KR101777911B1 - Biomarker for predicting of osteoporotic fracture risk - Google Patents

Biomarker for predicting of osteoporotic fracture risk Download PDF

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KR101777911B1
KR101777911B1 KR1020150078985A KR20150078985A KR101777911B1 KR 101777911 B1 KR101777911 B1 KR 101777911B1 KR 1020150078985 A KR1020150078985 A KR 1020150078985A KR 20150078985 A KR20150078985 A KR 20150078985A KR 101777911 B1 KR101777911 B1 KR 101777911B1
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base
human chromosome
polynucleotide
polymorphic site
nucleotide sequences
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KR20160143008A (en
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고정민
김신윤
이승훈
김범준
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울산대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The present invention relates to a biomarker, a single nucleotide polymorphism marker capable of predicting the risk of osteoporosis fracture, a method for predicting the occurrence of osteoporosis fracture using the marker, a method for providing information for predicting the occurrence of osteoporosis fracture using the marker And a probe capable of detecting the marker, and a composition for predicting osteoporosis fracture occurrence. According to the present invention, it is possible to effectively predict the risk of osteoporotic fracture of an individual, to diagnose and prevent osteoporotic fractures at an early stage, to prevent omission of excessive treatment or treatment due to inaccuracy of prediction of conventional osteoporotic fracture diagnosis method, An effective treatment method can be suggested.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for predicting osteoporotic fracture,

The present invention relates to a biomarker, a single nucleotide polymorphism marker capable of predicting the risk of osteoporosis fracture, a method for predicting the occurrence of osteoporosis fracture using the marker, a method for providing information for predicting the occurrence of osteoporosis fracture using the marker And a probe capable of detecting the marker, and a composition for predicting osteoporosis fracture occurrence.

Osteoporosis is a disease characterized by impaired bone strength, increasing the risk of osteoporotic fracture (OF).

Treatment of osteoporosis aims to prevent the occurrence of osteoporosis fractures, so it is important to find and treat patients who are at high risk for osteoporosis fractures in the management of osteoporosis. Currently, most of the diagnosis of osteoporosis is made by measuring bone mineral density (BMD). However, most patients with osteoporotic fractures are found to be not osteoporotic according to the BMD standard. Therefore, clinical risk factors (CRFs) themselves or the fracture risk assessment tool (FRAX® http: //www.shef. ), Including BMD data, may be used to increase the predictability of the risk of osteoporotic fracture . ac.uk/FRAX/ ) have been developed. Clinical treatment guidelines suggested that FRAX® could be used as a basis for determining whether to treat osteoporosis. However, FRAX® also has insufficient predictive accuracy and can lead to over treatment and unnecessary treatment. Therefore, there is a need for improved methods for predicting the occurrence of osteoporotic fractures.

Osteoporosis prevention, diagnosis, and therapy (JAMA 2001; 285 (6): 785-795)

It is an object of the present invention to provide a single base polymorphism marker that can effectively predict the risk of osteoporotic fracture.

The present invention provides information for predicting the occurrence of osteoporotic fracture of an individual comprising identifying a base of a polymorphic site of one or more single base polymorphic markers selected from the following single base polymorphic markers from a nucleic acid sample isolated from an individual The method provides:

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 113,051,074 base, wherein the 113,051,074 base of human chromosome 1 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,316,673 base, wherein the 40,316,673th base of human chromosome 11 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 29,070,533 base, wherein the 29,070,533 base of the human chromosome 13 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the above 42,845,500 base, wherein the 42,845,500 base of human chromosome 13 is T or C, or a polynucleotide complementary thereto; And

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 127,438,642 base, wherein the 127,438,642 base of the human chromosome 6 is C or A, or a polynucleotide complementary thereto.

The present invention also provides a composition for predicting osteoporotic fracture development comprising a probe capable of detecting at least one single base polymorphism marker selected from the single base polymorphism marker or a primer capable of amplifying the single base polymorphism marker. A kit for predicting osteoporotic fracture occurrence is provided.

According to the present invention, it is possible to effectively predict the risk of osteoporotic fracture of an individual, thereby to diagnose and prevent an osteoporotic fracture at an early stage, to reduce an excess treatment or omission due to the inaccuracy of a conventional osteoporotic fracture diagnosis method, And the like.

Figure 1 shows the process of identifying and identifying single base polymorphic markers of the present invention.
Figure 2 shows the distribution of genetic risk score-common variants (GRS-C) and genetic risk score-total variants (GRS-T) due to common mutations.

Glossary of Terms

Common mutations: Variants with minor allele frequency (MAF) = 1%

Clinical risk factors (CRFs): age, height, weight, current smoking status, alcohol consumption, and family history of fractures

Genetic risk scores-common variants (GRS-C): Genetic risk indexes due to common mutations

 Genetic risk scores-total variants (GRS-T): Genetic risk indexes due to common mutations and rare mutations

Functional variants: nsSNPs (non-synonymous single) predicted to be "benign", "possibly damaging" or "probably damaging" by sequence- and structure- nucleotide polymorphisms, or regulatory single nucleotide polymorphisms (rSNPs) within known transcription factor binding sites.

Rare mutations: Variants with a minority allele frequency <1%

Potentially harmful nsSNPs: nsSNPs designated as "possibly damaging" or "possibly damaging" by sequence- and structure-based prediction tools.

Acronym

BMD, bone mineral density; CMC method, combined multivariate and collapsing (CMC) method; CRFs, clinical risk factors; FRAX, fracture risk assessment tool; FN, femoral neck; GRS-C, genetic risk scores-common variants (GRS-T), genetic risk scores-total variants (GWASs), genome-wide association studies ; KBAC, kernel-based adaptive cluster methods; LS, lumbar spine; MAF, minor allele frequency; NRI index, net reclassification improvement index; nsSNPs, non-synonymous single nucleotide polymorphisms, NVF, non-vertebral fractures, OF, osteoporotic fracture, rSNPs, regulatory single nucleotide polymorphisms, SD, standard deviations, SKAT-O, test, type 1 diabetes mellitus, T2DM, type 2 diabetes mellitus, VF, vertebral fracture, WHO, World Health Organization, WSS method , weighted-sum statistics method

Common mutations in osteoporosis (minor allele frequency (MAF) ≥ 0) through genome-wide association studies (GWASs) to identify single nucleotide polymorphic markers that can predict osteoporotic fractures 1%] have been excavated. However, combining all of the 56 BMD loci identified by the large-scale GWAS meta-analysis showed that only 5.8% of the BMD changes were accounted for. This means that the mutations identified by GWAS do not fully account for the inheritance of osteoporosis and OF. Several hypotheses have been proposed to explain this "missing heritability". The first hypothesis is that non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region, which can alter the sequence of amino acids known as functional mutations, or rSNPs (regulated SNPs) within the dock region, It is likely that this is not a common variation. The second hypothesis is that it is difficult to identify rare mutations known to have a strong impact on disease outbreaks compared with common mutations by current genotyping methods.

Based on this hypothesis, the present inventors confirmed that common functional mutations affecting osteoporosis fracture occurrence and rare functional mutations can all affect osteoporotic fracture occurrence, thus completing the present invention. As a result, a total of 19 common functional mutations and 31 rare functional mutations were observed with single nucleotide polymorphism markers associated with osteoporotic fractures.

Thus, the present invention provides the use of a single nucleotide polymorphic marker that can predict the occurrence of osteoporotic fractures.

The present invention relates to a marker for predicting osteoporotic fracture occurrence comprising at least one single base polymorphism marker selected from single base polymorphism markers capable of predicting the occurrence of osteoporotic fracture; A method of predicting osteoporotic fracture occurrence using the marker; A method of providing information for predicting the occurrence of osteoporotic fracture using the marker; And a composition capable of detecting the marker or a primer capable of amplifying the marker, wherein the composition for predicting the occurrence of osteoporosis is provided.

Specifically, the present invention relates to a method for diagnosing osteoporotic fracture of an individual comprising identifying a base of a polymorphic site of one or more single base polymorphic markers selected from single base polymorphic markers capable of predicting the occurrence of osteoporotic fracture, Provides a method of providing information to predict occurrence.

The term &quot; polymorphism &quot; refers to the coexistence of one or more forms in a nucleic acid, including exons and introns, or a portion thereof. "Single nucleotide polymorphism" or "SNP" refers to the case where two or more single bases are present at a specific position in a gene. Most SNPs have two alleles. For one allele of the polymorphism, having two identical alleles means homozygosity (having the same base at the SNP position) for alleles and heterozygosity (SNPs for alleles) having two different alleles Lt; / RTI &gt; position). Preferred SNPs have two or more alleles that exhibit an incidence of 1% or more, more preferably 10% or 20% or more, in the selected population. In the present invention, the SNP name is denoted by rs ID. Rs ID means the official SNP (rs) ID assigned to each unique SNP in the National Center for Biotechnological Information (NCBI) .

The term &quot; allele &quot; refers to an alternative form of a gene comprising introns, exons, intron / exon junctions and 3 ' and / or 5 ' Generally, an allele refers to several types of genes that are located on the same gene locus on a homologous chromosome. Alleles of a particular gene may differ from each other by a single nucleotide or several nucleotides and may include substitution, deletion and insertion of nucleotides.

The term "marker", "polymorphic marker" or "single nucleotide polymorphic marker" refers to a genomic polymorphic site. Each polymorphic marker has two or more sequence variations characteristic of a particular allele at the polymorphic site.

The term &quot; individual &quot; refers to a subject who predicts osteoporosis fracture occurrence, and &quot; nucleic acid sample &quot; refers to a sample containing nucleic acid (DNA or RNA) obtained from an individual. The nucleic acid sample can be obtained from blood, urine, hair, amniotic fluid, cerebrospinal fluid collected from an individual; Or skin, muscles, oral mucosa or conjunctival mucosa; From a source including nucleic acids, such as tissue samples from the placenta, gastrointestinal tract or other organ, and the like. Methods for obtaining nucleic acid samples from an individual are well known in the art, and known methods can be used without limitation.

From a nucleic acid sample isolated from an individual, a polymorphic site of a single base polymorphism marker can be amplified or hybridized with a probe capable of detecting a single base polymorphism marker to identify a polymorphic site base.

For example, polymorphic sites can be amplified using PCR, ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, and nucleic acid-based sequence amplification (NASBA).

Sequence analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, SSCP , PCR-RFLP analysis, TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g., Sequenom's Mass ARRAY system), mini-sequencing method, Bio-Plex system (BioRad) SNP stream system (Beckman), Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadChip (Illumina's HumanOmni 2.5-8). For example, SNP chips can be used to identify bases at polymorphic sites. A SNP chip is one of a DNA microarray that can identify each base of hundreds of thousands of SNPs at a time.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) performing PCR using the DNA as a template and using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotides of step (1) from the analysis results.

Sequencing analysis can be performed using conventional methods for sequencing and can be performed using an automated gene analyzer. The allele-specific PCR means a PCR method in which a DNA fragment in which the SNP is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the SNP is located. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, In the case of performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, 3 'terminus does not perform complementary binding so that the amplification reaction can not be performed properly. DASH can be performed by a conventional method, preferably by a method such as Prince et al.

PCR extension analysis is performed by first amplifying a DNA fragment containing a base in which a single base polymorphism is located with a pair of primers and then inactivating all the nucleotides added to the reaction by dephosphorylation and adding SNP specific extension primer, dNTP mixture , Digoxinucleotide, reaction buffer, and DNA polymerase to perform primer extension reaction. At this time, the extension primer has a base immediately adjacent to the 5 'direction of the base in which the SNP is located as a 3' terminus, and a nucleic acid having the same base as the didyoxynucleotide is excluded in the dNTP mixture, and the didyoxynucleotide indicates a SNP Base type. For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base representing the SNP by comparing the lengths of the extended primers.

At this time, as a detection method, when the extension primer or the dideoxy nucleotide is fluorescence-labeled, the SNP is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 of ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the SNP can be detected by measuring the molecular weight using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique.

Examples of single nucleotide polymorphic markers for predicting osteoporotic fracture occurrence of the present invention are as follows.

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 113,051,074 base, wherein the 113,051,074 base of human chromosome 1 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,316,673 base, wherein the 40,316,673th base of human chromosome 11 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 29,070,533 base, wherein the 29,070,533 base of the human chromosome 13 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the above 42,845,500 base, wherein the 42,845,500 base of human chromosome 13 is T or C, or a polynucleotide complementary thereto; And

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 127,438,642 base, wherein the 127,438,642 base of the human chromosome 6 is C or A, or a polynucleotide complementary thereto.

The single nucleotide polymorphism markers correspond to common functional mutations that can predict the occurrence of osteoporotic fracture, as can be seen in the following examples.

In one embodiment, the single nucleotide polymorphism marker may be one or a combination of at least two of the markers, or both markers may be used in combination. As the number of combined markers increases, the accuracy of predicting the occurrence of osteoporosis increases. For example, two or more, three or more of the single nucleotide polymorphism markers may be used in combination.

In the above, when the base of the polymorphic site of the polymorphism of the single nucleotide polymorphism marker is as follows, the base is an indicator of possibility of osteoporotic fracture occurrence, that is, a possibility of osteoporotic fracture occurrence is higher than other bases.

The 113,051,074th base of human chromosome 1 is A;

The 40,316,673th base of human chromosome 11 is G;

29,070,533 base of human chromosome 13 is C;

The 42,845,500th base of human chromosome 13 is T; or

The 127,438,642 th base of the human chromosome 6 is C.I.

According to one embodiment of the present invention, it may further comprise identifying a base at a polymorphic site of one or more single base polymorphic markers selected from the following single base polymorphic markers.

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 68,367,943 base, wherein the 68,367,943 base of human chromosome 11 is G or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 2,185,956 base, wherein the 2,185,956 base of human chromosome 17 is T or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 65,885,357 base, wherein the 65,885,357th base of the human chromosome 1 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising 88,732,874 bases, wherein 88,732,874 base of human chromosome 4 is A or G, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the 52,082,846th base, wherein the 52,082,846th base of the human chromosome 5 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 29,322,363 base, wherein the 29,322,363th base of the human chromosome 3 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the 38th, 217th, 555th bases, wherein the 38th, 217th, 555th bases of the human chromosome 7 are A or G, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 119,965,024 base, wherein the 119,965,024 base of human chromosome 8 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 85,006,615 base, wherein the 85,006,615th base of human chromosome 3 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 120,969,769 base, wherein the 120,969,769th base of human chromosome 7 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 29th, 322nd, 297th bases, wherein the 29th, 322nd, 297th bases of the human chromosome 3 are C or A, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 28,126,245 base, wherein the 28,126,245th base of human chromosome 12 is C or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 120,968,674 base, wherein the 120,968,674 base of human chromosome 7 is A or G, or a complementary polynucleotide thereof; And

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,166,854 base, wherein the 27,166,854 base of the human chromosome 7 is A or G, or a polynucleotide complementary thereto.

The single nucleotide polymorphism markers correspond to common functional mutations that can predict the occurrence of osteoporotic fracture, as can be seen in the following examples.

In one embodiment, the single nucleotide polymorphism marker may be one or a combination of at least two of the markers, or both markers may be used in combination. As the number of combined markers increases, the accuracy of predicting the occurrence of osteoporosis increases. For example, two or more, three or more, five or more, seven or more, ten or more, or fourteen of the single nucleotide polymorphism markers may be used in combination.

In the above, when the base of the polymorphic site of the single nucleotide polymorphism marker is as follows, the base is an indicator of possibility of osteoporotic fracture, that is, an osteoporotic fracture is more likely to occur than other bases.

The 68,367,943th base of human chromosome 11 is G;

The 2,185,956th base of human chromosome 17 is T;

65,885,357 base of human chromosome 1 is A;

88,732,874 base of human chromosome 4 is A;

The 52,082,846th base of human chromosome 5 is T;

The 29,322,363th base of human chromosome 3 is T;

38, 217, 555 base of human chromosome 7 is A;

119,965,024 base of human chromosome 8 is T;

The 85,006,615th base of human chromosome 3 is C;

The 120,969,769th base of human chromosome 7 is G;

29, 322, 297 base of human chromosome 3 is C;

The 28,126,245th base of human chromosome 12 is C;

120,968,674 base of human chromosome 7 is A; or

The 27,166,854 th base of human chromosome 7 is A.

According to another embodiment of the present invention, it may further comprise identifying a base in a polymorphic site of one or more single base polymorphic markers selected from the following single base polymorphic markers.

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,131 base, wherein the 43,292,131 base of the human chromosome 10 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,146 base, wherein the 43,292,146th base of the human chromosome 10 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,337 base, wherein the 43,292,337th base of human chromosome 10 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,394 base, wherein the 43,292,394 base of human chromosome 10 is G or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,584 base, wherein the 43,292,584th base of the human chromosome 10 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,297,622 base, wherein the 43,297,622th base of human chromosome 10 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 27,147,763 base, wherein the 27,147,763th base of human chromosome 7 is G or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising 27, 166, 747 bases, wherein 27, 166, 747th bases of the human chromosome 7 are C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,169,034 base, wherein the 27,169,034 base of the human chromosome 7 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,183,762 base sequence, wherein the 27,183,762th base of human chromosome 7 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,196,304 base, wherein the 27,196,304 base of human chromosome 7 is G or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,273,202 base, wherein the 42,273,202 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,281,233 base, wherein the 42,281,233 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,281,312 base, wherein the 42,281,312 base of the human chromosome 2 is T or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42, 282, 136 base, wherein the 42,282,136th base of the human chromosome 2 is G or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,282,148 base, wherein the 42,282,148 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42,284,361 base, wherein the 42,284,361 base of human chromosome 2 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42,284,769 base, wherein 42,284,769 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 46,897,404 base, wherein the 46,897,404th base of human chromosome 11 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,898,044 base, wherein the 46,898,044th base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,900,719 base, wherein the 46,900,719 base of the human chromosome 11 is C or T, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,905,425 base, wherein the 46,905,425 base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,908,042 base, wherein the 46,908,042th base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,917,846 base, wherein the 46,917,846th base of the human chromosome 11 is T or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,920,476 base, wherein the 46,920,476th base of human chromosome 11 is G or A, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,660,881 base, wherein the 40,660,881 base of human chromosome 22 is T or C, or a complementary polynucleotide thereof;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,006 base, wherein the 40,661,006th base of human chromosome 22 is A or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,216 base, wherein the 40,661,216th base of human chromosome 22 is T or G, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,867th base, wherein the 40,661,867th base of human chromosome 22 is T or C, or a polynucleotide complementary thereto;

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,939 base, wherein the 40,661,939 base of the human chromosome 22 is C or G, or a polynucleotide complementary thereto; And

A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,662,533 base, wherein the 40,662,533 base of human chromosome 22 is C or A, or a polynucleotide complementary thereto.

The single nucleotide polymorphism markers correspond to rare functional mutations that can predict the occurrence of osteoporotic fracture, as can be seen in the following examples.

In one embodiment, the single nucleotide polymorphism marker may be one or a combination of at least two of the markers, or both markers may be used in combination. As the number of combined markers increases, the accuracy of predicting the occurrence of osteoporosis increases. For example, the single nucleotide polymorphism marker may be a polynucleotide comprising at least two, at least three, at least five, at least seven, at least ten, at least thirteen, at least fifteen, at least seventeen, at least twenty, at least twenty- 25 or more, 27 or more, or 31 may be used in combination.

In the above, when the base of the polymorphic site of the single nucleotide polymorphism marker is as follows, the base is an indicator of possibility of osteoporotic fracture, that is, an osteoporotic fracture is more likely to occur than other bases.

43,292,131 base of human chromosome 10 is C;

The 43,292,146th base of human chromosome 10 is A;

The 43,292,337 base of human chromosome 10 is A;

The 43,292,394th base of human chromosome 10 is G;

43,292,584 base of human chromosome 10 is T;

The 43,297,622th base of human chromosome 10 is G;

The 27,147,763th base of human chromosome 7 is G;

The 27,166,747th base of human chromosome 7 is C;

27,169,034 base of human chromosome 7 is C;

The 27,183,762th base of human chromosome 7 is T;

The 27,196,304th base of human chromosome 7 is G;

42,273,202 base of human chromosome 2 is A;

The 42,281,233 base of human chromosome 2 is A;

42,281,312 base of human chromosome 2 is T;

The 42,282,136th base of human chromosome 2 is G;

The 42,282,148th base of human chromosome 2 is A;

42,284,361 base of human chromosome 2 is G;

42,284,769 base of human chromosome 2 is A;

The 46,897,404th base of human chromosome 11 is A;

46,898,044 base of human chromosome 11 is T;

46,900,719 base of human chromosome 11 is C;

The 46,905,425th base of human chromosome 11 is T;

The 46,908,042th base of human chromosome 11 is T;

The 46,917,846th base of human chromosome 11 is T;

46,920,476 base of human chromosome 11 is G;

The 40,660,881 base of human chromosome 22 is T;

The 40,661,006th base of human chromosome 22 is A;

The 40,661,216th base of human chromosome 22 is T;

The 40,661,867th base of human chromosome 22 is T;

The 40,661,939 base of human chromosome 22 is C; or

The 40,662,533 base of the human chromosome 22 is C.I.

According to one embodiment of the present invention, in order to provide information for predicting the occurrence of osteoporotic fracture, clinical risk factors and / or bone mineral density can be additionally identified in addition to identifying bases in the polymorphic site of the single nucleotide polymorphism marker have. Clinical risk factors associated with osteoporotic fractures include, but are not limited to, family history of age, height, weight, smoking, alcohol intake, and family history of fracture. As can be seen in the following examples, the accuracy of prediction of the occurrence of osteoporotic fracture can be improved by confirming the clinical risk factors and / or the bone density together.

The present invention also provides a composition for predicting osteoporotic fracture development comprising a probe capable of detecting at least one single base polymorphism marker selected from the single base polymorphism marker or a primer capable of amplifying the single base polymorphism marker. A kit for predicting osteoporotic fracture occurrence is provided. All single nucleotide polymorphic markers described above can be used in kits.

The term &quot; probe &quot; refers to a substance capable of specifically hybridizing with a polymorphic site of a gene to predict occurrence of osteoporosis fracture. Specific methods of such gene analysis are not particularly limited, and those known in the art It may be by all gene detection methods.

The term 'primer' refers to a substance capable of specifically amplifying a polynucleotide of a single-nucleotide polymorphism marker. The term 'primer' refers to a nucleotide sequence having a short free 3 'hydroxyl group and a template complementary to a template base pair and function as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents for the polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at the appropriate buffer solution and temperature. PCR amplification can be used to predict the onset of osteoporotic fracture through the production of desired products. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. The appropriate length of the primer may vary depending on the purpose of use, and may be, for example, 15 to 30 nucleotides. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.

Probes or primers can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, the kit can be used without limitation as long as it can detect or identify a single base polymorphism marker. For example, the kit may be a DNA chip kit or a real time-polymerase chain reaction (RT-PCR) kit. The RT-PCR kits can be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerases and the like, in addition to the respective primer pairs specific for the polymorphic marker gene Enzymes such as reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control. Polymorphic markers can be identified by measuring mRNA expression levels of single base polymorphic markers using RT-PCR kits. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface and the complementary nucleic acid contained in the solution treated on the surface of the chip to enable a mass parallel analysis.

The kit of the present invention also includes a microarray. The microarray may be one comprising DNA or RNA polynucleotides. The microarray may be composed of a conventional microarray except that the polynucleotide of the polymorphic marker of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms.

Microarrays can be used with any known microarray without limitation, and methods for manufacturing microarrays are well known in the art. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are also well known in the art. For example, a nucleic acid sample may be labeled with a labeling substance capable of generating a detectable signal such as a fluorescent substance, followed by hybridization on a microarray, and detection of a hybridization result by detecting a signal generated from the labeling substance.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention. These embodiments are provided so that the disclosure of the present invention is not limited thereto and that those skilled in the art will fully understand the scope of the present invention and that the present invention is not limited by the scope of the claims Only.

Research group

The study subjects were three cohorts (Asan, Catholic, and Kangwon-Medical Centers) in Korea, including Asan Medical Center (AMC), Catholic Medical Center (CMC), and Kangwon Medical Center ; ACK-MC). All studies were approved by the ethics committee of each institution and received written consent from all participants. The AMC cohort included 2,922 Korean postmenopausal women who visited Asan Medical Center in Seoul. The CMC cohort included 1,206 postmenopausal women in the Chungju area. The KMC cohort consisted of 749 postmenopausal women who visited Kangwon National University Hospital. Menopause was defined as no menstruation for at least one year and was confirmed by serum follicle-stimulating hormone levels. The questionnaire was used to obtain information such as smoking history (current smoking status), alcohol consumption (≥3 units / day), medication being used, previous medical or surgical disease, family history of birth, fracture, and history of fracture. (Eg, glucocorticoids, sex hormones, bisphosphonates, or other osteoporosis agents) that are capable of affecting bone metabolism or premature menopausal women (<40 years of age), or taking these medicines within 12 months I have excluded anyone who has ever been. In the case of glucocorticoids, people who took more than one month were also excluded. We also excluded those who had experienced diseases that might affect bone metabolism such as T1DM, cancer, hyperparathyroidism, thyroid disease, or rheumatoid arthritis. Women with stroke or dementia were also excluded because of concerns about physical activity limitations. We also excluded women with severe degenerative changes in the lumbar spine (LS) with or without spondylolisthesis of the Nathan classification system at four or more steps and confirmed by conventional vertebral radiography.

At the excavation stage, targeted re-sequencing was performed on subjects with a bipolar phenotype from the AMC cohort. The subjects were divided into two groups using a femoral neck (FN) BMD value and a body mass index (BMI) to match the target for re-sequencing. As the BMI is known to have a high bone density, the BMI was corrected to amplify the genetic influence. As a result, a relatively low BMI (Interquartile range; IQR: 20.9-24.4 (IQR: 0.710-0.840 g / cm 2 ) were included in the FN BMD of the subjects who matched the age, even though the subjects had an average age of 6 years (kg / m 2 ). Even though the very low BMD group (n = 505) had a relatively high BMI (IQR: 21.5-25.1 kg / m 2 ), the FN BMD of the age matched subjects was lower than 10% (IQR: 0.594-0.723 g / cm &lt; 2 &gt;). After DNA extraction, quality monitoring, and concentration measurements, 501 control and 481 very low BMD groups were selected for targeted re-sequencing. Verification sequencing was performed on the remaining 3,895 subjects of the ACK-MC cohort.

DNA extraction, quality check and concentration measurement

The A260 / A280 ratio was measured to confirm that 1.8-2.0 was maintained, and the stability was confirmed by agarose gel electrophoresis. The concentration of gDNA was measured using Quant-iT PicoGreen dsDNA Assay kit (Invitrogen, Carlsbad, CA, USA).

LS  And FN At the site BMD  Measurement and fracture evaluation

BMD (g / cm 2 ) was measured by dual-energy X-ray absorptiometry. Lunar machine (Lunar, Expert XL, Madison, WI) was used in 1,138 women in the AMC cohort and 3,717 women in the remaining ACK- Were measured at the LS (L1-L4) and FN sites using a mechanical (Hologic, QDR 4500-A, Waltham, MA) According to the definition of WHO (World Health Organization), osteoporosis was defined as T-score ≤ -2.5 SD (standard deviation), and osteopenia was defined as -1.0 and -2.5 SD.

The cross-correction equation between the two measuring machines used the results of BMD measurements of 109 healthy Korean women (mean age 55 ± 11 years; range, 31-75 years) using two machines, and the formula was .

LS BMD (g / cm 2 ): Lunar = 1.1287 X Hologic - 0.0027

FN BMD (g / cm 2 ): Lunar = 1.1556 X Hologic - 0.0182

The variation coefficients representing the precision were 0.82% at the LS site and 1.10% at the FN site of the Lunar machine of AMC. The coefficient of variation of the Hologic machine was 0.85% and 1.08% for AMC, 1.36% and 1.42% for CMC, and 1.36% and 1.50% for KMC, respectively, for LS and FN, respectively. These figures were obtained by measuring the number of 17-25 applicants who did not participate in the study five times on the same day.

All subjects underwent lateral thoracolumbar spine radiographs to determine the presence of a vertebral fracture (VF). The VF assessment was based on the recommendations of the Working Group on Vertebral Fractures. The quantitative definition of VF was defined as a reduction of> 20% of the spine height (ie, front, center, or back). The questionnaires were used to evaluate non-vertebral fractures (NVFs), ie, fractures of the wrist, femur, lower arm, humerus, ribs, and pelvis. Except for severe trauma such as an automobile accident, or fracture caused by falling above the standing height, was excluded. Therefore, the fracture included only osteoporotic fractures that were confirmed to be caused by menopause or minor trauma after age 50 through questionnaires or counseling.

target  Re-sequencing resequencing )

198 genes for targeted re-sequencing (Table 1) were selected through literature. The target site included the exon encoding the protein, the exon-intron border, and the mammal-conserved control (10 kb upstream of the transcription start site). The total target site size of 198 genes for targeted re-sequencing was 1,751,078 bp. The Bait libraries were designed using the Agilent eArray website (https://earray.chem.agilent.com/earray/) and assessed for target site coverage. The online design process recommended that the target sequence be repeatedly masked to minimize off-target capture. The resulting bait was investigated in the human genome reference (hg19). If the bait mapped at more than one position has more than 90% sequence homology using BLAST (~ 12 mismatches across the bait), the bait was excluded from the design. Finally, 1,248,160 bp (71.3% of the target site) was captured by the Agilent SureSelect Sequence Enrichment Kit and sequenced using the Illumina HiSeq2000 analyzer (Illumina, San Diego, CA, USA). A random shear force was applied to genomic DNA (3 μg) using the Covaris System to generate an insert of approximately 150 bp. The ends of the fragmented DNA were end-repaired using T4 DNA polymerase and Klenow polymerase, and Illumina paired-end adapter oligonucleotides were ligated to the sticky ends. The ligation mixture was analyzed by agarose gel electrophoresis and a fragment of 200-250 bp was isolated. The purified DNA library product was hybridized with the SureSelect target Enrichment probes set (Agilent, Santa Clara, Calif., USA) according to the manufacturer's instructions to capture the target site. The HiSeq2000 paired-end flowcell was constructed using the captured library according to the manufacturer's protocol. Then clusters of PCR colonies were sequenced using the HiSeq2000 platform.

In order to select only qualitatively superior variations, the following four quality filters were applied. First, we excluded mutations that included only those with a call rate of ≥90% and those with significant deviations (P <1X10 -4 ) in the Hardy-Weinberg equilibrium test. Second, only sites with a minimum depth of 80 per target area were included. Third, allele frequencies were matched in forward and reverse strands. Finally, SNPs clustered within 5 bp around the SNP were excluded. "Functional rare variants" (rSNPs) that have the potential to alter amino acids in the exon (nsSNPs) or affect the regulation of gene expression by applying two functional filters have been selected. First, "benign", "possibly damaging", or "possibly" from the sequence- and structure-based prediction program using Polymorphism Phenotyping (PolyPhen-2) version 2 software (v2.2.2) Rare "nsSNP mutations that appear to be" likely damaging ". Second, a rare rSNP mutation in a known transcription factor binding site was selected using UCSC (Genome Browser database, http://genome.ucsc.edu/).

Figure 112015053900872-pat00001

In the above table, N represents the number of genes. Group I is a gene in which the researcher observes association with osteoporosis in Koreans; Group II has been associated with osteoporosis in Europe and genes identified in Asians; Group III is a gene that is associated with BMD and fracture in Asian (IIIA) or European (IIIB) genome wide association studies (GWAS). Group IV is a gene that has been associated with BMD in Asians (IVA) and Europeans (IVB) through GWAS; Group V refers to genes that have been associated with BMD in Asian (VA) and European (VB) through large-scale candidate gene association studies (CGAS).

Verification genotype analysis genotyping )

Genotype analysis was performed using the SNPtype TM assay (Fluidigm, San Francisco, CA, USA) according to the manufacturer's recommendations. In summary, 75 ng genomic DNA was used for PCR amplification of sites containing SNPs of interest using a STA primer set and a Qiagen 2 × Mutiplex PCR Master Mix (Qiagen, Valencia, CA, USA) at a reaction volume of 5 μL . The PCR conditions were as follows; After 1 cycle at 95 ° C for 15 minutes, 14 cycles at 95 ° C for 15 seconds and 60 ° C for 4 minutes. After amplification, the product was diluted 1: 100 in DNA suspension buffer. The diluted STA product (2.5 μL) was mixed with 3 μL 2 × Fast Probe Master Mix, 0.3 μL SNPtypeTM 20 × Sample Loading Reagent, 0.1 μL SNPtypeTM reagent, and 0.036 μL 50 × ROX ™ reference dye Invitrogen, Carlsbad, Calif., USA). After loading the Assay Pre-Mix and Sample Pre-Mix 9Sample Pre-Mix on a 96.96 Dynamic Array, the SNPtypeTM assay reaction was performed as follows: 95 ° C for 5 min, 95 ° C 15 seconds at 64 ° C, 15 seconds at 72 ° C, 15 seconds at 95 ° C, 45 seconds at 63 ° C, 15 seconds at 72 ° C, 15 seconds at 95 ° C, 45 seconds at 95 ° C 1 cycle at 72 ° C for 15 seconds, 95 ° C for 15 seconds, 61 ° C for 45 seconds, and 72 ° C for 15 seconds, followed by 15 seconds at 95 ° C, 45 seconds at 60 ° C, and 34 cycles at 72 ° C for 15 seconds, and last cycle for 10 seconds at 25 ° C. Analysis was performed using Fluidigm SNP Genotyping Analysis software (version 3.1.1; Fluidigm, San Francisco, Calif., USA).

SNP analysis using genotype data

The association between genotypes and consecutive data (bone mineral density) was evaluated by linear regression analysis. Age, body weight, and height were corrected by logistic regression analysis for the association of these data (osteoporosis, VF, NVF, osteoporosis fracture).

MAF <1% genetic burden analysis by rare variation

The following four methods were used to evaluate the total genetic load due to rare mutations. 1) combined multivariate and collapsing (CMC) method; 2) weighted-sum statistics (WSS); 3) kernel-based adaptive cluster (KBAC) method; 4) optimal sequence kernel association test (SKAT-O). This method was applied when there were at least two rare mutations.

Genetic risk score; GRS ) &Lt; / RTI &gt; Marker  Selection

GRS was calculated by selecting risky genes with risk alleles (MAF ≥ 1%) and rare mutations (MAF <1%) that were associated with traits associated with one or more osteoporosis. Risk allele and risk genes were defined as associated with decreased BMD, increased risk of osteoporosis, VF, NVF, and osteoporotic fracture. P values that were considered statistically relevant were α <P <0.05. α was 0.00008 (0.05 / 198 genes / 3 genetic models) when Bonferroni correction was applied for multiple tests. Weights of risk allele and risk genes were set. Common mutations are associated with a risk allele (MAF ≥ 1%), with a weight of 2 if there is two risk alleles for each gene, a weight of 1 if there is one risk allele, If not, the weight is given as 0. Rare mutations were calculated by multiplying the number of rare mutations and weights in each individual with respect to the risk gene.

The gene risk index was calculated to estimate cumulative effects. Common variants were calculated by summing the risk allele scores and GRS-C (GRS-C). All genetic risk scores were calculated by adding GRS-C to GRS-T.

statistics

Data are expressed as mean + SD or number, and percent. Multiple assays were used to confirm the association of osteoporosis-related traits (BMD, osteoporosis, VF, NVF, osteoporosis fractures) with gene risk index. The association of BMD with GRS-C or GRS-T with LS and FN was assessed by multiple regression analysis with correction of clinical risk factors (CRFs). CRFs included age, height, weight, current smoking status, alcohol consumption, and family history of fractures. The explanatory power of the BMD corrected for age and weight of GRS-C or GRS-T was calculated from the linear regression model with variance (corrected R 2 ). The association of GRS-C or GRS-T with osteoporosis was assessed by logistic regression analysis with corrected CRFs. The association of GRS-C or GRS-T with fracture (VF, NVF, osteoporotic fracture) risk was assessed by logistic regression analysis with corrections of BMD of CRFs and / or FN BMD.

To determine if genetic profiling can improve the predictability of fracture risk in patients with osteopenia (-1.0 <T-score <-2.5), the area under the curve of the receiver operating characteristic curve (ROC curve) (GRS-C or GRS-T) using the area under the curve (AUC). Three models for predicting fracture (VF, NVF, or osteoporotic fracture) were identified. Model I (CRFs + BMD), Model II (CRFs + BMD + GRS-C), and Model III (CRFs + BMD + GRS-T). CRFs included age, height, weight, current smoking status, alcohol intake, and family history of fractures. BMD included FN T-values included in most fracture risk prediction models. LS BMD data were not included because of technical limitations in measurement. However, in recent studies, it has been reported that AUC analysis is less sensitive and it is difficult to confirm the improvement of prediction ability by addition of marker. We therefore used a reclassification analysis to determine whether genetic profiling can improve the predictability of fracture risk in patients with osteopenia (-1.0 <T-score <-2.5). In a recent recommendation for osteoporosis treatment in Japan, the probability of major osteoporotic fracture was 15% or more for 10 years as a treatment standard for osteopenia patients. Therefore, the probability of occurrence of 15% was selected as the reference value for the reclassification analysis. Patients were classified into two groups as follows: <15% risk and ≥15% risk. In each model, the proportion of reclassified patients was calculated and compared.

GRS-C (Model II) or GRS-T (Model III), compared with models without GRS-C or GRS-T (Model I), if GRS is useful in predicting the probability of osteoporotic fracture The added probabilities were assumed to be increased in the fracture-incidence group or decreased in the non-fracture group. In the reclassification analysis, the improvement of the prediction probability was calculated by the net reclassification improvement index (NRI index)

NRI index = (probability increase in occurrence group) - (probability decrease in occurrence group) - (probability increase in non-occurrence group) + (probability decrease in non-occurrence group).

NRI index Statistical significance was estimated by Z-test. This is a simple asymptotic test for null hypothesis with NRI index = 0. If the net gain obtained from the reclassification analysis is greater than zero, then it is statistically significant. All statistical analyzes were performed using R (version 2.81) and SPSS statistical software (version 18; SPSS Inc. Chicago, IL, USA), and P ≤0.05 was considered statistically significant.

result

General

The study was carried out in three stages (Figure 1): 1) discovery set (n = 982); 2) the replication set (n = 3,895); And 3) reclassification analysis.

In the digestion stage, target sequence sequencing of 198 genes was performed in 982 individuals with bipolar phenotype. 12,087 mutations were identified, and through the four quality filters and two functional filters, 1,242 common functional mutations (457 nsSNPs in 134 genes and 785 rSNPs in 188 genes) and 1,560 rare functional mutations (1,329 nsSNPs in 189 genes and 231 rSNPs in 116 genes) were further screened for further analysis. Of these, 43 common functional mutations (9 nsSNPs in 9 genes and 34 rSNPs in 27 genes) and 150 rare functional mutations (119 nsSNPs in 15 genes and 31 rSNPs in 12 genes) The association with the phenotype associated with osteoporosis was observed. Of these, 72 mutations (9 common mutations in 9 genes and 63 rare mutations in 14 genes) could not be designed using the SNPtype TM assay (Fluidigm, San Francisco, CA, USA) We could not use it at the stage. In the validation phase, 34 common functional mutations (7 nsSNPs in 9 genes and 27 rSNPs in 21 genes) and 87 rare functional mutations (71 ns SNPs in 15 genes and 10 nS SNPs in 15 genes) from the remaining 3,895 individuals 16 rSNPs in dog genes) were associated with osteoporosis-related phenotypes. Finally, reclassification analysis was performed on 2,202 individuals with osteopenia to evaluate the impact of gene profile on fracture prediction.

Discovery set

Table 2 shows the characteristics of the patients at the excavation stage. In accordance with the criteria for selecting the bipolar phenotype (see study group), the two groups were similar in age and BMI was significantly higher in the very-low BMD group (P = 0.001 ). LS and FN BMD values were significantly higher in the control group (P <0.001).

Figure 112015053900872-pat00002

For sequencing, target sites containing 198 genes totaling 1,248 kb were captured. The captured fragments were sequenced to generate an average of 108,317 leads of ~ 101 bp each. Approximately 99.8% of the leads with an average fold coverage of 251 x (range, 89-730 x) were mapped against the target site. 10 × sequence coverage and Phred-like quality score of more than 30 were performed using the GATK unified genotyper module with quality criteria. The GATK Genomic Annotator module used UCSC hg19 and dbSNP 135 to provide data such as dbSNP rs ID, SNP location, and SNP function. A total of 12,087 mutations (mean 13.5 mutations per individual) were recalled, of which 10,654 successfully passed through four quality filters, 2,306 with common mutations (MAF ≥ 1%), 8,348 with rare mutations (MAF < 1%). 65% of the SNPs were new SNPs not reported in the dbSNP database. Of the 10,654 mutations, 3,481 (32.7%) occurred in the exon region, of which 982 SNPs (9.2%) were nsSNPs. Of the 10,654 mutations, 3,225 SNPs (30.3%) appeared in the upstream region.

Among 2,306 common mutations, 1,242 mutations in exon and regulatory regions were selected for further analysis. Of the 1,242 mutations, 457 of 134 genes were nsSNPs and 785 of 188 genes were rSNPs. Of the 8,348 rare mutations, 1,329 nsSNPs of 189 genes and 231 rSNPs of 116 genes were selected through two functional filters. In conclusion, 1,242 common functional mutations and 1,560 rare functional mutations were associated with osteoporosis. Of these, 43 common functional mutations in 32 genes (Table 3) and 150 functional mutations in 150 genes (Table 4) were observed to be associated with osteoporosis.

The replication set

Table 2 shows the patient characteristics in the verification phase. Using the Fluidigm BioMark system in the validation phase, nine common mutations in 9 genes and 63 rare mutations in 14 genes could not be designed. Thus, in the validation phase, only 34 common mutations (Table 3) of 26 genes and 87 rare mutations (Table 4 and Table 5) of 15 genes were possible.

Figure 112015053900872-pat00003

Figure 112015053900872-pat00004

Figure 112015053900872-pat00005

In the above table, A1 is a minor allele; AF1 is the frequency of minor alleles at the excavation stage; G a is the gene group (see Table 1); OR is ozbee; 95% CI represents the 95% confidence interval. b Relevance analysis adjusted for age, weight and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

C This mutation belonged to a rare mutation at the excavation stage.

Figure 112015053900872-pat00006

In the above table, G a is a gene group (see Table 1); OR is ozbee; 95% CI represents the 95% confidence interval.

a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

Figure 112015053900872-pat00007

In the above table, A1 is a minor allele; AF1 is a minor allele frequency based on excavation and verification steps; N represents the number of mutations.

Ten common mutations in eight genes (HOXA3, IBSP, ITGA1, PPP6R3, PTHLH, RSPO3, WNT16, and TNFRSF11B) were associated with BMD (β = 0.01-0.03) 6).

Figure 112015053900872-pat00008

Figure 112015053900872-pat00009

In the above table, A1 is a minor allele; AF1 represents the frequency of minority alleles based on excavation and validation steps.

a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

Eight common mutations were associated with osteoporotic fractures (Table 7), with 8 genes (AKAP11, FLT1, LEPR, LRRC4C, RSPO3, SMG6, STARD3NL, and WNT2B) 8 common mutations and VF were associated with 8 genes (FLT1, LEPR, RBMS3, RSPO3, SMG6, STARD3NL, and WNT2B) and two common mutations in two genes (CADM2 and IBSP) (Table 7).

Figure 112015053900872-pat00010

Figure 112015053900872-pat00011

In the above table, A1 is a minor allele; AF1 is a minor allele frequency based on excavation and verification steps; OR is ozbee; 95% CI represents the 95% confidence interval. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

As a result of the association analysis at the verification stage, 26 rare mutations of four genes (BMS1, LOC91461, LRP4, and TNRC6B) were associated with BMD (Table 8). The effect of rare mutations on bone mineral density (β = 0.03-0.06) was greater than that due to common mutations (β = 0.01-0.03).

Figure 112015053900872-pat00012

In the above table, n represents the number of rare mutations. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

As a result of the association analysis at the verification stage, 18 rare mutations of three genes (BMS1, HOXA3, and LOC91461) were observed to correlate with osteoporosis (Table 9).

Figure 112015053900872-pat00013

In the above table, n represents the number of rare mutations. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

Of the rare mutations, two rare mutations (LRP4, NFATC1) with greater effect than the common mutations in bone mineral density (β = 0.01-0.03) beta = 0.27-0.53) was observed (Table 10).

Figure 112015053900872-pat00014

In the above table, A represents a minor allele; MAF represents the frequency of minor alleles based on excavation and validation steps. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

Three rare mutations of three genes (CDK5RAP2, HOXA3, and LOC91461) showed osteoporosis and flare in one of the rare mutations, and one rare mutation in the BMS1 gene was associated with osteoporotic fracture and NVF risk (Table 11).

Figure 112015053900872-pat00015

In the above table, A represents a minor allele; MAF is the frequency of minor alleles based on excavation and validation steps; MAF2 is the minor allele frequency in the control; OR represents the odds ratio. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

To assess the roles of potentially harmful rare mutations, the rare mutations predicted to be "potentially compromised" or "likely to be compromised" by the PolyPhen-2 software were defined as "potentially harmful nsSNPs". Twenty-seven rare nsSNPs with five genes (BMS1, HOXA3, LOC91461, LRP4, TNRC6B) were associated with a phenotype associated with one or more osteoporosis. All rare nsSNPs in two genes (LOC91461 and HOXA3) and only one nsSNP in BMS1 appeared to be potentially deleterious and were excluded in identifying the role of potentially harmful rare mutations. On the other hand, 5 of 7 nsSNPs in LRP4 and 4 of 6 in TNRC6B were potentially harmful (Table 12A and 12B), and were used to identify the role of potentially harmful rare mutations after the addition.

Figure 112015053900872-pat00016

Figure 112015053900872-pat00017

Thus, only the five potentially harmful nsSNPs of LRP4 and the four potentially harmful nsSNPs of TNRC6B were further analyzed (Table 13). As a result of additional association analysis, it was observed that both the 5 potentially harmful rare mutations of LRP4 and the 3 potentially harmful rare mutations of TNRC6B were associated with bone mineral density (Table 13). The influence of potentially harmful rare mutations of LRP4 (β = 0.07-0.10) was greater than that of the rare mutations of LRP4 (β = 0.05-0.06) and common mutations of other genes (β = 0.01-0.03).

Figure 112015053900872-pat00018

In the above table, N represents the number of rare mutations. a Relevance analysis adjusted for age, weight, and height. When α <P <0.05, it is expressed as a bold. α represents the significance level (α = 0.05 / 198 genes / 3 genetic models = 0.00008) of the correction of the ferroni for multiple analyzes.

GRS  Wow BMD , osteoporosis, VF , NVF , Or osteoporosis fracture

GRS-C was calculated using 19 genes and 19 common functional mutations that are associated with osteoporosis-related phenotypes. GRS-T was calculated by including 5 genes and 31 rare functional mutations in GRS-C (Table 14).

Figure 112015053900872-pat00019

In the table above, a correlation analysis corrected age, weight, and height.

Figure 2 shows the GRS-C and GRS-T distribution of the subjects. The median values of GRS-C and GRS-T were 21.5 (range: 0-31.6) and 47.4 (range: 25.9-57.5), respectively. In all subjects (n = 4,877), GRS-C and GRS-T showed a significant correlation with BMD before and after CRFs correction (Table 15) (P = 0.007-0.023). GRS-C could account for 2.0% of the inheritance of the LS BMD region and 3.0% of the inheritance of the FN BMD region. GRS-T could account for 2.2% of the inheritance of the LS BMD region and 3.2% of the inheritance of the FN BMD region.

Figure 112015053900872-pat00020

In the table above, a association analysis corrected for age, weight, height, current smoking status, alcohol intake (≥ 3 units / d), and family history of fractures. Relevance analysis was adjusted for age, weight, height, current smoking status, alcohol intake (≥ 3 units / d), and family history of fractures. P < 0.05.

In all participants (n = 4,877), GRS-C and GRS-T were associated with VF, NVF, and osteoporotic fracture risk before and after correction of CRFs and BMD (P = 0.049- <0.001).

Figure 112015053900872-pat00021

In the table above, a association analysis corrected for age, weight, height, current smoking status, alcohol intake (≥ 3 units / d), and family history of fractures. b Relevance analysis was adjusted for age, weight, height, current smoking status, alcohol intake (≥ 3 units / d), family history of fractures, and T-value of femoral neck.

Osteopenia  there is In patients  Reclassification analysis

A retrospective analysis of osteopenia patients (n = 2,202) was performed to assess the impact of GRS-C or GRS-T addition on the predictive ability of the current fracture risk assessment tool (Table 17). Compared with Model I (CRFs + BMD), the model with GRS-C (Model II) reclassified 5.3% (n = 18) of fracture patients (n = 342) n = 4) were classified as low-risk groups. Therefore, the prediction rate improved by 4.1% (= 5.3% -1.2%). Compared with Model I, Model II reclassified 6.1% (n = 113) of low risk patients (n = 1,860) to low risk and 3.4% (n = 63) (= 6.1% -3.4%). Compared with Model I, Model II showed a predictive improvement of 6.8% (= 4.1% + 2.7%; P <0.001) in total. Likewise, Model III with GRS-T added to Model I showed a 9.6% prediction rate increase (P <0.001). The degree of improvement of the prediction rate was 0.36% for each common variation and about 1.56 times for the common variation of 0.56% depending on genetic load due to rare variation. For the VF prediction, the model II with GRS-C added to the existing prediction tool resulted in an improvement of 7.3% (P = 0.005) and the model III with GRS-C improved the prediction rate by 10.2% P < 0.001). The degree of improvement was 0.38% per common mutation and 0.58% by genetic load due to rare mutations, which was about 1.52 times the common mutation. For the NVF prediction, the model II with GRS-C added to the existing forecasting tool resulted in a limited prediction rate improvement of 3.0% (P = 0.091) and a prediction rate improvement of 4.9% in the model III with GRS-C (P = 0.008). The degree of improvement of the prediction rate was 0.16% per common variation and 0.38% by genetic load due to rare variation, which was about 2.38 times the common variation.

Figure 112015053900872-pat00022

Claims (11)

A method for providing information for predicting the occurrence of osteoporotic fracture of an individual comprising identifying a base at a polymorphic site of the following single base polymorphic marker from a nucleic acid sample isolated from the individual:
A polymorphic site in which the 40,316,673 base of human chromosome 11 is G or A.
The method according to claim 1, wherein, when the base of the polymorphic site of the polymorphism of the single nucleotide polymorphism marker is as follows, the base provides information for predicting the occurrence of osteoporotic fracture of an individual having a possibility of osteoporotic fracture:
The 40,316,673th base of human chromosome 11 is G.
4. The method of claim 1, further comprising identifying a base in a polymorphic site of at least one single base polymorphic marker selected from the group consisting of the following single base polymorphic markers: Way:
A polymorphic site in which the 113,051,074 base of human chromosome 1 is A or G;
A polymorphic site in which the 29,070,533 base of human chromosome 13 is C or T;
A polymorphic site in which 42,845,500 base of human chromosome 13 is T or C;
A polymorphic site in which the 127,438,642 base of human chromosome 6 is C or A;
A polymorphic site in which the 68,367,943 base of human chromosome 11 is G or C;
A polymorphic site in which the 2,185,956th base of human chromosome 17 is T or G;
A polymorphic site in which the 65,885,357 base of human chromosome 1 is A or G;
A polymorphic site in which the 88,732,874 base of human chromosome 4 is A or G;
A polymorphic site in which the 52,082,846th base of human chromosome 5 is T or C;
A polymorphic site in which the 29,322,363rd base of human chromosome 3 is T or C;
A polymorphic site in which the 38th, 217th, 555th bases of the human chromosome 7 are A or G;
A polymorphic site in which the 119,965,024 base of human chromosome 8 is T or C;
A polymorphic site in which the 85,006,615th base of human chromosome 3 is C or T;
A polymorphic site in which the 120,969,769 base of human chromosome 7 is G or A;
A polymorphic site in which the 29,322,297th base of human chromosome 3 is C or A;
A polymorphic site in which the 28,126,245th base of human chromosome 12 is C or A;
A polymorphic site in which the 120,968,674 base of human chromosome 7 is A or G; And
A polymorphic site in which the 27,166,854 base of human chromosome 7 is A or G.
The method according to claim 3, wherein when the base of the polymorphic site of the polymorphism of the single nucleotide polymorphism marker is as follows, the base provides information for predicting the occurrence of osteoporotic fracture of an individual having a possibility of osteoporotic fracture:
The 113,051,074th base of human chromosome 1 is A;
29,070,533 base of human chromosome 13 is C;
The 42,845,500th base of human chromosome 13 is T;
The 127,438,642 th base of the human chromosome 6 is C.I.
The 68,367,943th base of human chromosome 11 is G;
The 2,185,956th base of human chromosome 17 is T;
65,885,357 base of human chromosome 1 is A;
88,732,874 base of human chromosome 4 is A;
The 52,082,846th base of human chromosome 5 is T;
The 29,322,363th base of human chromosome 3 is T;
38, 217, 555 base of human chromosome 7 is A;
119,965,024 base of human chromosome 8 is T;
The 85,006,615th base of human chromosome 3 is C;
The 120,969,769th base of human chromosome 7 is G;
29, 322, 297 base of human chromosome 3 is C;
The 28,126,245th base of human chromosome 12 is C;
120,968,674 base of human chromosome 7 is A; or
The 27,166,854 th base of human chromosome 7 is A.
4. The method according to any one of claims 1 to 3, further comprising identifying a base at a polymorphic site of at least one single base polymorphic marker selected from the group consisting of the following single base polymorphic markers: How to provide information:
A polymorphic site in which the 43,292,131st base of human chromosome 10 is C or T;
A polymorphic site in which the 43,292,146th base of human chromosome 10 is A or G;
A polymorphic site in which the 43,292,337 base of human chromosome 10 is A or G;
A polymorphic site in which the 43,292,394 base of human chromosome 10 is G or C;
A polymorphic site in which the 43,292,584 base of human chromosome 10 is T or C;
A polymorphic site in which the 43,297,622 base of human chromosome 10 is G or A;
A polymorphic site in which the 27,147,763th base of human chromosome 7 is G or C;
A polymorphic site in which the 27,166,747 base of human chromosome 7 is C or T;
A polymorphic site in which the 27,169,034 base of human chromosome 7 is C or T;
A polymorphic site in which the 27,183,762th base of human chromosome 7 is T or C;
A polymorphic site in which the 27,196,304th base of human chromosome 7 is G or C;
A polymorphic site in which 42,273,202 base of human chromosome 2 is A or G;
A polymorphic site in which the 42,281,233 base of human chromosome 2 is A or G;
A polymorphic site in which the 42,281,312 base of human chromosome 2 is T or G;
A polymorphic site in which the 42,282,136th base of human chromosome 2 is G or C;
A polymorphic site in which the 42,282,148th base of human chromosome 2 is A or G;
A polymorphic site in which the 42,284,361 base of human chromosome 2 is G or A;
A polymorphic site in which the 42,284,769 base of human chromosome 2 is A or G;
A polymorphic site in which the 46,897,404th base of human chromosome 11 is A or G;
A polymorphic site in which the 46,898,044 base of human chromosome 11 is T or C;
A polymorphic site in which the 46,900,719 base of human chromosome 11 is C or T;
A polymorphic site in which the 46,905,425th base of human chromosome 11 is T or C;
A polymorphic site in which the 46,908,042 base of human chromosome 11 is T or C;
A polymorphic site in which the 46,917,846th base of human chromosome 11 is T or A;
A polymorphic site in which 46,920,476 base of human chromosome 11 is G or A;
A polymorphic site in which the 40,660,881 base of human chromosome 22 is T or C;
A polymorphic site in which the 40,661,006 base of human chromosome 22 is A or G;
A polymorphic site in which the 40,661,216th base of human chromosome 22 is T or G;
A polymorphic site in which the 40,661,867th base of human chromosome 22 is T or C;
A polymorphic site in which the 40,661,939 base of human chromosome 22 is C or G; And
A polymorphic site in which the 40,662,533 base of human chromosome 22 is C or A.
6. The method according to claim 5, wherein when the base of the polymorphic site of the polymorphism of the single nucleotide polymorphism marker is as follows, the base provides information for predicting the occurrence of osteoporotic fracture of an individual having a possibility of osteoporotic fracture:
43,292,131 base of human chromosome 10 is C;
The 43,292,146th base of human chromosome 10 is A;
The 43,292,337 base of human chromosome 10 is A;
The 43,292,394th base of human chromosome 10 is G;
43,292,584 base of human chromosome 10 is T;
The 43,297,622th base of human chromosome 10 is G;
The 27,147,763th base of human chromosome 7 is G;
The 27,166,747th base of human chromosome 7 is C;
27,169,034 base of human chromosome 7 is C;
The 27,183,762th base of human chromosome 7 is T;
The 27,196,304th base of human chromosome 7 is G;
42,273,202 base of human chromosome 2 is A;
The 42,281,233 base of human chromosome 2 is A;
42,281,312 base of human chromosome 2 is T;
The 42,282,136th base of human chromosome 2 is G;
The 42,282,148th base of human chromosome 2 is A;
42,284,361 base of human chromosome 2 is G;
42,284,769 base of human chromosome 2 is A;
The 46,897,404th base of human chromosome 11 is A;
46,898,044 base of human chromosome 11 is T;
46,900,719 base of human chromosome 11 is C;
The 46,905,425th base of human chromosome 11 is T;
The 46,908,042th base of human chromosome 11 is T;
The 46,917,846th base of human chromosome 11 is T;
46,920,476 base of human chromosome 11 is G;
The 40,660,881 base of human chromosome 22 is T;
The 40,661,006th base of human chromosome 22 is A;
The 40,661,216th base of human chromosome 22 is T;
The 40,661,867th base of human chromosome 22 is T;
The 40,661,939 base of human chromosome 22 is C; or
The 40,662,533 base of the human chromosome 22 is C.I.
The method according to any one of claims 1 to 3, further comprising predicting the occurrence of osteoporotic fracture of an individual comprising the further step of identifying at least one member selected from the group consisting of age, height, weight, smoking status, alcohol consumption, family history of fracture, How to provide information to do.
The method according to claim 5, further comprising the step of further identifying at least one selected from the group consisting of age, height, weight, smoking status, alcohol consumption, family history of fracture, and bone density, How to provide.
A probe capable of detecting a polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the above 40,316,673 base or a polynucleotide complementary thereto, wherein the 40,316,673th base of the human chromosome 11 is G or A, or a probe capable of detecting the polynucleotide complementary thereto A kit for predicting osteoporosis fracture comprising a primer capable of amplification.
10. The method of claim 9,
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 113,051,074 base, wherein the 113,051,074 base of human chromosome 1 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 29,070,533 base, wherein the 29,070,533 base of the human chromosome 13 is C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the above 42,845,500 base, wherein the 42,845,500 base of human chromosome 13 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 127,438,642 base, wherein the 127,438,642th base of the human chromosome 6 is C or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 68,367,943 base, wherein the 68,367,943 base of human chromosome 11 is G or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 2,185,956 base, wherein the 2,185,956 base of human chromosome 17 is T or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 65,885,357 base, wherein the 65,885,357th base of the human chromosome 1 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising 88,732,874 bases, wherein 88,732,874 base of human chromosome 4 is A or G, or a complementary polynucleotide thereof;
A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the 52,082,846th base, wherein the 52,082,846th base of the human chromosome 5 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 29,322,363 base, wherein the 29,322,363th base of the human chromosome 3 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleic acid sequences comprising the 38th, 217th, 555th bases, wherein the 38th, 217th, 555th bases of the human chromosome 7 are A or G, or a complementary polynucleotide thereof;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 119,965,024 base, wherein the 119,965,024 base of human chromosome 8 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 85,006,615 base, wherein the 85,006,615th base of human chromosome 3 is C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 120,969,769 base, wherein the 120,969,769th base of human chromosome 7 is G or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 29th, 322nd, 297th bases, wherein the 29th, 322nd, 297th bases of the human chromosome 3 are C or A, or a complementary polynucleotide thereof;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 28,126,245 base, wherein the 28,126,245th base of human chromosome 12 is C or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 120,968,674 base, wherein the 120,968,674 base of human chromosome 7 is A or G, or a complementary polynucleotide thereof; And
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,166,854 base, wherein the 27,166,854 base of the human chromosome 7 is A or G, or a polynucleotide complementary thereto; A probe capable of detecting at least one polynucleotide selected from the group consisting of a polynucleotide capable of amplifying the polynucleotide, and a primer capable of amplifying the polynucleotide.
11. The method according to claim 9 or 10,
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,131 base, wherein the 43,292,131 base of the human chromosome 10 is C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,146 base, wherein the 43,292,146th base of the human chromosome 10 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,337 base, wherein the 43,292,337th base of human chromosome 10 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,394 base, wherein the 43,292,394 base of human chromosome 10 is G or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,292,584 base, wherein the 43,292,584th base of the human chromosome 10 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 43,297,622 base, wherein the 43,297,622th base of human chromosome 10 is G or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 27,147,763 base, wherein the 27,147,763th base of human chromosome 7 is G or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising 27, 166, 747 bases, wherein 27, 166, 747th bases of the human chromosome 7 are C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,169,034 base, wherein the 27,169,034 base of the human chromosome 7 is C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,183,762 base sequence, wherein the 27,183,762th base of human chromosome 7 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 27,196,304 base, wherein the 27,196,304 base of human chromosome 7 is G or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,273,202 base, wherein the 42,273,202 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,281,233 base, wherein the 42,281,233 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,281,312 base, wherein the 42,281,312 base of the human chromosome 2 is T or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42, 282, 136 base, wherein the 42,282,136th base of the human chromosome 2 is G or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 42,282,148 base, wherein the 42,282,148 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42,284,361 base, wherein the 42,284,361 base of human chromosome 2 is G or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 42,284,769 base, wherein 42,284,769 base of human chromosome 2 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 46,897,404 base, wherein the 46,897,404th base of human chromosome 11 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,898,044 base, wherein the 46,898,044th base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,900,719 base, wherein the 46,900,719 base of the human chromosome 11 is C or T, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,905,425 base, wherein the 46,905,425 base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,908,042 base, wherein the 46,908,042th base of human chromosome 11 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,917,846 base, wherein the 46,917,846th base of the human chromosome 11 is T or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the above 46,920,476 base, wherein the 46,920,476th base of human chromosome 11 is G or A, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,660,881 base, wherein the 40,660,881 base of human chromosome 22 is T or C, or a complementary polynucleotide thereof;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,006 base, wherein the 40,661,006th base of human chromosome 22 is A or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,216 base, wherein the 40,661,216th base of human chromosome 22 is T or G, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,867th base, wherein the 40,661,867th base of human chromosome 22 is T or C, or a polynucleotide complementary thereto;
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,661,939 base, wherein the 40,661,939 base of the human chromosome 22 is C or G, or a polynucleotide complementary thereto; And
A polynucleotide consisting of 5 to 100 consecutive nucleotide sequences comprising the 40,662,533 base, wherein the 40,662,533 base of the human chromosome 22 is C or A, or a polynucleotide complementary thereto; A probe capable of detecting at least one polynucleotide selected from the group consisting of: a polynucleotide capable of amplifying the polynucleotide; and a primer capable of amplifying the polynucleotide.
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