according to the present invention detects the presence or absence of the above substitution mutation by the SMMD method, the fertility diagnostic kit includes a special primer 12, a counter primer 13, a mutant probe 20, and It is preferable that the wild-type probe 30 and the like are included. Only one of the mutant probe 20 and the wild-type probe 30 may be used.

  The special primer 12 includes an oligomer 12a having a base sequence complementary to a predetermined region downstream of the position 283 of the human Centrin1 gene shown in SEQ ID NO: 1, and a tag 12b. Tag 12b has a base sequence complementary to the region from position 284 of the human Centrin1 gene shown in SEQ ID NO: 1 to the site where oligomer 12a hybridizes, and is linked to the 5 ′ end of oligomer 12a. . On the other hand, the counter primer 13 has the same base sequence as the predetermined region upstream of the position 283 of the human Centrin1 gene.

  The mutant probe 20 is designed to have an oligomer having the same base sequence as the human mutant Centrin1 gene and having the base (T) at position 283 at the 3 'end. The wild-type probe 30 is designed to have an oligomer having the same base sequence as the human wild-type Centrin1 gene and having the base (C) at position 283 at the 3 'end.

  According to the above configuration, when the subject's Centrin1 gene is a substitution mutant type, the special target 15 can be ligated with the mutant probe 20 but cannot be ligated with the wild type probe 30. On the other hand, when the subject's Centrin1 gene is wild-type, the special target 15 can be ligated with the wild-type probe 30, but cannot be ligated with the mutant-type probe 20.

  In each of the above methods, the configuration for determining the presence or absence of a substitution mutation by examining the sense strand of the Centrin1 gene (the strand on the codon-encoding side) has been described in detail, but the same is true by examining the antisense strand. Needless to say, it is possible to make a judgment.

  In the present specification, the base sequence shown in SEQ ID NO: 1 is exemplified as a representative base sequence of the human wild-type Centrin1 gene. However, the “human Centrin1 gene” and the “human wild-type Centrin1 gene” described in the claims are exemplified. Is not limited to the nucleotide sequence shown in SEQ ID NO: 1. This is because many single nucleotide polymorphisms exist in human genes, and there is no exception in the human Centrin1 gene. Therefore, in the “human Centrin1 gene” and “human wild-type Centrin1 gene” described in the claims, one or several of the nucleotide sequence represented by SEQ ID NO: 1 as well as the nucleotide sequence represented by SEQ ID NO: 1 are used. A polynucleotide in which the base of is deleted, substituted, or added, and is involved in fertility (that is, a function equivalent to that of a polypeptide in which the polypeptide encoded by it consists of the amino acid sequence shown in SEQ ID NO: 2) In particular, a nucleotide sequence in which one or several bases are substituted by a single nucleotide polymorphism in the nucleotide sequence shown in SEQ ID NO: 1 is also included.

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, no more than 10, and even more preferably no more than 5 bases are contemplated. The same applies to the human mutant Centrin1 gene.

(2. Polynucleotides and polypeptides)
Next, the polynucleotide according to the present invention will be described. The polynucleotide according to the present invention is a base sequence in which the base at position 283 is substituted with T in the base sequence of the translation region of the human wild-type Centrin1 gene when adenine encoding a translation initiation codon is used as the base at position 1. It consists of

  In one embodiment, the polynucleotide according to the present invention may consist of the base sequence shown in SEQ ID NO: 3. However, the polynucleotide according to the present invention is not limited to the base sequence shown in SEQ ID NO: 3. The polynucleotide according to the present invention comprises not only the base sequence shown in SEQ ID NO: 3 but also a polynucleotide in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 3, In addition, a polynucleotide involved in hypofertility or infertility is also included, and in particular, a nucleotide sequence in which one or several bases are substituted by a single nucleotide polymorphism in the nucleotide sequence shown in SEQ ID NO: 3 is also included. .

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, no more than 10, and even more preferably no more than 5 bases are contemplated.

  As used herein, “polynucleotide” is synonymous with “gene”, “nucleic acid”, or “nucleic acid molecule” and refers to a polymer of nucleotides. In the present specification, “base sequence” is synonymous with “nucleic acid sequence” or “nucleotide sequence” and is shown as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

  The polynucleotide may be in the form of RNA (eg, mRNA) or DNA (eg, cDNA or genomic DNA). Furthermore, in the case of DNA, it may be either double-stranded or single-stranded. Furthermore, the single-stranded DNA or RNA may be a coding strand (also known as a sense strand) or a non-coding strand (also known as an antisense strand).

  The polynucleotide may be linked to a polynucleotide encoding a tag label (tag sequence or marker sequence) at the 5 'end or 3' end.

  Examples of the method for obtaining the polynucleotide include a method using PCR. Specifically, it can be obtained by PCR using a genomic DNA of a person having a mutant Centrin1 gene in which the base at position 283 is substituted with T, or by using a primer with a mutation. It can also be obtained from the wild-type Centrin1 gene by performing PCR.

  In addition, the polynucleotide may be inserted into a recombinant vector. This recombinant vector may be a vector used for in vitro translation or a vector used for recombinant expression.

  The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of host cell, an appropriate promoter sequence is selected from the above polynucleotide to ensure that the human mutant Centrin1 protein is expressed, and a vector incorporating this and the above polynucleotide in various plasmids is used as an expression vector. Use it.

  The expression vector contains an expression control region (for example, promoter, terminator, and / or replication origin) according to the type of host to be introduced. Conventional promoters (eg, trc promoter, tac promoter, lac promoter, etc.) are used as promoters for bacterial expression vectors, and examples of yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. Examples of the promoter for filamentous fungi include amylase and trpC. Examples of animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.). The expression vector can be prepared according to a conventional technique using a restriction enzyme and / or ligase. Transformation of the host with the expression vector can also be performed according to conventional techniques.

  A host transformed with the above expression vector is cultured, cultivated or bred, and then subjected to conventional techniques (eg, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography) from the culture. Etc.), the mutant Centrin1 protein can be recovered and purified.

  A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. . In addition, for example, when a polypeptide encoded by the polynucleotide is expressed in insects, an expression system using baculovirus may be used.

  If the recombinant vector is used, the polynucleotide can be introduced into an organism or cell, and as a result, the mutant Centrin1 protein can be expressed in the organism or cell. Furthermore, when the above recombinant vector is used in a cell-free protein synthesis system, a mutant Centrin1 protein can be selectively synthesized.

  Moreover, you may make it the form of the expression kit used in order to make a cell express the mutant type | mold Centrin1 protein mentioned above containing the recombinant vector mentioned above.

  The expression kit may contain a reagent other than the above recombinant vector. Other reagents include, for example, an introduction reagent for introducing a recombinant vector into a host cell, the host cell, a probe group for testing whether the recombinant vector has been introduced into the host cell, and the like. However, it is not limited to this. In other words, any reagent may be included as long as it supports the expression experiment of the mutant Centrin1 protein reliably and simply. By using the above expression kit, the mutant Centrin1 protein can be expressed reliably and simply in the target host cell.

  The polypeptide according to the present invention consists of an amino acid sequence encoded by the above-described polynucleotide.

  In one embodiment, the polypeptide may consist of the amino acid sequence shown in SEQ ID NO: 4. However, the polypeptide according to the present invention is not limited to the amino acid sequence shown in SEQ ID NO: 4. The polypeptide according to the present invention comprises not only the amino acid sequence shown in SEQ ID NO: 4, but also an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4. In addition, a protein involved in hypofertility or infertility is also included, and in particular, an amino acid sequence in which one or more amino acids are substituted by a single nucleotide polymorphism in the amino acid sequence shown in SEQ ID NO: 4 is also included.

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, no more than 10, and even more preferably no more than 5 amino acid residues are contemplated.

  In the present specification, “polypeptide” is synonymous with “peptide” or “protein”. The polypeptide according to the present invention may be isolated from a natural source or chemically synthesized.

  An “isolated” polypeptide refers to a polypeptide that has been removed from its natural environment. For example, a recombinantly produced polypeptide expressed in a host cell is considered isolated, as are natural or recombinant polypeptides that have been substantially purified by any suitable technique. The polypeptide according to the present invention can be obtained according to the method for using the polynucleotide according to the present invention described above.

  Polypeptides according to the present invention include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). ) Including products produced by recombinant techniques. The polypeptide may be glycosylated or non-glycosylated depending on the host used in the recombinant production procedure. Furthermore, the polypeptide may have a modified methionine residue that indicates translation initiation as a result of a host-mediated process.

  According to the polynucleotide, the expression vector, or the polypeptide of the present invention, a substitution mutant Centrin1 protein found in human infertility patients can be obtained. Therefore, a very useful tool can be obtained in developing a drug and a treatment method for the treatment of infertility caused by a substitution mutation at the position 283 of the human Centrin1 gene.

(3. Analysis of substitution mutations in the human Centrin1 gene)
(3-1. Materials and methods)
(3-1-a. Participants)
Infertile Japanese male subjects (N = 282) were divided into subgroups based on the degree of deficiency of spermatogenesis. Of these infertile subjects, 192 (68%) had nonocclusive azoospermia and 90 (32%) had severe hypospermia (<5 × 10 ⁶ cells / mL). When examined for genetic factors, all of these subjects had primary idiopathic infertility (Birmingham A et al. Fertil Steril 2004; 9: 2313-2317).

  On the other hand, as a control group (N = 96) of males whose fertility was confirmed, the father of a child born to a pregnant woman in a gynecology clinic was selected.

  The donor agreed to use blood for analysis of genomic DNA in this study.

(3-1-b. Identification of a mutation in the Centrin1 gene by direct sequencing of PCR amplified DNA)
In each of the following steps, unless otherwise specified, the treatment was performed according to a conventional method or an instruction manual attached to the kit. Genomic DNA was isolated from blood 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). In order to amplify the region containing the Centrin1 gene, PrimerF1 and PrimerR1 were designed as primer pairs used for PCR (see FIG. 1). As shown in FIG. 1, PrimerF1 has the same base sequence (SEQ ID NO: 5; 5′-GCGTTGGCAGTGGAGCGAGGGG-3 as the upstream region of the Centrin1 gene (region of −104 to −85 when the translation start point of the Centrin1 gene is +1). '). PrimerR1 is composed of a base sequence (SEQ ID NO: 6; 5′-TACATACTAGCCCTAGATAAAACCC-3 ′) complementary to the downstream region of the Centrin1 gene (region of +1148 to +1171).

  PCR was performed under the following conditions: 35 cycles of denaturation at 98 ° C for 10 seconds, annealing at 62 ° C for 30 seconds, and extension at 72 ° C for 1 minute. The PCR amplified fragment was purified using a SUPREC PCR spin column (Takara Bio).

  The fragment (about 1.3 kb) amplified by the primer pair consisting of PrimerF1 and PrimerR1 was sequenced using the above-described PrimerF1, PrimerR1, and each of the new PrimerF2 and PrimerR2. As shown in FIG. 1, PrimerF2 is composed of the same base sequence (SEQ ID NO: 7; 5'-AAGTGAACTCTCTGGGTTGCC-3 ') as the inner region of the Centrin1 gene (region of +537 to -556). PrimerR2 is composed of a base sequence (SEQ ID NO: 8; 5'-ATAGATCTAGACAAATTATGGGG-3 ') that is complementary to the inner region of the Centrin1 gene (region from +617 to +640). At this time, the reaction was performed using a thermal cycle sequencing kit (Applied Biosystems), and the reaction product was analyzed by ABI-PRISM 310 Genetic Analyzer (Applied Biosystems). The base sequence was determined by comparing the determination results in both directions.

(3-2. Results)
When the nucleotide sequence of the Centrin1 gene was analyzed for each of the infertile subject and the fertility confirmed subject, a subject in which the cytosine at position 283 of the Centrin1 gene was replaced with thymine in one allele was found to be an infertile subject. One person was found. This test subject had oligospermia (oligo-asthenozoospermia) with a sperm concentration of 6 million / ml and asthenozoospermia with a sperm motility rate of 0%. On the other hand, the above substitution mutations were not found at all from fertility confirmed subjects.

  When the cytosine base at position 283 of the human Centrin1 gene shown in SEQ ID NO: 1 is replaced with a thymine base, this site encodes a stop codon. As a result, the amino acid after glutamine at position 95 of the Centrin1 protein shown in SEQ ID NO: 2 is deleted (see SEQ ID NO: 5).

  Because the Centrin1 gene is widely conserved in mammals and the protein is particularly strongly expressed in testicular haploid germ cells, mutations (or decreased expression) of the Centrin1 protein cause (or cause) male infertility. Easy). Therefore, it was speculated that the subject in which the substitution mutation was found became infertile due to a reduction in the translation amount of normal Centrin1 protein.

  In this study, various single nucleotide polymorphisms were found in the Centrin1 gene in addition to the substitution mutations described above. These single nucleotide polymorphisms have patterns infertility and fertility confirmed subjects. There was no significant difference between the two and it was not found to affect fertility.

  The present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. That is, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

  In addition, it goes without saying that the present invention includes a numerical range other than the numerical range shown in the present specification as long as it is within a reasonable range that does not contradict the gist of the present invention.

  Since the fertility diagnostic kit according to the present invention can be used for estimation of fertility possibility and identification of the cause of infertility, it can be used in a medical field.

  The polynucleotide, expression vector and polypeptide according to the present invention provide a mutated polypeptide found in infertile patients based on the gene mutation described above. Therefore, establishment of effective drugs and treatment methods is expected by conducting research using this mutant polypeptide. These polynucleotides, expression vectors and polypeptides can be sold to research sites and are sufficiently industrially available. Moreover, industrial applicability improves further by making these into kits.

It is a schematic diagram which shows the structure of a human Centrin1 gene. It is a schematic diagram explaining the principle of the invader method. It is a schematic diagram explaining the principle of SMMD method.

Explanation of symbols

1 target gene 1a mutation detection site 2 invader probe 3 signal probe 3a nucleotide 3b flap 3c flap free body 4 flap endonuclease 5 fret probe 5a nucleotide 5b fluorescent dye 5c quencher 11 gene 11a / 11b mutation detection site 12 special primer 12a oligomer 12b Tag 13 Counter primer 14 PCR product 15 Special target 20 Mutant probe 25 Gold electrode 30 Wild type probe

Claims (12)

  A fertility diagnostic kit comprising a reagent for detecting the presence or absence of a mutation causing a functional defect in the human Centrin1 gene.   2. The reagent according to claim 1, wherein the reagent detects the presence or absence of a mutation at the position 283 of the human Centrin1 gene when the adenine encoding the translation initiation codon is the position 1 base. The fertility diagnostic kit as described.   The fertility diagnostic kit according to claim 2, wherein the reagent detects that the base at position 283 of the human Centrin1 gene is substituted with thymine. The reagent is
A set of primers for amplifying the region containing the base at position 283 of the human Centrin1 gene;
A base sequence determination reagent for determining the base sequence of the region,
The fertility diagnostic kit according to claim 2, comprising:   The fertility diagnostic kit according to claim 1, wherein the reagent detects the presence or absence of the mutation by an invader method.   The fertility diagnostic kit according to claim 1, wherein the reagent detects the presence or absence of the mutation by the SMMD method.   A mutation detection method comprising a step of detecting the presence or absence of a mutation that causes a functional defect of a human Centrin1 gene.   The mutation according to claim 7, wherein in the step, the presence or absence of a mutation in the position 283 of the human Centrin1 gene is detected when the adenine encoding the translation initiation codon is the position 1 base. Detection method.   A poly sequence comprising a base sequence in which the base at position 283 is substituted with thymine in the base sequence of the translation region of the human wild-type Centrin1 gene when adenine encoding the translation initiation codon is used as the base at position 1. nucleotide.   A polypeptide comprising an amino acid sequence encoded by the polynucleotide according to claim 9.   An expression vector comprising the polynucleotide according to claim 10.   An expression kit comprising the expression vector according to claim 11.

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