JP2018061453A - Method and system for assessing existence of genetic abnormalities in fertilized egg before implantation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To assess the existence of genetic abnormalities in a fertilized egg before implantation non-invasively.SOLUTION: The method of assessing the existence of a genetic abnormality in a fertilized egg before implantation comprising: (i) a step of preparing nucleic acid included in the supernatant of culture medium in which a mammalian fertilized egg is cultured in vitro; (ii) a step of acquiring base sequence information of the nucleic acid using a base sequence information acquisition means; (iii) a step of assessing the existence of genetic abnormalities by analyzing the acquired base sequence information. The culture supernatant is obtained from a fertilized egg cultured with no opening provided in the zona pellucida of the fertilized egg after fertilization. The present invention also provides a system for assessing the existence of genetic abnormalities in a fertilized egg before fertilization.SELECTED DRAWING: None

Description

  The present invention relates to a method for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation. The present invention also relates to a system for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation.

  In recent years, women's social advancement has been increasing, and there is a tendency toward late marriage and late birth. Along with this, an increasing number of women suffer from infertility. The main infertility treatment is assisted reproduction, represented by in vitro fertilization, but the technology has been developed at a remarkable pace. However, if the egg is aging due to late marriage or late birth, even if fertilization / early development is established by reproductive assistance, there is a possibility of causing implantation failure or miscarriage in early pregnancy. The cause of this is considered to be an abnormal number of chromosomes due to the fact that the egg's chromosomes cannot normally undergo meiosis due to aging of the egg. In addition, chromosomal abnormalities such as translocations, deletions, inversions, and severe genetic abnormalities can cause implantation failure and miscarriage, and can cause fatal severe genetic diseases after birth. .

  Currently, preimplantation genetic screening (PGS) is carried out as a method for examining such chromosomal abnormalities in fertilized eggs. PGS cultivates a fertilized egg for 2 to 3 days, and after the fertilized egg is divided into 4 to 8 blastomeres or cultured for 4 to 7 days and reaches a blastocyst, This is a method of collecting ~ 3 and selecting normal chromosomes of fertilized eggs. As a result, since only normal fertilized eggs are transplanted into the mother's uterus, it is considered possible to reduce the miscarriage rate and increase the pregnancy rate. In addition, as a method for examining a gene abnormality of a fertilized egg, a pre-implantation genetic diagnosis (PGD) is performed. PGD cultivates a fertilized egg for 2 to 3 days, and after the fertilized egg is divided into 4 to 8 blastomeres or cultured for 4 to 7 days and reaches a blastocyst, It is a method of collecting ~ 3 and selecting a fertilized egg having a normal gene. As a result, only fertilized eggs having normal genes are transplanted into the mother's uterus, and it is considered possible to prevent lethal genetic diseases.

  However, the effects on embryos and fetuses by collecting some blastomeres from fertilized eggs are unknown. Recently, it has been reported that the DNA contained in the fluid collected from the clonal or cleavage space of the embryo that has developed up to the blastocyst coincides with the DNA of the embryo (Non-patent Document 1 and Non-patent Document 2). In addition, a hole is made in a part of the zona pellucida that wraps the fertilized egg on the 3rd day of the initial culture of the fertilized egg, and the culture is continued until the 5th to 6th day when it reaches the blastocyst, and the DNA flows out into the culture solution It was reported that embryo-derived DNA can be detected from the culture supernatant (Non-patent Document 3).

Zhang Y, Li N, Wang L, Sun H, Ma M, Wang H, Xu X, Zhang W, Liu Y, Cram DS, Sun B Yao Y. , Molecular analysis of DNA in blastocoule fluid using next-generation sequencing. J Assist Reprod Genet. 33 (5), 637-645 (2016) Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferreretti AP, Gianaroli L. , Preimplantation genetic testing: polar bodies, blastomeres, tractorder cells, or blastocoelic fluid? Fertil Steril. 105 (3), 676-683. e5 (2016) Shamonki MI, Jin H, Haimoitz Z, Liu L. , Proof of concept: preimplantation genetic screening without embryo biopsy through cell-free DNA in emblem culture medium. Fertil Steril. 2016. doi: 10.016 / j. ferntstert. 2016.07.11.12.

  As described above, in the conventional preimplantation screening method, a part of the blastomere of the fertilized egg is collected, or a part of the zona pellucida surrounding the fertilized egg is artificially punched. This is a method for recovering DNA by invading the Therefore, it cannot be denied that invasion of a fertilized egg and / or part of an embryo may affect the subsequent embryogenesis process or cause embryo damage and death of the embryo. It also had ethical problems in invading some of the fertilized eggs and / or embryos. Therefore, there has been a demand for a method for safely and simply examining the presence or absence of a disease before implantation without invading the zona pellucida and part of the embryo of the fertilized egg.

  As a result of investigations by the inventors in order to solve the above-mentioned problems, unlike conventional methods, the presence or absence of genetic abnormality in a fertilized egg before implantation is determined without invading part of the zona pellucida and embryo. I found a way.

That is, the present invention includes the following inventions.
[1] A method for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation,
(I) a step of preparing a nucleic acid contained in a culture supernatant of a mammalian inseminated egg cultured in vitro;
(Ii) acquiring base sequence information of the nucleic acid using base sequence information acquisition means;
(Iii) analyzing the obtained base sequence information to determine the presence or absence of a gene abnormality,
Here, the culture supernatant is a culture supernatant obtained by culturing a fertilized egg in which no opening is provided in the zona pellucida of the fertilized egg after insemination.
[2] The method according to [1], wherein the step (i) includes a step of amplifying a nucleic acid by whole genome amplification means.
[3] The method according to [1] or [2], wherein the base sequence information acquisition unit is a next-generation sequencer or array CGH.
[4] The culture supernatant is a culture supernatant collected from 1 to 7 days after insemination, or a culture supernatant collected from the pronuclear stage (2PN) to the blastocyst stage of a fertilized egg. [1] The method according to any one of [1] to [3].
[5] The steps of [1] to [4], wherein the step of determining the presence or absence of a gene abnormality is a step of determining the presence or absence of a chromosomal abnormality and / or the step of determining the presence or absence of a gene mutation associated with a genetic disease The method according to any one of the above.
[6] The step of determining the presence or absence of the gene abnormality comprises detecting a chromosomal abnormality by detecting chromosomal aneuploidy, translocation, isochromosome, inversion, uniparental disomy, mosaic, deletion, or ploidy abnormality. Presence / absence determination and / or inborn errors of metabolism, primary immune deficiency syndrome, congenital skin disease, hereditary tumor, congenital malformation syndrome, congenital blood disease, congenital neuromuscular disease, congenital bone system disease Or the method of any one of [1]-[4] which is the process of detecting the gene abnormality relevant to a mitochondrial disease, and determining the presence or absence of the gene variation relevant to a genetic disease.
[7] The method according to any one of [1] to [6], wherein the mammal is a human.
[8] The step of determining the gene abnormality includes Turner syndrome, Patou syndrome, Edwards syndrome, Down syndrome, Klinefelter syndrome, Tripo-X syndrome, XYY syndrome, 5p monosomy (5p-syndrome), 5q monosomy (5q-syndrome) and 4p monosomy Wolf Hirschhorn syndrome, phenylketonuria, biopterin dysbolism, maple syrup urine disease, homocystinuria, xeroderma pigmentosum, sexual agammaglobulinemia, adenosine deami Nas deficiency, congenital epidermolysis bullosa, ichthyosis vulgaris, neurofibromatosis type 1, neurofibromatosis type 2, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, Angelman syndrome, Williams syndrome, Prader Willie Syndrome, Icardi Syndrome, Cornelia de Lange syndrome, Apert syndrome, hemophilia, muscular dystrophy, osteogenesis imperfecta, achondroplasia, achondroplasia, hypochondral dysplasia, fatal osteodysplasia, chronic progressive extraocular paralysis syndrome, Kearns Sayre Presence or absence of one or more diseases selected from the group consisting of syndrome, red rag fiber / myoclonic epilepsy syndrome, mitochondrial encephalomyopathy / lactic acidosis / stroke-like syndrome, Leigh's encephalopathy, Leber's disease, mitochondrial diabetes and Pearson's disease The method according to [7], which is a determination step.

[9] A system for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation,
Means for preparing a nucleic acid contained in the culture supernatant of a mammalian fertilized egg;
Base sequence information acquisition means for acquiring base sequence information of the nucleic acid prepared by the preparation means;
Means for analyzing the base sequence information obtained by the base sequence information acquisition means to determine the presence or absence of a gene abnormality;
Means for outputting a result obtained by the means for determining;
Including
Here, the culture supernatant is a culture supernatant obtained by culturing a fertilized egg in which an opening is not provided in the zona pellucida of the fertilized egg after insemination.
[10] The system according to [9], wherein the nucleic acid preparation means includes whole genome amplification means.
[11] The system according to [9] or [10], wherein the base sequence information acquisition unit is a next-generation sequencer or array CGH.
[12] The means according to [9] to [11], wherein the means for determining the presence / absence of a gene abnormality is a means for determining the presence / absence of a chromosomal abnormality and / or a means for determining the presence / absence of a gene mutation associated with a genetic disease. The system according to any one of the above.
[13] The means for determining the presence or absence of a genetic abnormality detects chromosomal anomaly by detecting chromosomal aneuploidy, translocation, isochromosome, inversion, uniparental disomy, mosaic, deletion, or ploidy abnormality. Means to determine presence and / or association with congenital metabolic disorders, primary immunodeficiency syndrome, congenital skin disease, hereditary tumor, congenital malformation syndrome, congenital neuromuscular disease, congenital bone system disease or mitochondrial disease The system according to any one of [9] to [11], which is means for detecting a genetic abnormality to determine whether or not there is a genetic mutation associated with a genetic disease.
[14] The system according to any one of [9] to [13], wherein the mammal is a human.
[15] The means for determining the presence or absence of the gene abnormality is Turner syndrome, Patou syndrome, Edwards syndrome, Down syndrome, Kleinfelter syndrome, Tripo-X syndrome, XYY syndrome, 5p monosomy (5p-syndrome), 5q monosomy (5q-syndrome) and 4p monosomy Wolf Hirschhorn syndrome, phenylketonuria, biopterin metabolism disorder, maple syrup urine disease, homocystinuria, xeroderma pigmentosum, sexual agammaglobulinemia, adenosine Deaminase deficiency, congenital epidermolysis bullosa, ichthyosis vulgaris, neurofibromatosis type 1, neurofibromatosis type 2, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, Angelman syndrome, Williams syndrome, Prader-Willi syndrome, Aicardy syndrome, Corne A de Lange syndrome, Apert syndrome, hemophilia, muscular dystrophy, osteogenesis imperfecta, achondroplasia, achondroplasia, hypochondral dysplasia, fatal osteodysplasia, chronic progressive extraocular palsy syndrome One or more selected from the group consisting of: Kearns-Sayer syndrome, red rag fiber / myoclonic epilepsy syndrome, mitochondrial encephalomyopathy / lactic acidosis / stroke-like syndrome, Leigh's encephalopathy, Leber's disease, mitochondrial diabetes and Pearson's disease The system according to [14], which is means for determining the presence or absence of a disease.

  According to the method and system of the present invention, unlike the methods reported so far, it is possible to determine the presence or absence of a gene abnormality in a fertilized egg before implantation by an extremely simple method and system. .

It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (positive control) which has 21 trisomy. The aneuploidy of autosomes (Nos. 1 to 22) and sex chromosomes (X chromosome and Y chromosome) was analyzed. Locus: Chromosome site, Length: Length, Copy number: Copy number, CytoBand: Location in chromosome, CNV Confidence: Reliability of existence of aneuploidy, CNV precision: Accuracy of experiment, Tiles: Number of tiles Yes. When CNV Confidence is “0”, it indicates that there is no aneuploidy. When CNV Confidence is> 10, it indicates that aneuploidy exists and the reliability of the data is high. Tiles indicates a reference destination that is already set in the region of 1 Mb to 2 Mb for each chromosome when the fragment data obtained by NGS is collated. It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (positive control) which has 21 trisomy. Each sequence data was normalized and the number of chromosomes was plotted for each chromosome. The vertical axis represents the number of chromosomes. The horizontal axis indicates the chromosome number (Nos. 1 to 22) and the sex chromosome (X and Y). When there is no aneuploidy, it is plotted around y = 2. Hereinafter, in FIGS. 2-5, it represents with the content similar to FIG. 1A and FIG. 1B. It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (6-day embryo, sample A). It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (6-day embryo, sample A). It is a figure which shows the result of having detected the aneuploidy of the culture supernatant origin genome (6th day embryo, sample A) of a fertilized egg. It is a figure which shows the result of having detected the aneuploidy of the culture supernatant origin genome (6th day embryo, sample A) of a fertilized egg. It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (6-day embryo, sample H). It is a figure which shows the result of having detected the aneuploidy of the fertilized egg origin genome (6-day embryo, sample H). It is a figure which shows the result of having detected the aneuploidy of the culture supernatant origin genome (6-day embryo, sample H) of a fertilized egg. It is a figure which shows the result of having detected the aneuploidy of the culture supernatant origin genome (6-day embryo, sample H) of a fertilized egg. It is the figure which compared the aneuploidy of the genome derived from a fertilized egg and the genome derived from the culture supernatant of a fertilized egg. The results of FIGS. 2-5 are listed. The numbers indicate the estimated number of chromosomes detected. It is a figure which shows the flowchart of one Embodiment of this invention.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the technical scope of this invention is not limited to the following form.

  The mammalian fertilized egg used in the present invention refers to a fertilized egg of a warm-blooded vertebrate, for example, primates such as humans and monkeys, rodents such as mice, rats, hamsters, guinea pigs and rabbits, dogs and This includes, but is not limited to, pets such as cats, livestock such as cattle, horses, pigs, and sheep. The mammal that provides a fertilized egg used in the present invention is preferably a human.

  The term “fertilized egg” used in the present invention means a fertilized egg obtained after artificially inseminating an egg and sperm in an in vitro environment, an embryo obtained by cleavage of the egg, and an egg obtained by freezing and thawing it It is meant to include any of (embryo), and unless otherwise specified, is meant to include any of them. In one embodiment of the present invention, the “fertilized egg” is obtained by, for example, in vitro fertilization (IVF) or micro insemination (ICSI) described below. Further, in this specification, the term “fertilized egg” is a term including the above-mentioned “fertilized egg”, and is the first formed by combining sperm and ovum regardless of whether or not an artificial insemination operation is performed. And embryos obtained by cleavage of the cells and eggs (embryos) obtained by freezing and thawing them.

  The fertilized egg of a mammal repeats cleavage after fertilization, increases the number of cells while forming small blastomeres through the 2 cell stage, 4 cell stage, and 8 cell stage, and forms a blastocyst through a morula . In general, cleavage begins about 30 minutes after fertilization, and reaches the 2-cell stage at about 30 hours after fertilization, the 4-cell stage at about 40 hours after fertilization, the 8-cell stage at about 60 hours after fertilization, and 3-4 after fertilization. Mulberry embryos form on day, and blastocysts form 4-6 days after fertilization. An egg before fertilization, a fertilized egg, and an embryo formed by splitting them (also referred to as a fertilized embryo) are covered with a membrane called a zona pellucida, and the zona pellucida protects the fertilized egg. In general, in a living body, a blastocyst that has reached the uterus through the fallopian tube hatches from the zona pellucida (hatching) while floating in the uterus and is then implanted into the endometrium. As a result, pregnancy is established and development proceeds.

  The fertilized egg used in the present invention is obtained by means known to those skilled in the art, and is obtained by, for example, in vitro fertilization (IVF: in vitro fertilization) or microinsemination (ICSI). Fertilized eggs are preferred.

  In vitro insemination is a method mainly performed in veterinary medicine and obstetrics and gynecology, and refers to a method in which fertilization that is normally established in vivo is artificially performed outside the body. Typically, in vitro, eggs are exposed to a solution containing sperm and fertilized. In humans, it is a method used as part of infertility treatment. Specifically, gonadotropin analogs are used to prevent ovulation in women being laid, and follicles are grown using menopausal gonadotropins or purified follicle stimulating hormone (FSH) or synthetic FSH, and then After confirming that the follicle is fully grown, ovulation is induced using a gonadotropin analog or chorionic gonadotropin (hCG). A thin needle is inserted into the follicle in the ovary, and the egg in the follicle is collected. This is a method in which the collected egg is inseminated with sperm collected from a man and inseminated or microinseminated. The fertilized egg is washed with a medium and cultured at a temperature close to body temperature, for example, around 37 ° C. The medium used for the culture may be a medium known to those skilled in the art and is not limited. Moreover, the culture medium used for the preculture of an egg, the preculture of a sperm, and the culture of a fertilized egg should just use the medium suitable for each culture | cultivation, and is not limited.

  Microinsemination is a method for artificially establishing insemination in vitro as in in vitro insemination. Typically, this is a method in which one sperm is directly injected into the cytoplasm of an egg using a micromanipulator or the like under a microscope. Microinsemination is used particularly when sperm of a patient having a disorder that reduces the insemination ability of sperm is used. For example, spermatozoa derived from a patient having a disorder such as severe sperm reduction, azoospermia, asthenozoospermia, sperm malformation, immobility spermatozoa. In the present invention, the sperm required for the formation of a fertilized egg is not particularly limited. Selection of the culture medium and culture method for culturing the fertilized egg obtained by microinsemination may be according to known methods, and is not limited.

  The method and system of the present invention makes it possible to determine whether or not an individual has a genetic abnormality before implantation when a fertilized egg obtained by in vitro fertilization occurs and grows into the individual. . In the method and the system of the present invention, the waste solution (culture supernatant) of the medium used when cultivating the fertilized egg before returning to the uterus in vitro is used. In the present invention, the term “culture supernatant” is used to include a concentrate obtained by purifying and concentrating the culture supernatant by a method known to those skilled in the art. The means for concentrating the culture supernatant used in the present invention is not particularly limited. Particularly preferred as the culture supernatant used in the present invention is a culture supernatant obtained by culturing a fertilized egg in which no opening is provided in the zona pellucida of the fertilized egg after insemination. The culture supernatant of the fertilized egg used in the present invention is preferably a culture supernatant of the fertilized egg before hatching. When the in vitro fertilized eggs are cultured in vitro, the present inventors have not invaded the zona pellucida that covers the fertilized eggs, but in the culture supernatant, the nucleic acid derived from the fertilized egg, particularly the genome derived from the fertilized egg. It was found that DNA or chromosome or genomic DNA or part of chromosome was included. Moreover, it discovered that the nucleic acid in the culture supernatant of the said fertilized egg corresponds with the nucleic acid which a fertilized egg has. According to the conventional method, it has been said that a nucleic acid derived from a fertilized egg cannot be examined unless a zona pellucida covering the fertilized egg is invaded and a hole is made in the zona pellucida. According to the present invention, there is no need to invade the zona pellucida of the fertilized egg, and the fertilized egg can be obtained by using the culture supernatant of the fertilized egg without using a part of the blastomeres generated by cleavage. It becomes possible to determine the disease and sex that the individual who has developed may have.

  The culture supernatant of the fertilized egg used in the present invention may or may not be stored frozen until use. When cryopreserved, for example, it may be stored at -10 ° C to -200 ° C, may be stored at -15 ° C to -150 ° C, and may be stored at -20 ° C to -90 ° C. . When not stored frozen, it may be room temperature (for example, 15 ° C to 30 ° C), 0 ° C to 25 ° C, 0 ° C to 10 ° C, or 2 ° C to 6 ° C. May be.

  The culture supernatant of the fertilized egg used in the present invention is preferably frozen and stored until use. If it can be cryopreserved, the nucleic acid contained in the culture supernatant can be stably stored until use, which is preferable. Moreover, even if the place where the fertilized egg is cultured and the place where the present invention is carried out using the culture supernatant of the fertilized egg are different, it can be easily and stably transported by cryopreservation. The cryopreserved culture supernatant can be used by thawing at room temperature or on ice or at 4 ° C. immediately before use.

  In the present invention, the culture supernatant of a fertilized egg is a culture supernatant collected 1 to 7 days after insemination. Preferably, it is a culture supernatant collected 2 to 6 days after insemination. More preferably, it is a culture supernatant collected 3 to 6 days after insemination.

  In the present invention, the culture supernatant recovered from the pronuclear stage (2PN (pronuclei)) to the blastocyst stage of the fertilized egg can be used as the culture supernatant of the fertilized egg. In the present invention, the culture supernatant collected from the 4 cell stage to the blastocyst stage of the fertilized egg can be used as the culture supernatant of the fertilized egg. In the present invention, the culture supernatant collected from the 8 cell stage to the blastocyst stage of the fertilized egg can be used as the culture supernatant of the fertilized egg.

  In the present invention, “no opening is provided in the zona pellucida of the fertilized egg after fertilization” means by physical means (eg, needle, laser, etc.) or chemical means (eg, proteolytic enzyme, acid treatment, etc.). The state that does not have artificially created holes in the zona pellucida of the fertilized egg after insemination. That is, the zona pellucida that covers the fertilized egg is in a state where it does not have an opening formed by tearing or thinning, and the inside and outside of the zona pellucida membrane are apparently isolated.

  In the present invention, a known sequencer (also called a sequencer) or array CGH can be used as the sequence information acquisition means.

  In this specification, a sequencer refers to a device that can read the base sequence of a nucleic acid, particularly DNA. In the present invention, as a sequencer, a sequencer using the dideoxy method (also referred to as Sanger method) (so-called first generation sequencer), a sequencer that performs massively parallel sequencing using sequential DNA synthesis / photodetection method, one molecule real time A sequencer using a sequencing method or a sequencer using a post-light sequencing method can be used.

  In one embodiment of the present invention, it is necessary to read a large amount of nucleic acid contained in the culture supernatant, particularly DNA constituting a wide range of the genome, in a short time and in large quantities. As information acquisition means, it is preferable to use a next-generation sequencer developed after the first generation. In this specification, the sequencers developed after the first generation are collectively referred to as the next generation sequencer (NGS), which has not been put into practical use at the time of filing this patent application. Those skilled in the art can naturally understand that the sequencer to be converted is also applicable to the present invention.

  Specific examples of the next-generation sequencer using sequential DNA synthesis / photodetection method applicable to the present invention include, for example, Illumina HiSeq system (Hisseq2000, HiSeq3000, HiSeq4000), MiSeq system, NextSeq system and Genome Analyzer IIx ( GAIIx), Roche's GS Junior, GsJunior Plus and GS FLX + system (GS FLX Titanium XL +, GS FLX Titanium XLR70), Thermo Fisher Scientific's SoLiD (TM) PG 2.0, Gene I Trademarked) system, but is not limited thereto.

  Examples of the next-generation sequencer using the single molecule real-time sequencing method include, for example, PacBio (registered trademark) RS II and PacBio (registered trademark) Sequal (trademark) system of Pacific Biosciences. It is not limited to this. An example of a next-generation sequencer using post-light sequencing technology includes, but is not limited to, the GridION system of Oxford Nanopore Technologies.

  The next-generation sequencer using the sequential DNA synthesis / photodetection method used as one embodiment of the present invention has some common features described below.

(1) Preparation of library DNA A sample DNA to be analyzed is fragmented to an arbitrary length, and a DNA library to which an adapter sequence is ligated is prepared. Through the ligated adapter sequence, the DNA library is fixed by hybridization to sequencing. The adapter sequence is a universal priming site for the sequencing primer.

(2) Preparation of template The library DNA fragment to which the adapter sequence is ligated is hybridized to a solid surface (bead or flat silicon material surface) having a linker of a complementary sequence of the adapter sequence. Polymerase chain reaction (PCR) is performed on the solid surface to clone and amplify each library DNA to form a cluster group of DNA derived from a single library fragment.

  In the method using library DNA immobilized on beads, a single bead in a water droplet is prepared so as to have a single library DNA fragment, and PCR (emulsion PCR) is performed to form a single bead. A cluster group derived from a library DNA fragment is formed. In the method using a flat silicon material surface, a cluster group derived from a single DNA fragment is formed on the silicon material surface by bridge PCR. Each cluster acts on an individual sequencing reaction.

(3) Sequencing In a sequencer, a reaction for incorporating a nucleic acid is performed using the base sequence of each cluster as a template. At that time, a light or fluorescent signal is produced depending on the type of base of the incorporated nucleic acid. These signals are read and the nucleotide sequence of the nucleic acid is taken as information.

(4) Data output:
Each device outputs raw data at the end of sequencing. This raw data is enormous data derived from the base sequence of each library DNA fragment. Any software is used to process the raw data and perform the desired analysis.

  In the present invention, the step of preparing a nucleic acid includes a step of amplifying a specific base sequence region by PCR using a set of forward and reverse primers sandwiching an arbitrary base sequence of the nucleic acid and a DNA polymerase, or whole genome amplification (WGA). : Whole Genome Amplification) means, for example, a step of amplifying a nucleic acid using a commercially available reagent or kit for whole genome amplification may be included. In the present invention, the step of preparing a nucleic acid preferably includes a step of amplifying the nucleic acid by whole genome amplification means. In the present invention, the step of amplifying the nucleic acid preferably uses a whole genome amplification means that covers almost the entire region of the genome from a sample containing a small amount of nucleic acid, and the bias of the region to be amplified is reduced. The whole genome amplification means that can achieve this object is not particularly limited. In the present invention, the step of preparing the nucleic acid may be performed by manual means, may be performed by automatic means using an automatic device or the like, or may be performed by mixing manual and automatic means.

  In one embodiment of the present invention, commercially available kits that can be used as a whole genome amplification means include, for example, GenomePlex Single Cell Whole Genome Amplification Kit provided by Sigma-Aldrich, PicoPLEXTM WGA Kit provided by New England Biolabs, Examples include, but are not limited to, REPLI-g Single Cell Kit provided by Qiagen, or True Prime Single Cell WGA Kit provided by Signis.

  In one embodiment of the present invention, the step of preparing a nucleic acid is appropriately selected according to the base sequence information acquisition means used in the present invention. For example, when using the Ion PGM (trademark) system provided by Thermo Fisher Scientific as the base sequence information acquisition means, the step of preparing the nucleic acid is the same as the step of amplifying the nucleic acid by the whole genome amplification means. It is necessary to further include a step of preparing a library nucleic acid, particularly a library DNA, using the prepared nucleic acid.

  For example, when the Ion PGM ™ system, which is a next-generation sequencer, is used as the base sequence information acquisition means, the library DNA is a kit provided by Thermo Fisher Scientific (for example, Ion Xpress Plus Fragment Library Kit and Ion). Xpress Barcode Adapters 1-16 Kit) and accompanying instructions. The method and reagents for preparing the library DNA are not limited to the above, and can be used alternatively if they can achieve the same purpose. More preferably, it is a kit or a reagent recommended by the company that provides the sequencer.

  The prepared library DNA is further prepared for decoding with a next-generation sequencer. For example, when using the Ion PGM ™ seek sensor provided by Thermo Fisher Scientific, Ion PGM ™ Template OT2 200 Kit, Ion PGM Sequencing 200 Kit v2, and Ion 316 Chip Kit v2 The base sequence of the nucleic acid is decoded using an Ion PGM ™ seek sensor. The reagents and chips described in the present specification are examples, and can be appropriately selected according to the sequencer used.

  According to the present invention, the base sequence information of the nucleic acid contained in the culture supernatant of a fertilized egg can be obtained. The base sequence information of the obtained nucleic acid is analyzed using any software, and when an inseminated egg obtained by insemination in vitro occurs and grows into an individual, the individual has a disease. It is possible to determine whether there is chromosomal aneuploidy or genetic abnormality. The software used is not particularly limited.

  In one embodiment of the present invention, in order to derive a highly reliable analysis result from a base sequence output from a next-generation sequencer, it is preferable to use a data amount that covers at least about 30 times the same base sequence. The base sequence fragments output from the next-generation sequencer are integrated on a computer, and a gene database (for example, DDBJ (DNA Data Bank of Japan), National Biotechnology Information Center (NCBI) in which various gene sequences are registered. It is possible to determine the presence or absence of a gene mutation by comparison with the base sequence of

  In one embodiment of the present invention, base sequence information obtained from a nucleic acid contained in a culture supernatant of a fertilized egg and information on a disease-specific gene mutation registered in a database (for example, a single nucleotide polymorphism ( It is possible to determine the presence or absence of a gene abnormality by comparison with a disease-specific gene mutation by SNP).

  In the present invention, the gene abnormality refers to an abnormality of a gene that causes a genetic disease, and includes, for example, a chromosomal abnormality, a gene mutation, a disease-specific single nucleotide polymorphism (SNP), and the like. In the present specification, the chromosomal abnormality includes chromosomal aneuploidy, translocation, homozygous chromosome, inversion, uniparental disomy, mosaic, deletion, or ploidy abnormality. In the present specification, gene mutation includes point mutation, missense mutation, nonsense mutation, frameshift mutation and the like. In the present specification, a disease-specific single nucleotide polymorphism (SNP) refers to a mutation of a base known to cause a disease.

  In one embodiment of the present invention, the aneuploidy of an autosome (for example, No. 1 to No. 22 in humans) based on nucleic acid contained in the culture supernatant of a fertilized egg, particularly genomic DNA, It becomes possible to identify the number of sex chromosomes (for example, the X chromosome and / or the Y chromosome in humans). Further, in one embodiment of the present invention, by analyzing the obtained base sequence information using arbitrary software, chromosome aneuploidy, translocation, homozygous chromosome, inversion, uniparental disomy, It becomes possible to detect the presence or absence of chromosomal abnormalities by detecting mosaics, deletions or ploidy abnormalities. As an example of software used in the present invention, for example, Ion (trademark) Reporter software (Thermo Fisher Scientific) can be used, but the present invention is not limited to this.

  In the present specification, aneuploidy refers to those in which the number of chromosomes (or part of chromosomes) is different from normal. The aneuploidy is known when the chromosome copy number is 3 (“trisomy”) or when the copy number is 1 (“monosomy”). These changes in the number of chromosomes caused by non-segregation of chromosomes during meiosis affect ontogeny and lead to various diseases (syndromes). Many individuals with trisomy and monosomy chromosomes can cause insemination problems, implantation problems, spontaneous abortion, and die early after birth. Also, some chromosomal aneuploidy results in various syndromes.

  Well-known aneuploidies are chromosome 21, chromosome 18, chromosome 13 trisomy, and abnormal number of sex chromosomes. Trisomy 21 is known to cause Down syndrome. Trisomy 18 is known to cause Edwards syndrome. Trisomy 13 is known as a cause of developing Patou syndrome (Pato syndrome). Sex chromosome aneuploidy (SCA) is, for example, X chromosome deletion (X-) is Turner syndrome, X chromosome addition is Kleinfelter syndrome (XXY) or tripo-X (Tripo-X) syndrome (XXX), Y Chromosome addition is known to cause XYY syndrome (XYY). Furthermore, 5q monosomy (5q-syndrome) and 4p monosomy Wolf-Hirschhorn syndrome are known as autosomal abnormalities. In this specification, diseases caused by chromosomal abnormalities are collectively referred to as chromosomal abnormalities. The above-mentioned diseases caused by chromosomal abnormalities are examples, and the diseases that can be determined by the method and system of the present invention are not limited to the above examples. Chromosome abnormalities are caused by chromosomal aneuploidy, translocations, isochromosomes, inversions, uniparental disomy, mosaics, deletions or ploidy abnormalities. In one embodiment of the present invention, the method and system of the present invention comprises chromosomal aneuploidy, translocation, homozygous chromosome, inversion, uniparental disomy, mosaic, deletion or polyploidy before implantation. An abnormality can be determined.

  According to the present invention, it is possible to determine the presence or absence of a disease caused by chromosomal aneuploidy before implantation of a fertilized egg without invading the fertilized egg. It is also possible to determine not only the presence or absence of a disease but also sex, for example, XX or XY.

  The present invention makes it possible to determine whether a fertilized egg before implantation has a genetic mutation associated with a hereditary disease. Examples of diseases that can be determined by the present invention include, for example, inborn errors of metabolism, primary immunodeficiency syndrome, congenital skin diseases, hereditary tumors, congenital malformation syndromes, congenital blood diseases, congenital blood diseases Neuromuscular diseases, congenital bone system diseases or mitochondrial diseases are mentioned. Examples of inborn errors of metabolism that can be determined according to the present invention include phenylketonuria, biopterin metabolism disorders, maple syrup urine disease, homocystinuria, xeroderma pigmentosum, and albinism. Examples of primary immunodeficiency syndromes that can be determined according to the present invention include, for example, concomitant agammaglobulinemia, adenosine deaminase deficiency, complex immunodeficiency, and the like. Examples of congenital skin diseases that can be determined according to the present invention include congenital epidermolysis bullosa, acne vulgaris and the like. Examples of hereditary tumors that can be determined according to the present invention include, for example, neurofibromatosis type 1, neurofibromatosis type 2, familial colon polyposis, hereditary nonpolyposis colon cancer, and the like. Examples of congenital malformation syndromes that can be determined according to the present invention include Angelman syndrome, Williams syndrome, Prader-Willi syndrome, Aicardy syndrome, Cornelia de Lange syndrome, Apert syndrome and the like. Examples of congenital blood diseases that can be determined according to the present invention include hemophilia and Fanconi anemia. Examples of congenital neuromuscular diseases that can be determined by the present invention include muscular dystrophy and Rett syndrome. Examples of congenital bone system diseases that can be determined according to the present invention include osteogenesis imperfecta, achondroplasia, achondroplasia, hypochondral dysplasia, and lethal osteodysplasia. Examples of mitochondrial diseases that can be determined according to the present invention include, for example, chronic progressive extraocular palsy syndrome, Kearns-Sayer syndrome, red rag fiber / myoclonic epilepsy syndrome, mitochondrial encephalomyopathy / lactic acidosis / stroke-like syndrome, Lee encephalopathy , Leber's disease, mitochondrial diabetes, and Pearson's disease. The above-mentioned diseases that can be determined by the present invention are exemplifications, and are not limited thereto.

  In one embodiment of the present invention, array CGH (comparative genomic hybridization (also referred to as microarray CGH)) can be used as the base sequence information acquisition means. The array CGH is a means for detecting a copy number change (excess or decrease) of nucleic acids, particularly DNA, in a sample. Array CGH can be implemented by techniques known to those skilled in the art. Specifically, human cot-1 for labeling the sample (specimen) to be investigated and the DNA extracted from the control sample with different fluorescent dyes, mixing the specimen and the DNA of the control sample, and blocking repeated sequences DNA (placenta-derived DNA containing a repetitive sequence such as Alu sequence and Kpn family) is added, and the resulting mixture is exposed and hybridized onto a slide containing a DNA probe having an arbitrary sequence. An epifluorescence microscope and quantitative image analysis are used to detect a change (increase / decrease) in fluorescence intensity, and an abnormal region contained in genomic DNA is determined. The reagents and equipment used for the array CGH may be those known to those skilled in the art and are not limited. By using array CGH, as in the case of using the above sequencer, genomic DNA, particularly chromosomal aneuploidy, translocation, isochromosome, inversion, uniparental disomy, mosaic, deletion, or ploidy abnormality is detected. Thus, it is possible to determine the presence or absence of chromosomal abnormalities.

  In one embodiment of the present invention, the system of the present invention comprises a nucleic acid preparation means contained in a culture supernatant of a mammalian fertilized egg, and a base sequence for obtaining the base sequence information of the nucleic acid prepared by the preparation means. A system comprising: information acquisition means; means for analyzing the base sequence information obtained by the base sequence information acquisition means to determine the presence or absence of a gene abnormality; and means for outputting a result obtained by the determination means The culture supernatant is characterized by using a culture supernatant obtained by culturing a fertilized egg without providing an opening in the zona pellucida of the fertilized egg after insemination. The system of the present invention is configured to carry out the above-described method of the present invention, and the configurations or means mentioned in the method of the present invention are all applicable to the system of the present invention.

  In one embodiment of the system of the present invention, nucleic acid preparation means comprises means for amplifying a specific base sequence region by PCR using a set of forward and reverse primers sandwiching an arbitrary base sequence of nucleic acid and DNA polymerase; Whole genome amplification (WGA) means, for example, nucleic acid amplification means using commercially available whole genome amplification reagents or kits may be included. The system of the present invention may be configured by manual means, may be configured by automated means, or may be configured by mixing manual and automatic means.

  In the present invention, the analysis of the base sequence information may be performed on a local server on which arbitrary software is installed, or may be performed on a cloud server via the Internet. Not.

  In the present invention, the means for determining the presence or absence of a gene abnormality may be a means for determining the presence or absence of a chromosomal abnormality and / or a means for determining the presence or absence of a gene mutation associated with a genetic disease. Further, in the present invention, the means for determining the presence or absence of a gene abnormality is a chromosomal abnormality by detecting chromosomal aneuploidy, translocation, homozygous chromosome, inversion, uniparental disomy, mosaic, deletion or ploidy abnormality Means for determining the presence or absence of diseases and / or congenital metabolic disorders, primary immune deficiency syndrome, congenital skin diseases, hereditary tumors, congenital malformation syndromes, congenital blood diseases, congenital neuromuscular diseases, congenital bones It may be a means for detecting the gene abnormality related to systematic disease or mitochondrial disease and determining the presence or absence of a gene mutation related to hereditary disease. The criteria for determining the presence or absence of a gene abnormality may be in accordance with a known method. For example, any base sequence of a nucleic acid contained in a sample is a base sequence of a disease-specific gene having a mutation registered in a database. If they match, it can be determined that the gene abnormality is “present”.

  In the present invention, the means for determining the presence or absence of a genetic abnormality may be performed on a local server on which arbitrary software is installed, for example, on a cloud server via the Internet. There is no limitation.

  In the present invention, the means for outputting the result obtained by the means for determining the presence or absence of a gene abnormality may be, for example, a monitor that displays the result, or a printer that prints and outputs the result. Well, not limited.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

1. Insemination method In this example, an in vitro fertilization (IVF) or microinsemination (ICSI) egg (destructive egg) whose development was stopped or abnormal after in vitro fertilization and its culture solution were used. The procedure of insemination is shown below.

1-1. In vitro insemination (IVF)
Eggs collected from the eggs were fertilized using Universal IVF Medium (Origio, 10315060), and insemination was confirmed the next day. After insemination, the inseminated ovum was washed and transferred to 50 μL of a global egg culture medium (LifeGlobal), and cultured to blastocysts for 5 to 6 days. The culture was performed in an incubator (ESPEC) under gas phase conditions of 37 ° C., 6% CO ₂ , 5% O ₂ , 89% air, and saturated humidity. A 35 mm Petri dish (Falcon, 351008) was used for the culture.

1-2. Microinsemination (ICSI)
The collected egg was transferred to a fertilized egg culture medium global total (LifeGlobal), and one sperm was directly injected into the cytoplasm of the egg with a glass pipette using a micromanipulator. The culture was performed under the same conditions as IVF.

2. Sample Collection Procedure Cultured according to the above procedure, embryos whose development was stopped or abnormal (waste embryos) and culture supernatants in which they were cultured (50 μL total amount) were transferred to PCR tubes and stored frozen at −30 ° C.

3. Whole genome amplification procedure <Sample used>
-Waste embryo (Day 6 embryo)
・ Waste embryo culture supernatant

<Reagent used>
・ GenomePlex Single Cell Whole Genome Amplification Kit (Sigma Aldrich)

  The whole genome amplification procedure for each sample was performed according to the instructions attached to the kit. The procedure is briefly described below.

  Each sample was transferred to a new PCR tube. The pre-amp reaction solution was added to the PCR tube containing the sample. The PCR tube to which the pre-amp reaction solution was added was set in a thermal cycler, and a pre-amp reaction was performed. A Whole Genome Amplification (WGA) reaction solution was added to the PCR tube after the reaction. The PCR tube to which the WGA reaction solution was added was set in a thermal cycler, and WGA reaction was performed. Nucleic acid purification was performed using the entire reaction solution after the WGA reaction. Gel electrophoresis was performed using the solution after nucleic acid purification, and the WGA reaction was confirmed by a gel photograph after the electrophoresis. The amount of nucleic acid contained in the solution after nucleic acid purification was quantified. What obtained sufficient amount of nucleic acid by quantification was handled as a purified nucleic acid solution after WGA reaction.

4). Next generation sequencer (NGS) library preparation procedure <Reagents used>
-Ion Xpress (TM) Plus Fragment Library Kit (Thermo Fisher Scientific, USA)
Ion Xpress (TM) Barcode Adapters 1-16 Kit (Thermo Fisher Scientific, USA)

  An NGS library was prepared using the sample prepared by the whole genome amplification procedure described above. The NGS library was carried out according to the instructions attached to the kit. The procedure is briefly described below.

After the WGA reaction, a shearing reaction was performed using the purified nucleic acid solution. A reaction stop solution was added to the solution after the fragmentation reaction. After stopping the fragmentation reaction, the solution was purified with magnetic beads for purification. After the fragmentation reaction, an adapter ligation reaction was performed using the purified eluate. After the adapter ligation reaction, the solution was purified with magnetic beads for purification. After the adapter ligation reaction, the purified solution was subjected to agarose gel electrophoresis. An arbitrary size was selected and collected from the electrophoresis pattern and size marker after gel electrophoresis. A library amplification (Amplify) reaction is performed using the recovered solution after gel electrophoresis.
After the library Amplify reaction, the solution was purified with purification magnetic beads. After the library amplification reaction, the amount and quality of the nucleic acid in the purified solution were measured with an Agilent 2100 bioanalyzer (Agilent Technology Co., Ltd.). The solution after the measurement was hereinafter handled as a library prepared solution.

5. NGS running procedure <used kit>
・ Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific, USA)
・ Ion PGM Sequencing 200 Kit v2 (Thermo Fisher Scientific, USA)
・ Ion 316 Chip Kit v2 (Thermo Fisher Scientific, USA)

  An NGS run was performed using the sample obtained in the above NGS library preparation procedure. The NGS run was performed according to the instructions attached to the kit. The procedure is briefly described below.

  The Ion OneTouch ™ 2 Instrument device (Thermo Fisher Scientific, USA) was set up. The concentration of the NGS library prepared solution was prepared. Thereafter, emulsion PCR was performed.

  The Ion OneTouch ™ ES instrument (Thermo Fisher Scientific, USA) was set up. After emulsion PCR, the solution was concentrated and purified.

  The Ion PGM ™ Sequencer instrument (Thermo Fisher Scientific, USA) was set up. A running plan was created on a personal computer (PC) attached to the apparatus.

  The purified concentrated sample after emulsion PCR was loaded onto a dedicated chip. NGS runs were performed on an Ion PGM ™ Sequencer instrument.

  After the run was completed, data was taken out on a PC, and genome mapping was performed using the Ion PGM ™ system (Thermo Fisher Scientific, USA, model number: 4462921). Upload the obtained mapping data to the cloud analysis service “Ion Reporter” provided by Thermo Fisher Scientific, and use “Low-pass whole-genome availability”, which is one of the analysis menus. The aneuploidy was analyzed.

<Result>
DNA could be extracted and amplified from waste embryos (Day 6 embryos) 6 days after culture of embryos obtained by microinsemination (ICSI) and external insemination (IVF) and culture supernatants thereof. In particular, chromosomal aneuploidy analysis was performed by a next-generation sequencer using two samples (A and H (both ICSI)) in which amplification of DNA in the culture supernatant was confirmed. As a result, it was revealed that the chromosome aneuploidy in the discarded embryo and the culture supernatant almost coincided with each other (FIGS. 2 to 6). From this result, it was revealed that the DNA in the culture supernatant in which the DNA amplification was remarkable was almost identical to the discarded embryo.

  By using the present invention, it has become possible to easily detect chromosomal aneuploidy without giving any invasiveness to the embryo.

Claims (15)

A method for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation,
(I) a step of preparing a nucleic acid contained in a culture supernatant of a mammalian inseminated egg cultured in vitro;
(Ii) acquiring base sequence information of the nucleic acid using base sequence information acquisition means;
(Iii) analyzing the obtained base sequence information to determine the presence or absence of a gene abnormality,
Here, the culture supernatant is a culture supernatant obtained by culturing a fertilized egg in which no opening is provided in the zona pellucida of the fertilized egg after insemination.   The method of claim 1, wherein step (i) comprises amplifying nucleic acid by whole genome amplification means.   The method according to claim 1 or 2, wherein the base sequence information acquisition means is a next-generation sequencer or array CGH.   The culture supernatant is a culture supernatant collected from 1 to 7 days after insemination, or a culture supernatant collected from the pronuclear stage (2PN) to the blastocyst stage of a fertilized egg. The method of any one of 1-3.   5. The method according to claim 1, wherein the step of determining the presence or absence of a gene abnormality is a step of determining the presence or absence of a chromosomal abnormality and / or the step of determining the presence or absence of a gene mutation associated with a genetic disease. The method described.   The step of determining the presence or absence of a genetic abnormality is to determine the presence or absence of a chromosomal abnormality by detecting chromosomal aneuploidy, translocation, isochromosome, inversion, uniparental disomy, mosaic, deletion or ploidy abnormality And / or congenital metabolic disorders, primary immunodeficiency syndrome, congenital skin disease, hereditary tumor, congenital malformation syndrome, congenital blood disease, congenital neuromuscular disease, congenital bone system disease or mitochondrial disease The method according to any one of claims 1 to 4, which is a step of detecting a gene abnormality associated with phenotype and determining the presence or absence of a gene mutation associated with a genetic disease.   The method according to any one of claims 1 to 6, wherein the mammal is a human.   The step of determining the gene abnormality includes Turner syndrome, Patou syndrome, Edwards syndrome, Down syndrome, Klinefelter syndrome, Tripo-X syndrome, XYY syndrome, 5p monosomy (5p-syndrome), 5q monosomy (5q- Syndrome) and 4p monosomy Wolf Hirschhorn syndrome, phenylketonuria, biopterin dysbolism, maple syrup urine disease, homocystinuria, xeroderma pigmentosum, companion agammaglobulinemia, adenosine deaminase deficiency , Congenital epidermolysis bullosa, ichthyosis vulgaris, neurofibromatosis type 1, neurofibromatosis type 2, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, Angelman syndrome, Williams syndrome, Prader-Willi syndrome, Icardi syndrome, Cornelia de Lan Syndrome, Apert syndrome, hemophilia, muscular dystrophy, osteogenesis imperfecta, achondroplasia, achondroplasia, hypocartilaginous dysplasia, fatal osteodysplasia, chronic progressive extraocular palsy syndrome, Kearns-Sayer syndrome Determining the presence or absence of one or more diseases selected from the group consisting of: red rag fiber / myoclonic epilepsy syndrome, mitochondrial encephalomyopathy / lactic acidosis / stroke-like syndrome, Lee encephalopathy, Leber's disease, mitochondrial diabetes and Pearson's disease 8. The method of claim 7, wherein: A system for determining the presence or absence of a genetic abnormality in a fertilized egg before implantation,
Means for preparing a nucleic acid contained in the culture supernatant of a mammalian fertilized egg;
Base sequence information acquisition means for acquiring base sequence information of the nucleic acid prepared by the preparation means;
Means for analyzing the base sequence information obtained by the base sequence information acquisition means to determine the presence or absence of a gene abnormality;
Means for outputting a result obtained by the means for determining;
Including
Here, the culture supernatant is a culture supernatant obtained by culturing a fertilized egg in which an opening is not provided in the zona pellucida of the fertilized egg after insemination.   The system according to claim 9, wherein the nucleic acid preparation means includes whole genome amplification means.   The system according to claim 9 or 10, wherein the base sequence information acquisition means is a next-generation sequencer or array CGH.   The means for determining the presence or absence of the genetic abnormality is a means for determining the presence or absence of a chromosomal abnormality and / or a means for determining the presence or absence of a gene mutation associated with a genetic disease. The described system.   The means for determining the presence or absence of a genetic abnormality is to determine the presence or absence of a chromosomal abnormality by detecting chromosomal aneuploidy, translocation, isochromosome, inversion, uniparental disomy, mosaic, deletion or ploidy abnormality And / or congenital metabolic disorders, primary immunodeficiency syndrome, congenital skin disease, hereditary tumor, congenital malformation syndrome, congenital blood disease, congenital neuromuscular disease, congenital bone system disease or mitochondrial disease The system according to any one of claims 9 to 11, which is a means for detecting a gene abnormality related to phenotype and determining the presence or absence of a gene mutation related to a genetic disease.   The system according to any one of claims 9 to 13, wherein the mammal is a human.   Means for determining the presence or absence of the gene abnormality are Turner syndrome, Patou syndrome, Edwards syndrome, Down syndrome, Klinefelter syndrome, Tripo-X syndrome, XYY syndrome, 5p monosomy (5p-syndrome), 5q monosomy ( 5q-syndrome) and 4p monosomy Wolf Hirschhorn syndrome, phenylketonuria, biopterin dysbolism, maple syrup urine disease, homocystinuria, xeroderma pigmentosum, sexual agammaglobulinemia, adenosine deaminase Deficiency, congenital epidermolysis bullosa, ichthyosis vulgaris, neurofibromatosis type 1, neurofibromatosis type 2, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, Angelman syndrome, Williams syndrome, Prader Willy Syndrome, Aicardy syndrome, Cornelia de Lange syndrome, Apert syndrome, hemophilia, muscular dystrophy, osteogenesis imperfecta, achondroplasia, achondroplasia, hypochondral dysplasia, fatal osteodysplasia, chronic progressive extraocular paralysis syndrome, Kearns Sayre A means for determining the presence or absence of one or more diseases selected from the group consisting of: Syndrome, red rag fiber / myoclonic epilepsy syndrome, mitochondrial encephalomyopathy / lactic acidosis / stroke-like syndrome, Lee encephalopathy, Leber's disease, mitochondrial diabetes and Pearson's disease 15. The system of claim 14, wherein