KR102343549B1 - KCNMB2 SNP markers for predicting responsiveness of Ritodrin administration in patients with Early Labor Contraction and use thereof - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphism (SNP) marker for predicting the responsiveness of ritodrine administration in patients with preterm labor and, more specifically, to an SNP marker specific to ritodrine administration, a composition for predicting the responsiveness of ritodrine administration in patients with preterm labor comprising the same, a kit or microarray comprising the composition, and a method for providing information predicting ritodrine administration responsiveness to a patient with preterm labor using the marker. By using the marker of the present invention, the responsiveness of ritodrine administration in patients with preterm labor can be predicted accurately and objectively.

Description

KCNMB2 SNP markers for predicting responsiveness of Ritodrin administration in patients with Early Labor Contraction and use thereof

The present invention relates to a single nucleotide polymorphism (SNP) marker for predicting the reactivity of ritodrin administration to patients with preterm labor, and more particularly, a ritodrin administration-specific KCNMB2 gene-derived SNP marker, It relates to a composition for predicting lithodrin administration responsiveness to lithodrin administration, a kit or microarray containing the composition, and an information providing method for predicting ritodrin administration responsiveness to a patient with preterm labor using the marker.

Preterm birth is defined as delivery before 37 weeks of gestation and is a major cause of morbidity and mortality (WHO Recommended definitions, terminology and format for statistical tables related to the perinatal period and use of a new certificate for cause of perinatal deaths. Acta Obstet Gynecol Scand. 1977;56:247-253.), accounts for 80% of neonatal deaths. Therefore, the use of appropriate drugs to delay premature birth is very important.

Ritodrin, used to prevent premature birth in pregnant women, is one of the adrenergic β2 receptor agonists, and is a uterine contraction inhibitor frequently used in Korea. When lithodrin binds to the adrenergic β2 receptor, adenylate cyclase (ADCY) is activated through G-protein, and the secondary signal transmitter cAMP (Cyclic adenosine monophosphate) concentration increases, causing uterine smooth muscle to relax. .

Adrenergic β2 receptor agonists induce hyperpolarization of smooth muscle by increasing potassium ion permeability, which is important for maintaining endometrium relaxation during pregnancy. Among BKca called macro-conductive Fig Ca ² + - activated ^{K + (large-conductance Ca 2} + -activated K +) channel is an important potassium channel in the endometrium of the pregnant women and nonpregnant women. Uterine contraction is mainly due to an increase in calcium concentration, and BKca channels are activated by depolarization or an increase in intracellular calcium concentration. Thus, contraction of the uterus and BKca channels are closely related.

Although ritodrin has a large and rapid uterine relaxation effect, many pregnant women have serious side effects, such as palpitations, pulmonary edema, and difficulty breathing, to ritodrin, so it is often necessary to change or discontinue the drug.

Therefore, before administering ritodrin to pregnant women, if it is possible to predict which patients will have a large effect and few side effects, it is thought that it will be possible to reduce the discomfort of the patient and the treatment team and to use ritodrin more efficiently.

Based on this clinical necessity, the present inventors conducted a study on SNPs related to ritodrin administration, and tried to find a marker that can predict the effect or side effects when ritodrin is administered to patients with preterm labor. As a result, KCNMB2 (potassium calcium-activated channel subfamily M regulatory beta subunit 2) It was confirmed that specific SNPs in the gene have a ritodrin administration-specific genotype, and the present invention was completed.

One object of the present invention is that in one or more polynucleotides selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, the 78th base in the nucleotide sequence of SEQ ID NO: 1 and the 70th nucleotide in the nucleotide sequence of SEQ ID NO: 3 is G or A; the 83rd base in the nucleotide sequence of SEQ ID NO: 2 and the 86th base in the nucleotide sequence of SEQ ID NO: 5 are C or T; or the 77th base in the nucleotide sequence of SEQ ID NO: 4 is T or C, the 78th base in the nucleotide sequence of SEQ ID NO: 1, the 83rd base in the nucleotide sequence of SEQ ID NO: 2, and the 70th base in the nucleotide sequence of SEQ ID NO: 3 , A polynucleotide consisting of 5 to 150 consecutive bases including the 77th base in the nucleotide sequence of SEQ ID NO: 4 or the 86th base in the nucleotide sequence of SEQ ID NO: 5, or a polynucleotide complementary thereto, including a polynucleotide complementary thereto, premature labor To provide a marker for predicting ritodrin administration responsiveness in patients.

Another object of the present invention is to provide a kit for predicting lithodrin administration responsiveness for patients with preterm labor comprising the composition.

Another object of the present invention is to provide a microarray for predicting ritodrin administration responsiveness to a patient with preterm labor comprising the composition.

Another object of the present invention is to (a) amplify or hybridize the polymorphic site of the marker of claim 1 from DNA of a sample isolated from an individual; and (b) determining the base of the amplified or hybridized polymorphic site of step (a).

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

In order to achieve the above object, one aspect of the present invention is at least one polynucleotide selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, the 78th base in the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3 the 70th base in the nucleotide sequence is G or A; the 83rd base in the nucleotide sequence of SEQ ID NO: 2 and the 86th base in the nucleotide sequence of SEQ ID NO: 5 are C or T; or the 77th base in the nucleotide sequence of SEQ ID NO: 4 is T or C, the 78th base in the nucleotide sequence of SEQ ID NO: 1, the 83rd base in the nucleotide sequence of SEQ ID NO: 2, and the 70th base in the nucleotide sequence of SEQ ID NO: 3 , A polynucleotide consisting of 5 to 150 consecutive bases including the 77th base in the nucleotide sequence of SEQ ID NO: 4 or the 86th base in the nucleotide sequence of SEQ ID NO: 5, or a polynucleotide complementary thereto, including a polynucleotide complementary thereto, premature labor A marker for predicting ritodrin administration responsiveness in a patient is provided.

As used herein, the term "ritodrin (Ritodrin)" is one of adrenergic β2 receptor agonists, and is a uterine contraction inhibitor frequently used in Korea. When lithodrin binds to the adrenergic β2 receptor, adenylate cyclase (ADCY) is activated through G-protein, and the secondary signal transmitter cAMP (Cyclic adenosine monophosphate) concentration increases, causing uterine smooth muscle to relax. . Although ritodrin has a large and rapid uterine relaxation effect, many pregnant women have serious side effects, such as palpitations, pulmonary edema, and difficulty breathing, to ritodrin, so it is often necessary to change or discontinue the drug.

Therefore, if it is possible to provide a ritodrin administration-specific marker, it is expected that more accurate ritodrin administration can be achieved. was provided

As used herein, the term "reactivity to ritodrin administration" means an effect or side effect according to ritodrin administration, and the effect according to ritodrin administration is prolonged delivery time to delivery (TTD) or increased gestational weeks. It may be that the risk of premature birth is lowered, but is not limited thereto. Side effects according to the ritodrin administration may include, but are not limited to, palpitations, pulmonary edema, and dyspnea. Prediction of the "reactivity to ritodrin administration" means predicting the effects or side effects of ritodrin administration, and if the TTD is prolonged or the risk of premature birth is lowered by ritodrin administration, it can be said that the ritodrin administration effect is high. , but not limited thereto. In addition, if the TTD is not long even if ritodrin is administered, or if there is a high risk of premature birth, or if there is a possibility that side effects may occur due to ritodrin administration, the effect of ritodrin administration may be low, but is not limited thereto.

In the present invention, the marker may include a single nucleotide polymorphism.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs from person to person. It is called (single nucleotide polymorphism, SNP). A specific polymorphic marker has two or more alleles that exhibit an incidence of at least 1%, more specifically at least 5% or 10%, in a selected population.

As used herein, the term "allele" refers to several types of a gene present at the same locus on homologous chromosomes. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.

As used herein, the term “rs_id” refers to rs-ID, which is an independent marker given to all SNPs initially registered by NCBI, which has been accumulating SNP information since 1998. In the present invention, it was described in the same form as rs150186451. rs_id described in this table means a SNP marker, which is a polymorphic marker of the present invention. Those skilled in the art will be able to easily identify the position and sequence of the SNP using the rs_id. The specific sequence corresponding to rs_id, which is the NCBI's dbSNP (The Single Nucleotide Polymorphism Database) number, may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also extends to such altered sequences.

SEQ ID NO: 1 to SEQ ID NO: 5 are polymorphic sequences including polymorphic sites. The polymorphic sequence refers to a sequence including a polymorphic site including a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

The marker for predicting lithodrin administration reactivity for the preterm labor patient is the 78th nucleotide in the nucleotide sequence of SEQ ID NO: 1, the 83rd nucleotide in the nucleotide sequence of SEQ ID NO: 2, the 70th nucleotide in the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: A polymorphic sequence comprising a polynucleotide comprising the 77th nucleotide in the nucleotide sequence of 4 or the 86th nucleotide in the nucleotide sequence of SEQ ID NO: 5 as a SNP, and a polynucleotide consisting of consecutive nucleotides before and after it, wherein the marker is a total of 5 to 150 consecutive It may include one or more polynucleotides selected from polynucleotides composed of multiple bases, or a complementary polynucleotide thereof.

The 78th base in the nucleotide sequence of SEQ ID NO: 1, the 83rd base in the nucleotide sequence of SEQ ID NO: 2, the 70th base in the nucleotide sequence of SEQ ID NO: 3, the 77th base in the nucleotide sequence of SEQ ID NO: 4, or the base of SEQ ID NO: 5 The marker including the 86th base in the sequence as an SNP may be derived from a potassium calcium-activated channel subfamily M regulatory beta subunit 2 (KCNMB2) gene, but is not limited thereto. Specifically, the nucleotide sequence of SEQ ID NO: 1 is rs10936979, a SNP in which the 178827878th nucleotide of chromosome 3 is mutated from G to A, and the nucleotide sequence of SEQ ID NO: 2 is a SNP in which the 178743983th nucleotide of chromosome 3 is mutated from C to T rs7624046 (MAF (major allele frequency) 0.38), the nucleotide sequence of SEQ ID NO: 3 is rs7625907 (MAF 0.48), which is a SNP in which the 178672670 nucleotide of chromosome 3 is mutated from A to G, the nucleotide sequence of SEQ ID NO: 4 is chromosome 3 rs9839376 (MAF 0.22), which is a SNP in which the 178741877 base of does not

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds.

The polynucleotide sequence may be obtained from various databases such as GenBank of NCBI, which is a known database, but is not limited thereto. For example, it may be derived from human, specifically KCNMB2 (potassium calcium-activated channel subfamily M regulatory beta subunit 2) gene, but is not limited thereto, and sequences having the same activity as the polynucleotide may be included without limitation. . The polynucleotide may include the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, specifically, the nucleotide sequence of SEQ ID NO: 1 is rs10936979, the nucleotide sequence of SEQ ID NO: 2 is rs7624046, the nucleotide sequence of SEQ ID NO: 3 is rs7625907, the sequence The nucleotide sequence of No. 4 may include rs9839376, and the nucleotide sequence of SEQ ID NO: 5 may include rs1382045, but is not limited thereto. In addition, the homology or identity may consist of a nucleotide sequence of 80%, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, or more specifically 99% or more, but this not limited

In addition, the polynucleotide of the present application may be included without limitation as long as it is a probe that can be prepared from a known gene sequence, for example, a sequence that hybridizes with a sequence complementary to all or part of the base sequence under stringent conditions. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity are 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and more specifically 99% or more. Conditions under which polynucleotides having homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or wash conditions of conventional Southern hybridization at 60° C., 1ХSSC, 0.1% SDS , specifically at a salt concentration and temperature equivalent to 60° C., 0.1ХSSC, 0.1% SDS, more specifically 68° C., 0.1ХSSC, 0.1% SDS. can

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (eg, J. Sambrook et al., supra).

In the present invention, when the 78th base of SEQ ID NO: 1 is GG or GA in the allele, it can be predicted that the ritodrin administration effect is higher than that of AA, but is not limited thereto.

When the 83rd base of SEQ ID NO: 2 is CC or CT in the allele, it can be predicted that the ritodrin administration effect is higher than that of TT, but is not limited thereto.

When the 70th base of SEQ ID NO: 3 is AA or AG in the allele, it can be predicted that the ritodrin administration effect is higher than that of GG, but is not limited thereto.

When the 77th base of SEQ ID NO: 4 is TT or TC in the allele, it can be predicted that the ritodrin administration effect is higher than in the case of CC, but is not limited thereto.

When the 86th base of SEQ ID NO: 5 is CC or CT in the allele, it can be predicted that the ritodrin administration effect is higher than that of TT, but is not limited thereto.

In one embodiment of the present invention, rs10936979, rs7624046, rs7625907, rs9839376, and rs1382045 had shorter delivery delay times compared to the wild-type allele (Table 2). rs7624046 was 1230.6 hours with the wild-type allele (CC, CT) and 859.2 hours with the mutant allele homozygous (TT). rs9839376 delayed births in the presence of the wild-type allele at 1230.6 hours with the wild-type allele (TT, TC) and 614.0 hours with the variant allele homozygous (CC).

In another embodiment of the invention, homozygous for the variant allele of rs9839376 had a 2.68 fold higher risk of premature birth compared to those with the T allele (Table 3). Those who were homozygous for the variant allele of rs7624046 (TT) had a 2.1 times higher risk of preterm birth than those without it.

The above results provide evidence that rs10936979, rs7624046, rs7625907, rs9839376 or rs1382045 among the genetic mutations of KCNMB2 affect the efficacy and safety of ritodrin, as in the Examples rs10936979, rs7624046, Effective use of ritodrin can be expected by confirming the genetic mutation of rs7625907, rs9839376 or rs1382045. The present inventors have demonstrated as described above that rs10936979, rs7624046, rs7625907, rs9839376 or rs1382045 is a marker for predicting the reactivity of ritodrin administration to patients with preterm labor.

Another aspect of the present invention provides a composition for predicting lithodrin administration responsiveness to a patient with preterm labor, comprising an agent capable of detecting or amplifying the marker.

The terms used herein are the same as described above.

The composition of the present invention may include a probe capable of binding to any one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 5 and combinations thereof or a complementary polynucleotide thereof or a primer capable of specifically amplifying , but not limited thereto.

As used herein, the term "probe" refers to an allele-specific probe, in which a polymorphic site exists in nucleic acid fragments derived from two members of the same species, and thus hybridizes to a DNA fragment derived from one member, but a fragment derived from another member. does not hybridize to In this case, the hybridization conditions must be sufficiently stringent to hybridize to only one of the alleles, showing a significant difference in the intensity of hybridization between alleles. This can lead to good hybridization differences between different allelic forms.

The probe according to the present invention may be included in a composition capable of predicting reactivity according to ritodrin administration by confirming through a specific hybridization reaction with the polymorphic site of the SNP of the present invention. The specific method of gene analysis as described above is not particularly limited, and may be by any gene detection method known in the art to which the present invention pertains.

The probe may be used in a method of predicting lithodrin administration responsiveness by detecting an allele. The prediction methods include detection methods based on hybridization of nucleic acids, such as Southern blotting, and may be provided in a form previously bound to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be usually performed under stringent conditions, for example, a salt concentration of 1M or less and a temperature of 25° C. or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 °C may be suitable for allele-specific probe hybridization.

As used herein, the term "primer" refers to a nucleotide sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and prevent template strand copying. A short sequence that serves as a starting point for Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. By performing PCR amplification, the responsiveness of ritodrin administration can be predicted by whether the desired product is produced. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

The primer according to the present invention may be included in a composition capable of predicting reactivity according to ritodrin administration by confirming the polymorphic site of the SNP of the present invention through amplification. Specifically, the primer refers to a primer capable of specifically amplifying a polynucleotide of a marker capable of predicting reactivity according to lithodrin administration to a premature labor patient.

The primers used for amplifying the polymorphic marker are prepared under suitable conditions (eg, 4 different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and template-directing DNA under appropriate temperature. It can be a single-stranded oligonucleotide that can serve as a starting point for synthesis. The appropriate length of the primer may vary depending on the intended use, but may be usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template.

The probes or primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods, and such nucleic acid sequences can also be modified using many means known in the art. have. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

The markers of the present invention may be a marker set including one or more, two or more, three or more, four or more, or all five of each of the above markers. The marker is the 78th nucleotide in the nucleotide sequence of SEQ ID NO: 1, the 83rd nucleotide in the nucleotide sequence of SEQ ID NO: 2, the 70th nucleotide in the nucleotide sequence of SEQ ID NO: 3, the 77th nucleotide in the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 Depending on the genotype at the 86th base in the nucleotide sequence of can

Another aspect of the present invention provides a kit for predicting lithodrin administration responsiveness to a patient with preterm labor comprising the composition.

The terms used herein are the same as described above.

The kit may be an RT-PCR kit or a DNA analysis (eg, DNA chip) kit, but is not limited thereto.

The kit can predict the responsiveness of administration of ritodrin to patients with preterm labor by confirming the amplification of the marker of the present invention, or by checking the expression level of the mRNA by the expression level of the marker.

In a specific embodiment, the kit of the present invention may be a kit for predicting the responsiveness of ritodrin administration to a patient with preterm labor contraction, including essential elements necessary for performing a DNA chip. A DNA chip kit is a method in which nucleic acid species are attached in a gridd array to a generally flat solid support plate, typically a glass surface no larger than a microscope slide, and the nucleic acids are uniformly arranged on the chip surface, and the DNA chip It is a tool that allows multiple hybridization reactions to occur between the nucleic acids on the chip surface and the complementary nucleic acids contained in the solution treated on the chip surface, enabling massively parallel analysis.

Another aspect of the present invention provides a microarray for predicting lithodrin administration responsiveness to a patient with preterm labor comprising the composition.

The terms used herein are the same as described above.

The microarray may include DNA or RNA polynucleotides. The microarray consists of a conventional microarray except that it contains a probe capable of detecting the marker of the present invention or an agent capable of amplifying.

A method of manufacturing a microarray by immobilizing a probe on a substrate is well known in the art, and the process of immobilizing a probe on a substrate related to the prediction of lithodrin administration responsiveness for preterm labor patients of the present invention also uses this prior art. so that it can be easily manufactured. In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. For the detection, for example, a nucleic acid sample is labeled with a fluorescent substance, for example, a label capable of generating a detectable signal including a substance such as Cy3 and Cy5, and then hybridized on a microarray and the labeling substance. A hybridization result can be detected by detecting a signal generated from

Another aspect of the present invention comprises the steps of (a) amplifying or hybridizing the polymorphic site of the marker from DNA of a sample isolated from an individual; and (b) determining the base of the amplified or hybridized polymorphic site of step (a).

The terms used herein are the same as described above.

As used herein, the term "subject" may be a preterm labor patient for predicting ritodrin administration responsiveness. DNA may be obtained from samples such as hair, urine, blood, various body fluids, tissues, cells, or saliva isolated from the subject, but is not limited thereto.

Any method known to those skilled in the art can be used for the step of amplifying the polymorphic site of the marker from the DNA of step (a) or hybridizing it with a probe. For example, it can be obtained by amplifying the target nucleic acid through PCR and purifying it, and other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) )), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), self-maintaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874). (1990)) and nucleic acid-based sequence amplification (NASBA) may be used, but are not limited thereto.

Any method known to those skilled in the art may be used for the step of determining the base of the polymorphic site in step (b). For example, sequence analysis, hybridization by microarray, allele specific PCR (allele specific PCR), dynamic allele-specific hybridization (DASH), PCR extension analysis, PCR-SSCP ( PCR-single strand conformation polymorphism), PCR-restriction fragment length polymorphism (PCR-RFLP) analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. Sequenom's MassARRAY system), mini-sequencing (mini- sequencing) method, Bio-Plex system (BioRad), CEQ and SNPstream system (Beckman), Molecular Inversion Probe array technology (eg Affymetrix GeneChip) and BeadArray Technologies (eg, Illumina GoldenGate and Infinium assay), etc. may be used. can, but is not limited thereto. One or more alleles in polymorphic markers can be identified, including microsatellites, SNPs, or other types of polymorphic markers, by the above methods or other methods available to those skilled in the art. Determination of the base of such a polymorphic site can be specifically performed through a SNP chip, which is one of the DNA microarrays that can check each base of hundreds of thousands of SNPs at once.

For example, the sequence analysis may use a conventional method for determining the nucleotide sequence, and may be performed using an automated gene analyzer. In addition, allele-specific PCR refers to 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 3' end of the base in which the SNP is located. The principle of the method is, for example, when a specific base is substituted from A to G, a primer containing A as a 3' end base and a reverse primer capable of amplifying a DNA fragment of an appropriate size are designed and PCR When performing the reaction, when the base at the SNP position is A, the amplification reaction is normally performed and a band at the desired position is observed, and when the base is substituted with G, the primer can bind to the template DNA, but This takes advantage of the fact that the amplification reaction is not performed properly because the 3' end does not perform complementary binding. DASH may be performed by a conventional method, specifically, by a method by Prince et al.

On the other hand, PCR extension analysis first amplifies a DNA fragment containing a base in which a single nucleotide polymorphism is located with a pair of primers, and then inactivates all nucleotides added to the reaction by dephosphorylation, and here a SNP-specific extension primer; A primer extension reaction is performed by adding a dNTP mixture, dideoxynucleotide, reaction buffer and DNA polymerase. In this case, the extension primer uses the 3' end of the base immediately adjacent to the 5' direction of the base at which the SNP is located, and the nucleic acid having the same base as the dideoxynucleotide is excluded from the dNTP mixture, and the dideoxynucleotide represents the SNP It is selected from one of the base types. For example, when there is a substitution from A to G, when a mixture of dGTP, dCTP and TTP and ddATP are added to the reaction, the primer is extended by DNA polymerase at the base where the substitution has taken place, and after a few bases, A The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution does not occur, since the extension reaction is terminated at that position, it is possible to determine the type of base representing the SNP by comparing the lengths of the extended primers.

The TaqMan method comprises the steps of (1) designing and manufacturing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling the probes of different alleles with FAM dye and VIC dye (Applied Biosystems); (3) using the DNA as a template and performing PCR using the primers and probes; (4) analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer after the PCR reaction is completed; and (5) determining the genotype of the polynucleotide of step (1) from the analysis result.

In this case, as a detection method, when an extension primer or dideoxynucleotide is fluorescently labeled, the SNP can be detected by detecting fluorescence using a gene analyzer (eg, ABI's Model 3700, etc.) used for general sequencing. In the case of using a non-labeled extension primer and dideoxynucleotide, the SNP can be detected by measuring the molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique.

In the method, when the 78th base of SEQ ID NO: 1 among the nucleotide sequences determined in step (b) contains GG or GA in the allele, the effect of ritodrin administration is It may be predicted to be high, but is not limited thereto.

In the above method, among the nucleotide sequences determined in step (b), when the 83rd nucleotide of SEQ ID NO: 2 includes CC or CT in the allele, the effect of ritodrin administration when the subject of step (a) is a patient with preterm labor contraction It may be predicted to be high, but is not limited thereto.

In the above method, among the nucleotide sequences determined in step (b), when the 70th nucleotide of SEQ ID NO: 3 contains AA or AG in the allele, the effect of ritodrin administration when the subject of step (a) is a preterm labor contraction patient It may be predicted to be high, but is not limited thereto.

In the above method, among the nucleotide sequences determined in step (b), when the 77th nucleotide of SEQ ID NO: 4 includes TT or TC in the allele, the effect of ritodrin administration when the subject of step (a) is a patient with preterm labor contraction It may be predicted to be high, but is not limited thereto.

In the above method, among the nucleotide sequences determined in step (b), when the 86th nucleotide of SEQ ID NO: 5 includes CC or CT in the allele, the effect of ritodrin administration when the subject of step (a) is a preterm labor contraction patient It may be predicted to be high, but is not limited thereto.

The SNP marker of the present invention is a single nucleotide polymorphic marker derived from the ritodrin administration-specific KCNMB2 gene, and can accurately and objectively predict the responsiveness of ritodrin administration to patients with preterm labor.

1 is a diagram showing linkage disequilibrium (LD) relationships in test subjects for each SNP.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Example 1. SNP (single nucleotide polymorphisms) selection

Among SNPs derived from KCNMB2 (potassium calcium-activated channel subfamily M regulatory beta subunit 2) with a MAF of 25% or higher in Japanese and Chinese, HaploReg v4.1 and Tagger's "best N" method (Ofir D, Gad K, Eran H, Ron S. Increasing the power of association studies by imputation-based sparse tag SNP selection. Communications in Information & Systems. 2009;9;269-82.) was used. Linkage disequilibrium (LD) values were calculated through Haploview 4.2 (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;22;263-5.) (Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplotype blocks in the human genome. Science. 2002;296;2225-9.).

As a result, as SNPs, rs10936979, rs7624046, rs7429015, rs7625907, rs6443559, rs9839376, rs9637454, rs11918114, rs1382045 and rs1042719 in the ADRB2 (Adrenoceptor Beta 2) gene in the KCNMB2 gene were selected as SNPs, and subsequent experiments were performed.

Example 2. Selection of test subjects

Among patients recruited from Ewha Womans University Mokdong Hospital from January 2010 to December 2014, mothers aged 18 years or older from 20 to 36 weeks of gestation had preterm labor with intact membrane and changes in the cervix. If the uterine contractions accompanied by uterine contractions were 3 or more every 10 minutes, and informed consent was given in advance, subjects were included. In the case of severe placental abruption, preeclampsia, fetal distress, severe oligohydramnios, placenta previa, or severe premature membrane rupture, Subjects were excluded from the study, even if the continuation of pregnancy was dangerous to the mother or the fetus, if ritodrin was taken to suppress uterine contractions during McDonald's surgery, or if other contraction inhibitors were used. The patient data were collected through documents or electronic medical records, and the collected information included the pregnant woman's age, height, weight, BMI, gestational age at the time of drug administration, and modified Bishop score.

As a result, 163 of 236 patients met the above conditions and were selected as test subjects. The average of the subjects was 31.0 ± 4.0 years old, 30.0 ± 4.0 weeks of dosing, 62.5 ± 8.5 kg body weight, and 24.0 ± 3.1 kg/m ² body mass index. Basic data such as the mother's age, height, weight, body mass index, gestational weeks at the time of drug administration, and Bishop score are shown in Table 1 below.

The experiment of this example passed the Ewha Womans University Mokdong Hospital Bioethics Review Committee based on the Declaration of Helsinki.

number of patients Median TTD (95% CI) P value age 0.499 <31 years old 77 1168.57 (753.10-1584.03) ≥31 years old 86 1040.03 (670.96-1409.11) Weeks of gestation at the time of drug administration <0.001 <30 weeks 75 1850.67 (1752.89-1948.45) ≥0 weeks 88 701.53 (504.11-898.95) Modified Bishop score ^a 0.001 <2 89 1298.93 (1152.05-1445.82) ≥2 39 613.95 (70.61-1157.29) weight 0.983 <60 kg 66 978.55 (476.07-1481.03) ≥60 kg 97 1230.62 (964.51-1496.72) BMI 0.934 <25 kg/m ² 112 1247.82 (940.71-1554.92) ≥25 kg/m ² 51 1040.03 (656.05-1424.02)

^a Modified Bishop score is the sum of cervix dilation and effacement scores. Dilatation score: 0, <l cm; 1, 1 to 3 cm; 2, 3 to 5 cm; 3, ≥ 5 cm. Effacement score: 0, 0% to 40%; 1, 40% to 60%; 2, 60% to 80%; 3, ≥80%.

Lithodrin was administered by intravenous infusion (IV) at an initial infusion rate of 0.05 mg/min, and increased by 0.05 mg/min every 10 minutes until an appropriate uterine response was obtained. After that, patients who entered the uterine rest period received a maintenance regimen of 0.05 mg/min for 12 to 48 hours.

Example 3. Assay of association between SNP and time to delivery (TTD)

After the patient's blood was collected and treated with ethylenediaminetetraacetic acid (EDTA), DNA was extracted using a kit (QIAamp DNA Blood Mini Kits, QIAGEN GmbH, Hilden, Mettmann, Germany). KCNMB2 and ADRB2 genotypes were analyzed from the extracted DNA. KCNMB2 and ADRB2 genotypes were analyzed using the ABI PRISM SNaPShot Multiplex kits (ABI PRISM SNaPShot Multiplex kits, Applied Biosystems, Foster City, CA, USA).

TTD was evaluated through Kaplan-Meier survival data-analysis using log-rank. In univariate analysis, factors with a P value less than 0.05 were subjected to multivariate analysis using Cox's proportional hazards model. It was said that there was statistical significance when the P value was less than 0.05, and the data were analyzed using SPSS statics version 20.0 for Windows.

Through the 'best N' method, 9 SNPs satisfying the Hardy-Weinberg equilibrium were selected as SNPs to be analyzed (representing 77% of KCNMB2 SNPs), and rs7624046 showed a linkage disequilibrium relationship with rs9839376 (r ² = 0.423, D' = 0.973) (Fig. 1).

As the first outcome evaluation index, the time to delivery (TTD) after drug administration was set.

As a result, rs10936979 (G>A), rs7624046 (C>T), rs7625907 (A>G), rs9839376 (A>G), and rs1382045 (C>T) had shorter delivery delay times compared to the wild-type allele (Table). 2). rs7624046 was 1230.6 hours with the wild-type allele (CC, CT) and 859.2 hours with the mutant allele homozygote (TT). In particular, rs9839376 is 1230.62 hours (95% confidence interval: 1008.00-1453.24) in people carrying the wild-type allele (TT, TC) and 613.95 hours (95% confidence interval: 0.00- 1969.76), it was found that the presence of wild-type alleles delayed fertility.

It can be seen that the above results are similar to those of ADRB2 (JE Chung, et al. BMC Genet. 2017 Nov 13;18(1):96.) of the previous study.

In addition, even with the same KCNMB2 gene-derived SNP, there was no difference between the TTD values in the case of rs7429015 and the high P value confirmed that the correlation with TTD was low.

Minor Allele Frequency (%) genotype Number of patients (%) Median TTD (95% CI) P value ADRB2 rs1042719
(c.1053 G>C) 43.3
GG
GC, CC 54 (33.3)
108 (66.7) 1338.5 (1022.87-1654.20)
978.6 (710.45-1246.65) 0.013
KCNMB2 rs10936979
(c.228-300 G>A) 31.2 GG, GA
AA 151 (93.2)
11 (6.8) 1168.57 (944.61-1392.52)
568.75 (0.00-1775.29) 0.030 rs7624046(c.-67-63360 C>T) 41.7 CC, CT
TT 137 (84.0)
26 (16.0) 1230.62 (988.43-1472.81)
859.17 (453.25-1265.08) 0.015 rs7429015(c.-68+97352 A>G) 40.1 AA, AG
GG 101 (62.3)
61 (37.7) 1027.17 (664.53-1389.80)
1190.70 (863.92-1517.48) 0.144 rs7625907(c.-68+113686 A>G) 50.0 AA, AG
GG 120 (73.6)
43 (26.4) 1230.62 (933.15-1528.09)
859.17 (445.48-1272.86) 0.013 rs6443559(c.-68+118214 C>A) 41.1 CC, CA
AA 105 (64.4)
58 (35.6) 1072.63 (737.37-1407.90)
1103.78 (626.46-1581.11) 0.106 rs9839376 (c.-67-65466 T>C) 23.9 TT, TC
CC 152 (94.4)
9 (5.6) 1230.62 (1008.00-1453.24)
613.95 (0.00-1969.76) <0.001 rs9637454(c.-68+3063 G>A) 45.3 GG
GA, AA 48 (29.8)
113 (70.2) 1072.63 (660.37-1484.90)
1230.62 (897.14-1564.09) 0.211 rs11918114(c.-68+13309 C>T) 28.5 CC, CT
TT 150 (92.0)
13 (8.0) 1103.78 (823.32-1384.25)
991.47 (337.87-1645.07) 0.205 rs1382045(c.-68+78852 C>T) 45.4 CC, CT
TT 112 (68.7)
51 (31.3) 1230.62 (916.15-1545.08)
991.47 (468.24-1514.69) 0.037

Example 4. Assay for association between SNP and risk of premature birth

We performed a multivariate analysis that controlled for factors that could affect TTD and ritodrin drug responsiveness, such as gestational age at the time of first administration of the drug, Bishop's score, and genotype of the beta2-adrenergic receptor.

As a result, the risk of premature birth was 2.68 times (95% confidence interval: 1.16-6.20) higher in the rs9839376 mutant homozygote than in the T allele (Table 3). Those who were homozygous for the rs7624046 variant allele (TT) had a 2.1-fold (95% confidence interval: 1.14-3.73) risk of premature birth compared to those who did not (Table 4).

Factor Unadjusted Hazard Ratio (95% CI) Adjusted Hazard Ratio (95% CI) Age (≥31 years) 1.13 (0.79-1.63) 1.53 (0.98-2.37) Gestational age at start of drug therapy (≥30 weeks) 6.34 (3.81-10.52) ^*** 7.62 (3.95-14.70) ^** Modified Bishop score (≥2) 2.03 (1.30-3.17) ^** ADRB2 rs1042719
(G>C) GG One GC, CC 1.64 (1.11-2.42) ^* KCNMB2 rs10936979
(G>A) GG, GA One AA 2.20 (1.06-4.54) rs7625907
(A>G) AA, AG One GG 1.69 (1.11-2.56) ^* rs9839376
(T>C) TT, TC One One CC 3.52 (1.67-7.45) ^** 2.68 (1.16-6.20) ^* rs1382045
(C>T) CC, CT One TT 1.51 (1.02-2.22) ^*

^* P <0.05, ^** P <0.01, ^*** P <0.001.

Factor Model I Model II Hazard Ratio (95% CI) Hazard Ratio (95% CI) Gestational age at start of drug therapy (≥30 weeks) 6.84 (3.57-13.09) ^** 7.62 (3.95-14.70) ^** Modified Bishop score (≥2) 1.70 (1.08-2.67) ^* KCNMB2 rs7624046
(C>T) CC, CT One TT 2.06 (1.14-3.73) ^* rs9839376
(T>C) TT, TC One CC 2.68 (1.16-6.20) ^*

^* P <0.05, ^** P <0.01, ^*** P <0.001.

In Table 4, Rs7624046 and rs9839376 have a linkage disequilibrium (LD) relationship, and were included in models I and II, respectively.

The results of the above examples provide evidence that the genetic mutation of KCNMB2 affects the effectiveness and safety of ritodrin, and effective use of ritodrin can be expected through the genetic mutation confirmation of pregnant women at risk of premature birth as in the example.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims and their equivalents.

<110> Ewha University - Industry Collaboration Foundation <120> KCNMB2 SNP markers for predicting responsiveness of Ritodrin administration in patients with Early Labor Contraction and use it <130> KPA191718-KR <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs10936979 <400> 1 agaggtaggg gctggagcat gtcaagaagc tgcctacaat gtagaaatac agggtgagga 60 caggtgatga ttttaaagta tgcctcatgt taccagtgag ttccataagg gaaaaaagaa 120 cttaagtcaa gcactggaga ataccatatc 150 <210> 2 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs7624046 <400> 2 tgtttgaaga gaatgtaaca ttggacagaa ttaaagcatc tatttctgga gatttgtgaa 60 atatcattag caaattttag ttcagagggt ataggcatat gaccctcaag aaggtcagta 120 ttcactagaa tatggtttgc tatagcgata 150 <210> 3 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs7625907 <400> 3 ccgaacattc acaagggtct atgctaacac tgacttcgtg acttcataca cccacagaga 60 aatgtgccca tgaacatttg atttgaggaa gctgatggta catcctaagc taatgtcttc 120 aatattttta ctagggaagt tctttaaatt 150 <210> 4 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs9839376 <400> 4 acttgccctt atgaactatt taaatatttc tctgataaat cccaactctg catctaccca 60 aggctcatag gatagattta gtcttctatt tattctggag aaatccaaca ttaatgggta 120 atgaataagc acctatctag ctcctaagat 150 <210> 5 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs1382045 <400> 5 aggctcataa caaaccctgc atatggtagt tattttctgt ctctgataga ccattctccc 60 tttacaacat cttactggac atgttcctac ttgggtgttc tataagataa attcaaactt 120 attaagtccc acaatgattt cagtatttct 150 <210> 6 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs7429015 <400> 6 ataattccca aataagtccc aaaacgattc tccaggaaca ggcaagcttt ctttcactag 60 ctagcaggca ttcatgctaa caaacactga attcaaatga tattcctata aagtagaaat 120 tatcacctga gaattcttct tgaataatga 150 <210> 7 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs6443559 <400> 7 gcccaacttt catttcctca tggtgtaaac tttgcctcag ttaatataag taaaaccagc 60 taaaacaaaa gcacttgcca caactctggg agtggaacat caaactcccc acgctgacct 120 cagttgacag agggcttcct gactcagaga 150 <210> 8 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs9637454 <400> 8 ctgaaaaaag gggaaagaga gagagagagt gtgtgtgtgc gtgtgtgtgt gttgagtgtg 60 catgtgtgcg tgtgtgtgtg atttctcctt attttataaa cagagacttt ttcaatctcc 120 tgctggttcc ttattatttc ctaaaccttt 150 <210> 9 <211> 150 <212> DNA <213> Unknown <220> <223> KCNMB2 rs11918114 <400> 9 gtatgacagt ttagtataga gagaagagac tttctggcag aatcatgcca cttgcttact 60 taactggctc aacaaagcta tacaattata tccatacata gaatgcatga ctacctattt 120 aacagagtag ctaaggaaac agaaaatcac 150 <210> 10 <211> 150 <212> DNA <213> Unknown <220> <223> ADRB2 rs1042719 <400> 10 cttatctact gccggagccc agatttcagg attgccttcc aggagcttct gtgcctgcgc 60 aggtcttctt tgaaggccta tgggaatggc tactccagca acggcaacac aggggagcag 120 agtggatatc acgtggaaca ggagaaagaa 150

Claims (19)

In one or more polynucleotides selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5,
the 78th nucleotide in the nucleotide sequence of SEQ ID NO: 1 and the 70th nucleotide in the nucleotide sequence of SEQ ID NO: 3 are G or A;
the 83rd base in the nucleotide sequence of SEQ ID NO: 2 and the 86th base in the nucleotide sequence of SEQ ID NO: 5 are C or T; or
In the nucleotide sequence of SEQ ID NO: 4, the 77th base is T or C,
The 78th nucleotide in the nucleotide sequence of SEQ ID NO: 1, the 83rd nucleotide in the nucleotide sequence of SEQ ID NO: 2, the 70th nucleotide in the nucleotide sequence of SEQ ID NO: 3, the 77th nucleotide in the nucleotide sequence of SEQ ID NO: 4, or the base of SEQ ID NO: 5 A marker composition for predicting lithodrin administration responsiveness to a preterm labor patient, comprising a polynucleotide consisting of 5 to 150 consecutive bases including the 86th base in the sequence, or a polynucleotide complementary thereto.
The marker composition of claim 1, wherein the responsiveness to administration of ritodrin is an effect or side effect according to administration of ritodrin.
According to claim 1, When the 78th base of SEQ ID NO: 1 is GG or GA in the allele, it is predicted that the ritodrin administration effect is higher than that of AA, the marker composition.
According to claim 1, wherein when the 83rd base of SEQ ID NO: 2 is CC or CT in the allele, it is predicted that the ritodrin administration effect is higher than that of TT, the marker composition.
According to claim 1, wherein, when the 70th base of SEQ ID NO: 3 is AA or AG in the allele, it is predicted that the effect of ritodrin administration is higher than that of GG, the marker composition.
According to claim 1, When the 77th base of SEQ ID NO: 4 is TT or TC in the allele, it is predicted that the ritodrin administration effect is higher than that of CC, the marker composition.
According to claim 1, wherein when the 86th base of SEQ ID NO: 5 is CC or CT in the allele, the ritodrin administration effect is predicted to be higher than that of TT, the marker composition.
The marker composition according to any one of claims 3 to 7, wherein the high effect of administering ritodrin increases the delay time of delivery or lowers the risk of premature birth.
A composition for predicting lithodrin administration reactivity for patients with preterm labor, comprising an agent capable of detecting or amplifying the marker composition of claim 1 .
The method of claim 9, wherein the composition comprises a probe capable of binding to any one or more polynucleotides selected from the group consisting of SEQ ID NOs: 1 to 5 and combinations thereof, or a complementary polynucleotide thereof, or a primer capable of specifically amplifying comprising a composition.
A kit for predicting lithodrin administration responsiveness for preterm labor patients comprising the composition of claim 9.
The kit according to claim 11, wherein the kit is an RT-PCR kit or a DNA chip kit.
A microarray for predicting lithodrin administration responsiveness to a patient with preterm labor comprising the composition of claim 9.
(a) amplifying or hybridizing the polymorphic site of the marker composition of claim 1 from DNA of a sample isolated from an individual; and
(b) comprising determining the base of the amplified or hybridized polymorphic site of step (a),
Information provision method for predicting ritodrin administration responsiveness in patients with preterm labor.
15. The method of claim 14, wherein the method comprises the nucleotide sequence determined in step (b),
When the 78th base of SEQ ID NO: 1 includes GG or GA in the allele, the method comprising the step of predicting that the ritodrin administration effect will be high when the subject of step (a) is a preterm labor contractile patient.
15. The method of claim 14, wherein the method comprises the nucleotide sequence determined in step (b),
When the 83rd base of SEQ ID NO: 2 includes CC or CT in the allele, (a) comprising the step of predicting that the effect of administering ritodrin will be high when the subject of step (a) is a preterm labor contractile patient.
15. The method of claim 14, wherein the method comprises the nucleotide sequence determined in step (b),
When the 70th base of SEQ ID NO: 3 contains AA or AG in the allele, the method comprising the step of predicting that the ritodrin administration effect will be high when the subject of step (a) is a patient with preterm labor contraction.
15. The method of claim 14, wherein the method comprises the nucleotide sequence determined in step (b),
When the 77th base of SEQ ID NO: 4 includes TT or TC in the allele, the method comprising the step of predicting that the ritodrin administration effect will be high when the subject of step (a) is a preterm labor contractile patient.
15. The method of claim 14, wherein the method comprises the nucleotide sequence determined in step (b),
When the 86th base of SEQ ID NO: 5 includes CC or CT in the allele, the method comprising the step of predicting that the ritodrin administration effect will be high when the subject of step (a) is a patient with preterm labor contraction.

Images