KR101850065B1 - Information providing method for drug treatment decisions of hypothyroidism patients by TSHR mutations and DIO2 T92A single nucleotide polymorphism detection - Google Patents

Abstract

The present invention relates to hypothyroidism treatment, and more specifically, to a microarray and kit for diagnosing a patient with hypothyroidism that needs, other than mono-therapy of administering L-T4 (levothyroxine) alone, co-administration of L-T3 (liothyronine), and a diagnosis method using the same. In the present invention, it was confirmed that the expression and activity of DIO2 protein due to thyroid stimulating hormone (TSH) are significantly inhibited when C.274A>G (A on the DIO2 gene encoding the 274th base of the mRNA sequence encoding the DIO2 protein changes to G) homozygote SNP is present in type 2 iodothyronine deiodinase (DIO2) gene, and C.1349G>A (A on the TSHR gene encoding the 1349th base of the mRNA sequence encoding the TSHR protein changes to G) heterozygote loss-of-function mutation is present in thyroid stimulating hormone receptor (TSHR) gene. Thus, the SNP of the DIO2 gene and TSHR mutation may be beneficially used in the diagnosis of a patient with hypothyroidism that needs the co-administration of L-T3.

Description

[0001] The present invention relates to a method of providing information for determining a drug treatment method for hypothyroidism patients using SNP detection of a thyroid stimulating hormone receptor (TSHR) gene mutation and DIO2 (type 2 iodothyronine deiodinase) mutations and DIO2 T92A single nucleotide polymorphism detection}

The present invention relates to a method for providing information for determining a drug treatment method for hypothyroidism patients using SNH (Thyroid Stimulating Hormone Receptor) mutation and SNP (single nucleotide polymorphism) detection of DIO2 (type 2 iodothyronine deiodinase) gene.

Hypothyroidism is a condition in which the thyroid hormone is not well formed in the thyroid gland, so the thyroid hormone level in the body is lowered or deficient. The cause is that the thyroid itself has a problem and the thyroid hormone production is decreased. And a decrease in thyroid hormone production due to signal problems. The most common cause is Hashimoto's thyroiditis (autoimmune thyroiditis), which reduces thyroid hormone production in the thyroid itself. Hypothyroidism may also cause hypothyroidism because thyroid stimulating hormone (TSH) is not secreted. If the thyroid gland is removed because of thyroid cancer, thyroid hormone can not be produced, so hypothyroidism can occur. Key symptoms include chronic fatigue, anorexia, weight gain, cold riding, and constipation. In addition to this, the skin becomes dry, a woman's menstrual cycle changes, and menstrual overload is accompanied. Increased levels of prolactin (prolactin) in the blood can lead to milk secretion. If thyroid hormone is severely deficient, neurological symptoms such as coma may accompany. Thyroid hormone (thyroxine (T4) or triiodothyronine (T3) is needed for the treatment.

L-T4 (Levothyroxine) monotherapy has been applied as a standard therapy in relation to the treatment of hypothyroidism. Even if L-T4, which is an inactive form of thyroid hormone, is administered, triiodothyronine (triiodothyronine, T3 ) Will be fully converted in vivo. However, there is also a question about the efficacy of L-T4 monotherapy because 10% to 15% of patients treated with this therapy still exhibit symptoms of hypothyroidism such as neurocognitive dysfunction and dysfunction. Although clinical relevance has not yet been shown to be consistent in humans, studies in several animal models have shown that maintaining a normal level of T3 in the treatment of hypothyroidism is a priority, and therefore L-T4 and L-T3 liothyronine in combination with raising the level of T3 in serum may be useful in some patients. However, it has not been studied in which patients whether L-T4 alone is ineffective and concomitant administration of L-T3 is necessary.

It is well known that thyroid metabolism is tightly regulated by the hypothalamus-pituitary-thyroid (HPT) axis through a positive and negative feedback mechanism from a macroscopic point of view. However, it is known that deiodinases (DIOs) are involved in the regulation of thyroid hormone at the microscopic level, that is, at the cellular level, but there are few known mechanisms involved in the regulation of thyroid hormones.

Type II diadinase (DIO2) is an enzyme that converts inactive thyroxine (T4) into an active thyroid hormone, triiodothyronine (T3), which is the final concentration determinant of T3 that is active in vivo It plays an important role and is known to be upregulated by TSH. In the case of the DIO2 gene, a specific SNP, ie, a DIO2 gene corresponding to the 274th base of the mRNA sequence coding for the DIO2 protein, has a homozygous SNP changed to A in the case of hypothyroidism, . However, there are some claims that L-T3 can be regarded as a patient requiring simultaneous administration of L-T3. However, the SNP is frequently observed in about 40% of all human species There is a difficulty in describing the SNP alone.

Therefore, the present inventors confirmed that the skin-derived fibroblasts of patients with hypothyroidism, in which the increase of DIO2 activity is significantly suppressed compared to other normal human fibroblasts, despite treatment of TSH, which is generally observed in hypothyroid patients, Analysis of the genotypes revealed that C.274A> G of DIO2 gene (G on A / DIO2 gene encoding 274 base of mRNA sequence encoding DIO2 protein changed to A) homozygous SNP and C1349G> A of TSHR gene (Mutation of A on TSHR gene encoding 1349 base of mRNA sequence coding for TSHR protein to G), the SNP and TSHR of the DIO2 gene Mutation can be used for the diagnosis of hypothyroidism patients requiring simultaneous administration of L-T3, thereby completing the present invention.

The present invention provides a method for providing information for effectively diagnosing a patient requiring simultaneous administration of L-T3 (liothyronine) in the determination of hormone therapy for hypothyroidism, and a kit for the diagnosis.

In order to achieve the above object, the present invention provides a polynucleotide comprising SEQ ID NO: 1 encoding a DIO2 (type 2 iodothyronine deiodinase) protein, wherein the base at position 274 is changed from A to G by a single base polymorphism (C.274A> G), or a polynucleotide comprising its complementary polynucleotide and a polynucleotide comprising SEQ ID NO: 2 encoding a Thyroid Stimulating Hormone Receptor (TSHR) protein, wherein the polynucleotide comprises 10 to 100 consecutive bases L-T4 (Levothyroxine) and L-T3 (SEQ ID NO: 2), which comprise a polynucleotide comprising 10 to 100 contiguous bases, including mutations (C.1349G > A) The present invention provides a microarray for diagnosing hypothyroidism patients who need to be coadministered with lysozyme or liothyronine.

In addition, the present invention provides a diagnostic kit for hypothyroidism patients, which is required to be administered in combination with T4 (Levothyroxine) and L-T3 (liothyronine) containing the microarray.

The present invention also relates to a polynucleotide comprising SEQ ID NO: 1 encoding the DIO2 protein, wherein the nucleotide at position 274 is a single nucleotide polymorphism (C.274A> G) changed from A to G and a nucleotide polymorphism L-T4 (levothyroxine) and L-T3 (polymorphism), which contain a complementary probe or primer set capable of detecting a mutation (C.1349G > A) (liothyronine), which is used for the diagnosis of hypothyroidism.

In addition,

1) separating the nucleic acid from the biological sample separated from the subject; And

2) determining L-T4 (Levothyroxine) comprising the SNP base sequence located at the 274th position of the polynucleotide of SEQ ID NO: 1 from the nucleic acid and the 1349th mutant nucleotide sequence of the polynucleotide of SEQ ID NO: 2 L-T3 (liothyronine) is needed for the diagnosis of hypothyroidism.

In addition,

1) separating the protein from the biological sample separated from the subject; And

2) TSHR protein from the N-terminal to arginine (R) changed to histidine (H) (R450H) and threonine (T) from amino acid N-terminal to alanine (A) (Levothyroxine) and L-T3 (liothyronine), which comprises the step of detecting a DIO2 protein in a sample of a patient with hypothyroidism.

In the present invention, in relation to the diagnosis of hypothyroidism, C.274A> G of DIO2 (type 2 iodothyronine deiodinase) gene (A is changed to G on the DIO2 gene encoding the 274th base of the mRNA sequence encoding the DIO2 protein) A homozygous SNP and TSHR (Thyroid Stimulating Hormone Receptor) gene C.1349G > A (loss of heterozygosity of the TSHR gene encoding a TSHR gene encoding the 1349th nucleotide of the TSHR gene) of-function mutation, the expression and activation of DIO2 protein by thyroid stimulating hormone (TSH) was markedly inhibited, thereby reducing the ability to convert T4 to T3, resulting in the L -T4 (Levothyroxine) alone, in addition to the L-T3 (liothyronine) combined with the need to prescribe a patient with hypothyroidism to diagnose hypothyroidism hypothyroidism By checking for very importance to, SNP and mutation of TSHR the DIO2 gene can be usefully employed in hypothyroidism patients diagnosed require co-administration of T4 (Levothyroxine) and L-T3 (liothyronine).

FIG. 1A shows DIO2 C.274A> G SNP, DIO2 activity, and TSHR gene mutation as a result of a family tree and thyroid function test of hypothyroid patients A and their families.
FIG. 1B shows the mutation of the SNP and TSHR genes of the DIO2 gene according to the result of the nucleotide sequence analysis of the hypothyroid patient A:
TSHR R450H: C.1349G > A (A on the TSHR gene encoding the 1349th base of the mRNA sequence encoding the TSHR protein changes to G); And
DIO2 T92A: C.274A> G (A on the DIO2 gene encoding the 274th base of the mRNA sequence encoding the DIO2 protein changes to G).
FIG. 1C shows thyroid hormone levels in hypothyroid patients A; FIG.
FIG. 2A shows immunohistochemical staining of TSHR in human pituitary tissue. FIG.
FIG. 2B shows the expression of DIO2 and TSHR proteins expressed in C2C12 cells, TaT1 cells, and primary fibroblasts.
FIG. 3A shows the expression of TSHR and DIO2 proteins by TSH or T4 treatment in TaT1 cells and primary fibroblasts:
TSH: TSH No processing (0), 1, 10 IU processing and
T4: T4 No treatment (0), 20, 100 pM treatment.
FIG. 3b shows changes in DIO2 mRNA expression and relative DIO2 activity in fibroblasts of hypothyroid patients with homozygous DIO2 gene (C.274A> G) SNP and heterozygous TSHR mutant gene (C.1349G> A) Fig.
WT (DIO2 WT / WT, TSHR WT / WT): normal DIO2 and normal TSHR gene types of fibroblasts;
Hetero (DIO2 WT / VT, TSHR WT / WT): heterozygous DIO2 gene of fibroblast (C.274A> G) and normal TSHR gene type;
Hemo (DIO2 VT / VT, TSHR WT / WT): homozygous DIO2 gene of fibroblasts (C.274A> G) and normal TSHR gene type; And
Patient (DIO2 VT / VT, TSHR WT / MT): homozygous DIO2 gene (C.274A> G) and heterozygous TSHR mutant gene (C.1349G> A) type of fibroblasts.
FIG. 4A shows relative DIO2 mRNA expression by cAMP treatment in fibroblasts in hypothyroid patients with homozygous DIO2 gene (C.274A> G) SNP and heterozygous TSHR mutant gene (C.1349G> A) .
FIG. 4B is a graph showing the amount of cAMP produced by TSH stimulation in fibroblasts in hypothyroid patients with homozygous DIO2 gene (C.274A> G) SNP and heterozygous TSHR mutant gene (C.1349G> A) to be.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a polynucleotide comprising a polynucleotide comprising SEQ ID NO: 1 encoding a DIO2 (type 2 iodothyronine deiodinase) protein, wherein the nucleotide located at position 274 is a polynucleotide having a base polymorphism (C.274A> G) A polynucleotide comprising 100 consecutive bases or a complementary polynucleotide thereof, and a polynucleotide comprising SEQ ID NO: 2 encoding a TSHR (Thyroid Stimulating Hormone Receptor) protein, wherein a mutation of base G at position 1349 to A (Levothyroxine) and L-T3 (liothyronine), which comprises a polynucleotide comprising 10 to 100 consecutive bases, or a complementary polynucleotide thereof, including the polynucleotide of the present invention (C.1349G> A) A microarray for diagnosing hypothyroidism patients is provided.

More preferably, the continuous base is 10 to 70 contiguous bases, but is not limited thereto.

The microarray comprises a conventional microarray except that the polynucleotide 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 term " 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. Such probes include peptide nucleic acids described in the literature (Nielsen et al., Science, 254, 1497-1500 (1991)). 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 must be sufficiently stringent to hybridize to only one of the alleles, showing significant differences in the hybridization intensity between alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for a diagnostic method or the like for detecting alleles. The diagnostic methods include detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M 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 [deg.] C may be suitable for allele-specific probe hybridization.

The present invention also provides a kit for diagnosing hypothyroidism patients, which is required to be administered in combination with L-T4 (Levothyroxine) and L-T3 (liothyronine) containing the microarray.

The present invention also relates to a polynucleotide comprising SEQ ID NO: 1 encoding a DIO2 (type 2 iodothyronine deiodinase) protein, wherein the nucleotide at position 274 is changed from A to G by single base polymorphism (C.274A> G) and TSHR Thyroid Stimulating Hormone Receptor) A complementary probe or primer set capable of detecting a mutation (C.1349G > A) in which the base G at position 1349 in the polynucleotide consisting of SEQ ID NO: 2 encoding the protein is changed to A (L-T4) (Levothyroxine) and L-T3 (liothyronine).

The kit may contain a reagent necessary for amplification of the primer, and may further include a reagent necessary for amplification of the nucleic acid, wherein the nucleic acid may be labeled with a detectable label. In addition, the detectable label is any one selected from the group consisting of a streptavidin-like phosphatease conjugate, a chemiluminescent substance, and a chemiluminescent substance, But is not limited thereto. The hybridization may further include at least one selected from the group consisting of buffer solution, reverse transcriptase, dNTP, rNTP (pre-mixed or separate feed type), marker reagent, and washing buffer solution But is not limited thereto. In addition, the primer can amplify the SNP as a target site, its size and position to bind to the template are not limited, and those skilled in the art can easily design a primer using conventional primer selection software.

The kit for diagnosing hypothyroidism patients may be prepared by a conventional method known to those skilled in the art. Such a suitable primer set can be easily designed by those skilled in the art with reference to the sequence of the present invention. As used herein, the term "probe" means a linear oligomer having a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a particular nucleotide sequence. Preferably, the probes are single strands for maximum efficiency in hybridization. The probe is preferably a deoxyribonucleotide.

The kit may further comprise a fluorescent material, wherein the fluorescent material is selected from the group consisting of a strepavidin-like phosphatease conjugate, a chemiluminescent substance, and a chemiluminescent substance But it is not limited thereto.

The reaction reagent may further include a buffer solution used for hybridization, a reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (pre-mixed or separate feed type), a chemical inducer for fluorescent dye And a washing buffer solution. However, the present invention is not limited thereto. Any reaction reagent necessary for hybridization reaction of a DNA microarray chip known to a person skilled in the art can be included.

In addition,

1) separating the nucleic acid from the biological sample separated from the subject; And

2) determining L-T4 (Levothyroxine) comprising the SNP base sequence located at the 274th position of the polynucleotide of SEQ ID NO: 1 from the nucleic acid and the 1349th mutant nucleotide sequence of the polynucleotide of SEQ ID NO: 2 L-T3 (liothyronine) is needed for the diagnosis of hypothyroidism.

The expression of TSHR (C.1349G> A) protein and DIO2 (C.274A> G) protein in the step 2) is preferably measured using a mutant protein-specific antibody, but not limited thereto, But are not limited to, blood, tissues, cells, serum, plasma, saliva, sputum and urine.

In the above method, the step of extracting the genomic DNA from the sample isolated from the subject can be carried out by a method known in the art. For example, in order to prepare pure DNA when a DNA of interest is present in a cell, a cell extract may first be prepared and then further subjected to differential precipitation, column chromatography, organic solvent extraction, etc., And can be manufactured by standard techniques in the field. DNA isolated from cells or tissues can be directly purified or amplified by specific amplification of a specific region using amplification methods such as PCR or RT-PCT (real time PCR) I never do that. The DNA includes not only DNA but also cDNA synthesized from mRNA. In addition, PCR or RT-PCR may be performed on the entire DNA sequence of an individual, but may also be performed only around a region known as a single base polymorphism.

In this method, determination of the base sequence of the separated DNA can be performed by various methods known in the art. For example, a probe comprising a sequence of a single nucleotide polymorphic site or a probe complementary thereto, which hybridizes through the nucleotide sequence of DNA directly determined by the dideoxy method, is hybridized with the DNA, and the hybridization degree obtained therefrom is measured, A method of determining the nucleotide sequence of the nucleotide sequence of SEQ ID NO: The degree of hybridization can be detected by marking a detectable label on the target DNA and specifically detecting only the hybridized target DNA, but other electrical signal detection methods can be used. Specifically, a hybridization with a microarray, an allele-specific hybridization, an allele-specific amplification, a sequencing, a 5 'nuclease digestion (allele-specific hybridization, 5 'nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-stranded conformation polymorphism. But is not limited thereto.

In addition,

1) separating the protein from the biological sample separated from the subject; And

2) Thyroid Stimulating Hormone Receptor (TSHR) protein whose arginine (R) as the 450th amino acid from the N-terminal is changed to histidine (H) (C.1349G> A) and threonine The combination of L-T4 (Levothyroxine) and L-T3 (liothyronine), which involves the step of detecting a DIO2 (type 2 iodothyronine deiodinase) protein that has been changed to alanine (A) (C.274A> G) Provides a method of providing information necessary for the diagnosis of a hypotonia patient.

The expression of TSHR (C.1349G > A) protein and DIO2 (C.274A > G) protein in the step 2) is preferably measured using a mutant protein specific antibody, but is not limited thereto.

The sample is preferably selected from the group consisting of hair, blood, tissue, cells, serum, plasma, saliva, sputum and urine, but is not limited thereto.

In a specific example of the present invention, the present inventors analyzed the DIO2 and TSHR gene mutations in patients with hypothyroidism, and found that the G base on the genomic DNA corresponding to the 1349th base of the TSHR cDNA sequence mutated from one allelic gene to A (Heterozygous mutation for TSHR C.1349G> A) and also has a SNP (Single Nucleotide Polymorphism) with a G base on the genomic DNA corresponding to nucleotide 274 of the DIO2 cDNA sequence in both alleles (homozygous SNP for DIO2 C.274A > G) (see FIG. 1B). Due to the C1349G> A mutation of the TSHR gene, the 450th amino acid arginine (R) is changed to histidine (H) (R450H) from the N-terminus, and the C2274A> G SNP of the DIO2 gene causes N- The threonine (T), which is the 92nd amino acid, changes to alanine (A) (T92A).

Thus, the present inventors performed immunochemical staining using a human pituitary tissue slide sample to examine the expression of TSHR protein in human pituitary gland tissue. As a result, TSHR protein was expressed in human pituitary tissue (see Fig. 2a). In addition, DIO2 and TSHR protein expression was examined in various cell lines and primary skin fibroblasts. As a result, it was confirmed that DIO2 protein and TSHR protein were expressed in both C2C12 cell line and TαT1 cell as well as primary skin fibroblast (see FIG. 2b).

The present inventors also investigated the change of DIO2 activity according to C1349G> A mutation of TSHR gene and C.274A> G SNP of DIO2 gene. First, TSHR and DIO2 protein expression in the TaT1 and IMR90 fibroblasts were examined by TSH or T4 treatment. As a result, TSHR decreased in TSH treated group (TaT1 and fibroblasts) Protein expression was increased. On the other hand, in the group treated with T4 (TaT1 and fibroblasts), it was confirmed that the protein expression of both TSHR and DIO2 was decreased in a T4 concentration-dependent manner (see FIG. In addition, the expression of DIO2 mRNA and DIO2 activity in TSF-treated skin fibroblasts of patients with hypothyroidism were examined by the TSH-stimulated DIO2 mRNA expression in fibroblasts with TSHR gene regardless of the presence of T92A SNP And DIO2 activity were increased. However, it was confirmed that DIO2 mRNA expression and activity were not significantly increased in fibroblast (heterozygous TSHR R450H + homozygous DIO2 T92A) derived from a hypothyroid patient A (see FIG. 3B).

In addition, the present inventors tried to confirm the regulation of the production of cAMP by TSH in fibroblasts derived from patients with hypothyroidism. We investigated whether the change of DIO2 activity by TSH treatment was related to the degree of cAMP synthesis by mutation of TSHR. The promoter of the DIO2 gene contains a cAMP response element, which can induce the expression of DIO2 by cAMP alone without TSH stimulation. The mRNA expression of the DIO2 gene was significantly increased by the cAMP treatment regardless of whether the DIO2 gene SNP or the TSHR gene was mutated in the irradiated cells (see FIG. 4A). In addition, the degree of synthesis of cAMP by mutation of SNP and TSHR gene of DIO2 gene during TSH treatment was examined. As a result, in case of cells derived from heterozygous TSHR R450H + homozygous DIO2 T92A, it was confirmed that the synthesis of cAMP was inhibited (see Fig. 4B). This corresponds to the results of the mRNA expression and activity change of DIO2 by TSH in FIG. 3c. It is considered that the lack of sufficient cAMP in the fibroblasts of patients with hypothyroidism during TSH treatment is a cause Respectively.

Thus, C.274A> G of DIO2 (type 2 iodothyronine deiodinase) gene (homozygous SNP and Thyroid Stimulating Hormone Receptor (TSHR) (the A on the DIO2 gene encoding the 274th base of the mRNA sequence encoding the DIO2 protein is changed to G) ) Mutation of the gene C.1349G > A (loss of function mutation of A on the TSHR gene encoding the 1349th base of the mRNA sequence encoding the TSHR protein to G) It was confirmed that the expression and activation of DIO2 protein by thyroid stimulating hormone (TSH) was significantly inhibited. The SNP and TSHR mutations of the DIO2 gene were useful for the diagnosis of hypothyroidism patients requiring concurrent administration of L-T3 Lt; / RTI >

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Thyroid function test and genetic analysis in hypothyroid patients and their families

<1-1> Genetic profile of patients with hypothyroidism (A) and their families

A 68-year-old Korean male (I-1 in Fig. 1a) visited a tertiary care facility with hypothyroidism. The main symptom of this patient was anorexia and weight gain for 3 months. Thyroid stimulating hormone (TSH) and serum free T4 (free T4, fT4) were measured at the local hospital in the range of 85.50 mIU / L (0.27-4.20) and 0.16 ng / dL (0.93-1.70), respectively. This patient was prescribed to take 100 μg of L-T4 daily for 3 months and the drug intake improved symptoms associated with hypothyroidism, but serum TSH levels were not suppressed through the treatment (FIG. 1c).

The thyroid function test and thyroid autoantibody test showed TSH 98.74 mIU / L, fT4 1.10 ng / dL, T3 64.93 ng / dL (80.00-200.00), Tg antibody 114.8 IU / mL (0.0-115.0) 16.34 IU / mL (0.00-34.00), and TSH-receptor antibody 3.0 IU / L (0.0-1.5). Initially, immunological analysis of serum TSH levels was considered to be erroneous due to heterophile antibody interference, but all three tests for this interference were negative. When 30 μg of L-T3 was administered three times a day instead of 100 μg of L-T4 per day, the serum TSH level rapidly decreased to 5.53 mIU / L. Thus, L-T4 was prescribed with L-T4 and the patient's thyroid function remained within the normal range for 5 years (Figure 1c).

The patient's eldest son (II-1) had asymptomatic thyroid dysfunction and TSH 4.97 mIU / L, fT4 1.41 ng / dL, T3 103.00 ng / dL, Tg antibody 16.3 IU / mL, TPO antibody 16.4 IU / mL, and TSH-receptor antibody 0.8 IU / L. (TSH 2.78 mIU / L, fT4 0.99 ng / dL, T3 112.50 ng / dL, Tg 112.5 ng / dL, and Tg of the patient were found to be positive for thyroid disease antibody 55.7 IU / mL, TPO antibody 294.2 IU / mL, and TSH-receptor antibody 2.5 IU / L. Both the patient's daughter (II-4) and the youngest son (II-5) were asymptomatic hypothyroidism and the patient's grandchild (III-1) was taking L-T4 as congenital hypothyroidism. In addition, TSH of grandchildren was appropriately inhibited by administration of L-T4, indicating that it has normal DIO2 (type 2 iodothyronine deiodinase) activity.

FIG. 1A shows gene family diagrams and results of thyroid function tests showing the allele genotype of DIO2 activity, TSHR (Thyroid Stimulating Hormone Receptor) and DIO2 genes of patient A and his family. Serum TSH, free T4 and T3, TPO antibody, Tg antibody and TSHR antibody levels were measured by immunochemiluminescence and serum selenium was measured by plasma - mass spectrometry.

<1-2> Patients with hypothyroidism (B)

A 42 - year - old Korean man with a well - differentiated papillary thyroid cancer underwent thyroidectomy and radical neck node dessection. Because thyroid malignancy was found in multiple lymph nodes, radioactive iodine ablation (100 mCi) was also administered. Because L-T4 did not inhibit increased TSH even though it was increased stepwise up to 250 μg, dose of L-T4 was reduced to 100 μg to prevent thyrotoxic symptom and L-T3 30 &Lt; / RTI &gt; Subsequently, the level of TSH decreased within the normal range (Table 1).

<1-3> Patients with hypothyroidism (C)

A 75 - year - old female patient has been complaining of a huge goiter (diameter 10 cm) since her early 20s. The patient's thyroid function test results showed extremely high TSH levels, high fT4 levels and relatively low T3 levels (TSH 82 mIU / L, fT4> 7.70 ng / dL, and T3 64 ng / dL without thyrotoxic symptoms) . When 30 μg of L-T3 was administered twice a day, the size of the goiters was significantly reduced and the levels of fT4 were not different but the level of TSH was decreased and the level of T3 was increased (Table 2A). Thyrotropin-releasing hormone (TRH) stimulation test results showed that abnormally increased TSH was not caused by pituitary adenomas secreting RTH or TSH.

<1-4> Hypothyroidism  Patient DIO2  And TSHR  Gene analysis result

The TSHR and DIO2 gene mutations were analyzed for the above patients. Specifically, for gene analysis, gDNA was isolated from blood samples by standard extraction method and whole exome sequencing was performed. The pre-enrichment DNA library was prepared according to the Illumina TrueSeq DNA sample preparation guide (Illumina TrueSeq DNA sample preparation, Illumina, Inc., San Diego, CA, USA). Exome enrichment was performed using Illumina TrueSeq Exome Enrichment probes and streptavidin beads. The enriched exome library was loaded into the Illumina cBot for cluster formation and the flow cells with clusters of the enriched exome libraries were transferred to the Illumina HiSeq2000fh and the captured sequencing depths of approximately 40 times the average sequencing depth (High-throughput sequencing) was performed. TSHR mutation and DIO2 SNP were also confirmed by Sanger seqencing method. gDNA is obtained by using a primer for the exon of the TSHR gene described in SEQ ID NO: 3 and SEQ ID NO: 4 below and a primer for exon 2 of the DIO2 gene described in SEQ ID NO: 6 and SEQ ID NO: 6 below The PCR products were analyzed by ABI 3500xL DNA Analyzer.

As a result, patient A (I-1 in FIG. 1A) had mutated G bases on the genomic DNA corresponding to the 1349th base of the TSHR cDNA sequence shown in SEQ ID NO: 2 from one allelic gene to A (heterozygous mutation for TSHR C.1349G > A) also has a SNP (Single Nucleotide Polymorphism) with a G base on the genomic DNA corresponding to nucleotide 274 of the DIO2 cDNA sequence described in SEQ ID NO: 1 in both alleles (homozygous SNP for DIO2 C.274A > G) (FIG. 1B). Due to the C1349G> A mutation of the TSHR gene, the 450th amino acid arginine (R) is changed to histidine (H) (R450H) from the N-terminus, and the C2274A> G SNP of the DIO2 gene causes N- The threonine (T) which is the 92nd amino acid changes to alanine (A) (T92A)).

Patients B and C also carried the homozygous SNP for DIO2 C.274A> G in the DIO2 gene and C.274A> G SNP in the heterozygous for the TSHR gene (Patient B is D727E (GAC> GAG) and Patient C is F525S (TTC> TCC).

order SEQ ID NO: TSHR exon primer (F) 5'-CTGGGCAATGTCTTTGTCCTGCTTATTC-3 ' SEQ ID NO: 3 TSHR exon primer (R) 5'-TATGTGTTGGGGGTGTCATGGGATTG-3 ' SEQ ID NO: 4 DIO2 exon2 primer (F) 5'-TGTGAATTCAAGTGGCAATG-3 ' SEQ ID NO: 5 DIO2 exon2 primer (R) 5'-CCAATAGGGCTCTGTTGAAA-3 ' SEQ ID NO: 6

Human pituitary tissue and cell line ( C2C12  Cells and TaT1  Cells), skin fibroblasts (primary cells) TSHR  Protein expression

The role of DIO2 in voice regulation by TSH-mediated hypothalamic-pituitary-thyroid (HPT) axis is well known. In this connection, DIO2 and TSHR are known to be expressed together in human pituitary gland, mouse thyroid stimulating cell TαT1 cells, and DIO2 and TSHR are known to be expressed in various cells, especially myoblast C2C12 cells, human osteoblasts and human fibroblasts It has also been reported to be expressed together in fibroblasts. Accordingly, the present invention has been made to confirm this.

&Lt; 2-1 > TSHR  Confirm

Immunohistochemical staining was performed using human pituitary tissue slide samples to investigate TSHR protein expression in human pituitary tissue.

Specifically, human pituitary tissues were fixed with cold 4% paraformaldehyde in 0.1 M PBS, pH 7.4, and paraffin blocks were prepared. After 4-5 탆 sections were mounted on glass slides, (Xylene 3 times for 5 minutes, 2 times for 10 minutes in 100% ethanol, 2 times for 10 minutes in 95% ethanol, twice for 10 minutes in 70% ethanol and twice for 5 minutes in water) process). Samples of prepared tissue slides were prepared by treating the primary antibody against DIO2 or TSHR (DIO2 antibody (1: 1000 dilution, Abcam) and TSHR antibody (1: 250 dilution, Abbiotec) at room temperature for 2 hours, Secondary antibody, FITC goat anti-rabbit IgG antibody (1: 500 dilution, Invitrogen) was treated with DAPI for one hour at room temperature and then observed with fluorescence microscope.

As a result, as shown in FIG. 2A, TSHR protein was expressed in human pituitary gland tissue.

<2-2> Cell line ( C2C12  Cells and T? T1  Cells) and dermal fibroblasts DIO2  And TSHR protein expression

In addition to the tissue of Example <2-1>, DIO2 and TSHR protein expression was also examined in various cell lines and primary skin fibroblasts.

Specifically, C2C12 cells, TαT1 cells, and primary dermal fibroblasts were each divided into 2 × 10 ⁴ cells / well (6-well plate), cultured for 24 hours, and then the cells were recovered. The protein obtained by lysis with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris buffer, pH 8.0) was electrophoresed on 12% (1: 2000, Abcam), anti-TSHR antibody (1: 1500, Abbiotec (1: 2000), and anti-CD30 antibody ) And anti-actin antibody (1: 2000, Abcam) overnight, washed with PBS containing 0.1% Tween-20, treated with secondary antibody for one hour at room temperature and then treated with WEST-ZOL plus ECL Western blotting detection system (iNtRON BioTechnology).

As a result, it was confirmed that both DIO2 protein and TSHR protein were expressed in C2C12 cell line, TαT1 cell as well as primary skin fibroblast, as shown in FIG. 2B.

TSHR  C.1349G > A mutation of the gene and DIO2  Changes in DIO2 activity according to C.274A> G SNP of a gene

<3-1> TSH  Or T4 treatment T? T1  In cells and fibroblasts TSHR  And DIO2  Identification of Protein Expression

We performed western blotting to investigate changes in TSHR and DIO2 protein expression in TaT1 cell lines and IMR90 fibroblasts treated with TSH or T4.

Specifically, TaT1 cells and primary dermal fibroblasts were each divided into 2 × 10 ⁴ cells / well (6 well plate), cultured for 24 hours, and then cultured in serum-free medium for 18 hours. Then, TSH (1, 10 IU) was treated for 8 hours or T4 (20, 100 pM) for 16 hours and western blotting was performed as described in <2-2> above.

As a result, it was confirmed that TSHR decreased in TSH-treated group (TaT1 and fibroblasts), but protein expression of DIO2 increased. On the other hand, in the T4-treated group (TaT1 and fibroblasts), it was confirmed that the protein expression of both TSHR and DIO2 was decreased in a T4 concentration-dependent manner.

<3-2> In skin fibroblasts of hypothyroid patients TSH  Depending on the treatment DIO2  mRNA expression and DIO2  Check for changes in activity

Recently, increased TSH in children has been reported to be associated with the conversion of T3 / T4 and T4 to T3 suggesting that TSH functions in the conversion of T4 to T3 via DIO2. Therefore, the present inventors investigated DIO2 mRNA expression and DIO2 activity according to TSH stimulation.

Specifically, hypothyroidism patients (heterozygous TSHR C.1349G> A + homozygous DIO2) and control (Wild type (WT) (TSHR WT + DIO2 WT), Hetero (TSHR WT + heterozygous DIO2 C.274A> G), Homo ( The skin tissue extracted from TSHR WT + homozygous DIO2 C.274A > G) was cut very finely and washed with PBS, and then cultured in DMEM medium supplemented with 40% FBS, changing the medium once a week. Were cultured in DMEM medium supplemented with 15% FBS, penicillin (100 U / mL) and streptomycin (100 ug / mL) for up to 10 times. All cultured cells were cultured at 5% CO ₂ and 37 ° C., In the experiment, cells cultured from No. 5 to No. 10 were used. The fibroblasts cultured by the above method were treated with TSH 1 and 10 IU (including untreated control group), respectively, and 3 hours, 6 hours, Relative DIO2 mRNA expression and DIO2 activity Total RNA (total RNA) was extracted from the obtained cells and quantitatively RT-PCR was performed to measure the mRNA expression amount. Total RNA was purified using a TRIZol (Invitrogen, USA) The RNA extracted with oligo dT primer was reverse transcribed using the RevertAid (TM) H Minus First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, USA), and ligated with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, All PCR amplification reactions were performed in a total volume of 25 μl containing 150 ng of cDNA using the SYBR Green I qPCR kit (TaKaRa, Shiga, Japan). 5'-TCCAGTGTGGTGCATGTCTC-3 '(Forward Primer) and 5'-CTGGCTCGTGAAAGGAGGTC-3' (Reverse Primer) described in SEQ ID NO: 7 and SEQ ID NO: 8 as the primers for amplification of DIO2 were used, As primers for Gapdh amplification for comparison, 5'-TGTTGCCATCAATGACCCCTT-3 '(Forward Primer) and 5'-CTCCACGACGTACTCAGCG-3' (Reverse Primer) described in SEQ ID NO: 9 and SEQ ID NO: 10 below were used. Normalization using the expression level of Gapdh was performed by the comparative threshold (Ct) method by the manufacturer (Applied Biosystems). For the DIO2 activity measurement, the skin fibroblasts obtained from the medium were washed with cold PBS, centrifuged (4C, 3000 rpm, 10 minutes), and the cell lysate buffer (100 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 20 mM DTT) and then reacted with a certain amount of [125 I] T4 or [125 I] rT3 by sonication. 100 μl 2% BSA and 800 μl 10% trichloroacetic acid was added to terminate the reaction. The 125 I released was separated by column chromatography using AG 50W-X2 resin, and 125I was counted. The de-iodination activity was calculated as femtomoles of iodine released per mg of protein per hour.

As a result, as shown in FIG. 3B, the expression of DIO2 mRNA and DIO2 activity were increased by TSH stimulation in fibroblasts in which the TSHR gene was WT, irrespective of the presence or absence of C.274A> G SNP in the DIO2 gene. ( Heterozygous TSHR C.1349G> A + homozygous DIO2 C.274A> G) derived from a hypothyroid patient with a hypothyroidism of -1> did not significantly increase DIO2 mRNA expression and activity.

order SEQ ID NO: DIO2 amplification primer (F) 5'-TCCAGTGTGGTGCATGTCTC-3 ' SEQ ID NO: 7 DIO2 amplification primer (R) 5'-CTGGCTCGTGAAAGGAGGTC-3 ' SEQ ID NO: 8 Gapdh amplification primer (F) 5'-TGTTGCCATCAATGACCCCTT-3 ' SEQ ID NO: 9 Gapdh amplification primer (R) 5'-CTCCACGACGTACTCAGCG-3 ' SEQ ID NO: 10

Hypothyroidism  In patient-derived fibroblasts TSH  by cAMP  Generation control

The expression of DIO2 gene by TSH is known to be mediated by cAMP synthesized through activation of TSHR receptor protein. Thus, it was examined whether the change of DIO2 activity according to the genotype by the TSH treatment described in Example 3 was related to the degree of cAMP synthesis according to mutation of TSHR.

<4-1> to cAMP  by DIO2  gene mRNA  Regulation of expression

The promoter of the DIO2 gene contains a cAMP response element, which can induce the expression of DIO2 by cAMP alone without TSH stimulation.

First, primary dermal fibroblasts were divided into 2 × 10 ⁴ cells / well (6 well plate), cultured for 24 hours, and incubated for 18 hours in serum-free medium. Then, 1 mM DB-CAMP (Sigma) To obtain cells. Total RNA (total RNA) was extracted from these cells and quantitative RT-PCR was performed. Specifically, the total RNA was extracted according to the manufacturer's protocol using a triazole (TRIzol, Invitrogen, USA) reagent. RNA extracted with oligo dT primers was reverse transcribed using the RevertAid (TM) H Minus First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, USA), and the RNA was reverse transcribed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, USA) Real-time quantitative RT-PCR was performed. All PCR amplification reactions were performed with SYBR Green I qPCR kit (TaKaRa, Shiga, Japan) and in a total volume of 25 μl containing 150 ng of cDNA. 5'-TCCAGTGTGGTGCATGTCTC-3 '(Forward Primer) and 5'-CTGGCTCGTGAAAGGAGGTC-3' (Reverse Primer) described in SEQ ID NO: 7 and SEQ ID NO: 8 as the primers for amplification of DIO2 were used, As primers for Gapdh amplification for comparison, the expression amounts of 5'-TGTTGCCATCAATGACCCCTT-3 '(Forward Primer) and 5'-CTCCACGACGTACTCAGCG-3' (Reverse Primer) Gapdh described in SEQ ID NO: 9 and SEQ ID NO: Normalization was performed using a comparative threshold (Ct) method by a manufacturer (Applied Biosystems).

As a result, as shown in FIG. 4A, the mRNA expression of the DIO2 gene was significantly increased by the cAMP treatment irrespective of whether the DIO2 gene SNP or the TSHR gene was mutated in the irradiated cells.

<4-2> TSH  At processing DIO2  Gene SNP, TSHR  Regulation of the synthesis of cAMP by mutation of the gene

We investigated whether the difference in the activity of DIO2 according to genotype during TSH treatment was related to the degree of cAMP synthesis.

Specifically, primary skin fibroblasts were divided into 2 × 10 ⁴ cells / well (6 well plates), cultured for 24 hours, and cultured in serum-free medium for 18 hours. TSH was treated with 1 IU for 30 minutes. For cAMP analysis, the cells were first treated with 0.1 N hydrochloric acid, phosphodiesterase activity was inhibited, and the cells were lysed. After centrifugation at 600 g at room temperature, supernatant ). cAMP was measured using a cAMP direct immunoassay kit (Millpore) according to the manufacturer's protocol and repeated three times in total.

As a result, as shown in FIG. 4B, in the case of cells derived from hypothyroid patients (heterozygous TSHR C.1349G> A + homozygous DIO2 C.274A> G), the synthesis of cAMP by TSH was inhibited there was. This corresponds to the results of the mRNA expression and activity change of DIO2 by TSH in FIG. 3c. It is considered that the lack of sufficient cAMP in the fibroblasts of patients with hypothyroidism during TSH treatment is a cause Respectively.

<110> Gachon University Gil Medical Center <120> Information providing method for drug treatment decisions          hypothyroidism patients by TSHR mutations and DIO2 T92A single          Nucleotide polymorphism detection <130> 2016P-07-055 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 5985 <212> DNA <213> Homo sapiens <400> 1 atgggcatcc tcagcgtaga cttgctgatc acactgcaaa ttctgccagt ttttttctcc 60 aactgcctct tcctggctct ctatgactcg gtcattctgc tcaagcacgt ggtgctgctg 120 ttgagccgct ccaagtccac tcgcggagag tggcggcgca tgctgacctc agagggactg 180 cgctgcgtct ggaagagctt cctcctcgat gcctacaaac aggtgaaatt gggtgaggat 240 gcccccaatt ccagtgtggt gcatgtctcc agtacagaag gaggtgacaa cagtggcaat 300 ggtacccagg agaagatagc tgagggagcc acatgccacc ttcttgactt tgccagccct 360 gagcgcccac tagtggtcaa ctttggctca gccacttgac ctcctttcac gagccagctg 420 ccagccttcc gcaaactggt ggaagagttc tcctcagtgg ctgacttcct gctggtctac 480 attgatgagg ctcatccatc agatggctgg gcgataccgg gggactcctc tttgtctttt 540 gaggtgaaga agcaccagaa ccaggaagat cgatgtgcag cagcccagca gcttctggag 600 cgtttctcct tgccgcccca gtgccgagtt gtggctgacc gcatggacaa taacgccaac 660 atagcttacg gggtagcctt tgaacgtgtg tgcattgtgc agagacagaa aattgcttat 720 ctgggaggaa agggcccctt ctcctacaac cttcaagaag tccggcattg gctggagaag 780 aatttcagca agagatgaaa gaaaactaga ttagctggtt aaaggtatga ttataagaga 840 gcttattgtt ttaaaaagtt atataaaggc aaggaaatta agaactgaat ccatatttca 900 acagagccct attggcttac tgaaagacag gagtttatct atcggaagaa catgaatctc 960 taacagctcc atacttcttt cactactcaa atggcattgg gctgagtaag taaccatatc 1020 acctctcttc ttagtaaaaa gccctatgtg aaaagatccc aagatggaga ggaagaaacg 1080 ctaattcagc atgtgttcat tctgcattga gaaggaactg atacatctga tgcatgcttt 1140 gagaccagaa gaaaagactt acctgaataa ttactacatt agggaagcta ctgtctacgt 1200 taagataaag ggtattgcct tggctctatt tggcatggat ggagcccagt tggaaaattc 1260 ccaaatatta caacaagtcc ttgaacccag gccatgtggt tagacgttgg tgttaaggtt 1320 agaccttatg ttagagtcat ttctgatgtt ccagcttcta gccatgtagt gctctcagtc 1380 ttcatacccc agaaattatt ggtatatttg tagataccga gaatgatccc tcagtctgag 1440 aggttagaat gatcatctgt aatctgaggg ttaatttcta ggcaggtgga gagagtggta 1500 aaaaagaaat gaaattgaca agctaggaaa gaggaggcag aaagatttgg aaaattcaca 1560 gagtttcacc cttaagctgt agagagtggg tcacatttgt tagccacgga aacatagaaa 1620 catacacaag gccagaaaaa gaagaaggag ctcaactaaa agtggcatag agaatacaca 1680 tataaaaaca atatatttgt catatgctcc tagagaggag aaaggggtga ttgaaagaaa 1740 aaaaaatact taaatatttg taattgtgag gggtttcttt tggaaataat tacttttgaa 1800 ccatgtatgt ggtatgtata ttttcagtgg gttaattata ccccatgata cctattaaag 1860 gaaaaccagt gggtctggtg gtgctggtct tttcctcccc attcctacaa tttctatgtg 1920 gcccaagtca ttcctaatct tggtctctat agcagtgttc tctctgaatg ctgagctgaa 1980 gaaattatac gtacatacac acatacatac atacatacaa atatatgtat atatattctc 2040 agctgctgcg ggaggtaggt accatggcca ttcagcacag ccttgatttc ctcccaaagt 2100 aggtgagcta tagtgaagaa taggtgcaaa caaacaagct tacttccatt gcaaaataga 2160 agaagaggaa gttagagata attctgatca atcattttgg aggctttgtt ataaggcaac 2220 ccccggtata tcatggaatt tccattgaca tttgaatttg gacttggatc ttcccttggt 2280 cccattagct gaggtttagt aatctaaagt ccctatagta tatgattata atgctatttt 2340 aaaaaatata tatataaaat atttttttct ttttaaaata gacactatag ttttacccat 2400 aagtaatatt taaagattat agctcccaaa agaatggacc aaccactttc gtatcataat 2460 ttctttttgg taaatatgag actattatga aatcatagta tatgattgta tttaaaggta 2520 caatcaaagg atcttttgtc cattccatta ataactgaat aaaaaataaa taaaatggat 2580 agaaaaaaac taaagttgaa aatacattct taaactagtt gtctgaattg agaaaagagt 2640 gagaactagg tgtgcaagaa ccaaacgtat tttattttat tttttaaatg ggagcaacat 2700 atcagtcgtg tcaccagctg gtatattgtg taaatattaa agctccattg ggactgattt 2760 ttcatggcaa catcagcttt ctaatgttct aaattctata aaaaccaccc acaaagaaac 2820 aaagcaaatt tcattatcta atgagttgct ggaaaatcat attgagaata attatttcag 2880 attcctcagt tgttaacttc tacattcaag gcttatctct gcccccattg atttttaacc 2940 tcaaaatggt gttgagattt acgtggaacc ctaaagcagt aaaataaaaa acctggttgc 3000 agcacattca cactgttgtc cttaaaattc cccttttttc tctatgtacg ataaagtaac 3060 agtatgtcag ataagccggt ggggggatga gattaggctg aggcagtgct agtcaactgg 3120 ggaaaaggat gatggaaaaa tcacccagtt gtgctatatt tttaaagaag gaggtcgttt 3180 atgtgtgcag acaattctcc ctgaggttag cccaatggag aaatgaagca gaggaaggaa 3240 acatagaaag acatgggcta tcagggagga agatgttcaa tagaacatgc aagaatttct 3300 ggaagaaagg ctgtggaagg gccaatggag aaaatgaatg gacaaagctc aggaatccta 3360 cgctatgtag aatgtcttgg tgttatcagg gttaagcctg taattatgta acctatttat 3420 cgcaacatga atttttatga tttcttgtga tgtattcttt tatgaaatta acaagaactc 3480 attattttga ggtagaggaa aatcaatgct ttatctgata tgctgagaaa ttattagatt 3540 gccaatactc atgtgcgttt catgtgtttt ataaggtttg ttcctttgaa gaattgtagt 3600 tcttagtccc acagggaaat gtgtatctat ttatatatca tagtataaat ctatgatata 3660 tttatatcat atataaaagt ctgagttctc tttcttagtc cctaatcatg tttctcccat 3720 aggctgtgtt tacatggagc tatcggttta gccttttaag cttcattagc ttgtctatta 3780 ttgaaatagt ttccaagaaa ttttagatat tatcataaca tctgggtcta ctcaaacact 3840 tattgtttga aagacttatg tcttggacct atcaaaaact gactttattt attgcttagt 3900 gaaaatacta gtgggatcaa caatgatttt cttgaatggg catgaatgga gatgcccgca 3960 cagtaatgta gaaatgtttc atacagctat taaaatgtaa ctgacctcct tagaggcaga 4020 ttagtaactg ttcctacttt gtatagctaa gtgacagtca cttaacttac atgactttct 4080 tttttcacat tgggtctctg gtcctgtgtc ttcacctcat ttatagcacg tctccttgat 4140 ttttggtagt atcaacttcc cagtgatctg ttcagttaag ttcttctccc gttaaccagg 4200 aagtgcttat tctctcatca cagtgggaag aatagcctat tgtctttcat tttgcctgag 4260 tgtattttac tatttgggct ctgaaataaa aattatgaaa tatggtgagg tcacatgttg 4320 gtgctgcctt gctgcataaa attctagagg gcaggttaga gacagtatgt atgccttcgg 4380 gaaaattcaa aggtggatta caaggtgtcc tcagcatgcc ctatggccta tgtgcgaagc 4440 aagaagaatt gactgattta caggacttct ctttatgtca atcttaagag gatggatgaa 4500 tctggacatt tgttccaccc gacctctgac tgatggtttg gaaaataact ttaattagga 4560 tcatatgacc attgaaaaag gaaaaatgta gactctgact tccgtcccac tgaaggatta 4620 atgaaaacct ttactagcat ttagagcttt tcagaacatc cccactgtca tgtgtctcag 4680 cagtggagac tgcaagtaag gcttttaatt ttaggaggtt tttttttttt tttttttttc 4740 cctaaatggt atggccaaaa gtcagagtta aaatatatat agttagattc caacttcctc 4800 cttcactcta aaaatagaat ccaaacccac tcttcatata tgcttccaga atggggctta 4860 agtaccaatc tctgctttgc aatgggcaca atcttggtca tgtcctgagg ctctctaaga 4920 aaagagagga tctaggatgg gagagctaga aagttgctaa ctgggaagaa caaggccctg 4980 agggcttggt ctaccaatct gggaagattt gaaaacaaac ttctcgcaac tgaaggaagg 5040 ctgaaggctg ctgcaagtca ttgagtgact ttaggatgag caaaacattg ggccacttcc 5100 taatgcccta tgtgtatagt accagaagca aggtctcaga cttaacagac ccagctctgt 5160 tccaaggtga gtctgaacca atagaaagca aacatgtgca gatatccaaa caagactgct 5220 catgcaagtc ggggctggct acccgtctta ggcagcaaca gcagagctcc agggagctta 5280 ttcaatattt actgagactt cgaagaccca gcagatgttt aatgaagtca ctattttggc 5340 tcaaaccctc cacttctccc cctcccctca aaaagccaac aggtaaacac ataaatgaaa 5400 gaaacccaca gaaggggatg ggaaataaag aaaattctct caagacttct ccaggcccat 5460 gtcactggtc agcgtggttt ttatgtgtat taggattggg ggatgtgaag aaataagtat 5520 ccagtacttt ataaccaaag caattaaatg atattgggta gggaatgttg gccagttttg 5580 tttagttttg catcacattg tcaccagact cactagccca agtaatcggg cgcccgaaga 5640 gggagacaga gatgtgcaga gttgaccagt gtgcggatga taactactga cgaaagagtc 5700 atcgactcag ttagtggttg gatgtagtca cattagtttg cctctcccca tctttgtctc 5760 cctggcaagg agaatatgcg ggacatgatg ctaagagtcc tgggtaaatg tggtgagaat 5820 gcacgcgtgc atatgctaca catatgtgct tctcagttgc agaaaatgaa ctgctttggg 5880 agattatcag tagaaagagt gttatcatat tggtgctgag tgctatgtgt gcttatacaa 5940 tttgttcttg tattttaata aactttgaat aaaagaataa agcta 5985 <210> 2 <211> 4244 <212> DNA <213> Homo sapiens <400> 2 atgaggccgg cggacttgct gcagctggtg ctgctgctcg acctgcccag ggacctgggc 60 ggaatggggt gttcgtctcc accctgcgag tgccatcagg aggaggactt cagagtcacc 120 tgcaaggata ttcaacgcat ccccagctta ccgcccagta cgcagactct gaagcttatt 180 gagactcacc tgagaactat tccaagtcat gcattttcta atctgcccaa tatttccaga 240 atctacgtat ctatagatgt gactctgcag cagctggaat cacactcctt ctacaatttg 300 agtaaagtga ctcacataga aattcggaat accaggaact taacttacat agaccctgat 360 gccctcaaag agctccccct cctaaagttc cttggcattt tcaacactgg acttaaaatg 420 ttccctgacc tgaccaaagt ttattccact gatatattct ttatacttga aattacagac 480 aacccttaca tgacgtcaat ccctgtgaat gcttttcagg gactatgcaa tgaaaccttg 540 acactgaagc tgtacaacaa tggctttact tcagtccaag gatatgcttt caatgggaca 600 aagtggatg ctgtttacct aaacaagaat aaatacctga cagttattga caaagatgca 660 tttggaggag tatacagtgg accaagcttg ctggacgtgt ctcaaaccag tgtcactgcc 720 cttccatcca aaggcctgga gcacctgaag gaactgatag caagaaacac ctggactctt 780 aagaaacttc cactttcctt gagtttcctt cacctcacac gggctgacct ttcttaccca 840 agccactgct gtgcttttaa gaatcagaag aaaatcagag gaatccttga gtccttgatg 900 tgtaatgaga gcagtatgca gagcttgcgc cagagaaaat ctgtgaatgc cttgaatagc 960 cccctccacc aggaatatga agagaatctg ggtgacagca ttgttgggta caaggaaaag 1020 tccaagttcc aggatactca taacaacgct cattattacg tcttctttga agaacaagag 1080 gatgagatca ttggttttgg ccaggagctc aaaaaccccc aggaagagac tctacaagct 1140 tttgacagcc attatgacta caccatatgt ggggacagtg aagacatggt gtgtaccccc 1200 aagtccgatg agttcaaccc gtgtgaagac ataatgggct acaagttcct gagaattgtg 1260 gtgtggttcg ttagtctgct ggctctcctg ggcaatgtct ttgtcctgct tattctcctc 1320 accagccact acaaactgaa cgtcccccgc tttctcatgt gcaacctggc ctttgcggat 1380 ttctgcatgg ggatgtacct gctcctcatc gcctctgtag acctctacac tcactctgag 1440 tactacaacc atgccatcga ctggcagaca ggccctgggt gcaacacggc tggtttcttc 1500 actgtctttg caagcgagtt atcggtgtat acgctgacgg tcatcaccct ggagcgctgg 1560 tatgccatca ccttcgccat gcgcctggac cggaagatcc gcctcaggca cgcatgtgcc 1620 atcatggttg ggggctgggt ttgctgcttc cttctcgccc tgcttccttt ggtgggaata 1680 agtagctatg ccaaagtcag tatctgcctg cccatggaca ccgagacccc tcttgctctg 1740 gcatatattg tttttgttct gacgctcaac atagttgcct tcgtcatcgt ctgctgctgt 1800 tatgtgaaga tctacatcac agtccgaaat ccgcagtaca acccagggga caaagatacc 1860 aaaattgcca agaggatggc tgtgttgatc ttcaccgact tcatatgcat ggccccaatc 1920 tcattctatg ctctgtcagc aattctgaac aagcctctca tcactgttag caactccaaa 1980 atcttgctgg tactcttcta tccacttaac tcctgtgcca atccattcct ctatgctatt 2040 ttcaccaagg ccttccagag ggatgtgttc atcctactca gcaagtttgg catctgtaaa 2100 cgccaggctc aggcataccg ggggcagagg gttcctccaa agaacagcac tgatattcag 2160 gttcaaaagg ttacccacga gatgaggcag ggtctccaca acatggaaga tgtctatgaa 2220 ctgattgaaa actcccatct aaccccaaag aagcaaggcc aaatctcaga agagtatatg 2280 caaacggttt tgtaagttaa cactacacta ctcacaatgg taggggaact tacaaaataa 2340 tagtttcttg aatatgcatt ccaatcccat gacaccccca acacatagct gccctcactc 2400 ttgtgcaggc gatgtttcaa tgtttcatgg ggcaagagtt tatctctgga gagtgattag 2460 tattaaccta atcattgccc ccaagaagga agttaggcta ccagcatatt tgaatgccag 2520 gtgaaatcaa aataatctac actatctaga agactttctt gatgccaagt ccagagatgt 2580 cattgtgtag gatgttcagt aaatattaac tgagctatgt caatatagag cttctcagtt 2640 ttgtataaca tttcatacta aagattcagc aaatggaaaa tgctattaat ttggttggtg 2700 accacaagat aaaatcagtc ccacgttggc tcagttcaac tagatgttcc ctgatacaaa 2760 gagaacttga tttccttaaa actgaaaagc caaacacagc tagctgtcat acaagaaaca 2820 gctattatga gacatgaagg agggtaagaa ttagctttaa gttttgtttt gctttgtttt 2880 gttttttaac tcaacctatt aatcatctct tcacaagaat ccacctgatg tgaccaagct 2940 attatgtgtt gcctggaaaa actggcaaga tttcagctta tgtggcctag caaactaaga 3000 attgctcttc ttggccagcc tcatagcata aaagatgtga actctaggaa gtctttctga 3060 gtagcaataa gtgggaatta tgggcagagc acactcaatc ccctgttgat taataaaaca 3120 ggctggacac taattaacta tgggacttaa atctgtagaa atgaaggagt ccaatagctt 3180 cttccaattt taaaactcta gtacatccct ttccctcaaa tatatatttc taagataaag 3240 agaaagaaga gcactaagta agtagaatct gtttttccta ttttgtaggg ctgctgactc 3300 ctagtccttg aagcctagac acatgaccca ggaaattttt cctttgtttc acttttgatt 3360 atgatgtctg agccaaaaat tcaattaagt aaacatactc gcctggatct gaatcattca 3420 tttaattact agatctaccc agctgttata tcaggccaaa aacagattcg tgtttatata 3480 aaagagtaaa cgatggttgc aaattttggc tatttagagt tgctacttca ctatgaagag 3540 tcacttcaaa acacttcgct tgtctttagg gatgattttt gccatttcca gtccacggta 3600 tgatactaaa gctgtcaaga gaggtttctt cttttctgaa actgccagct ctttccagcc 3660 ctgttgatca ctggacataa agcttctttt ccccaataat tcttctttac ttaaaatagt 3720 caggatcttt atctacagat gtactctcca ggttacctgt gatgatagcc ccctaatgtc 3780 ctgctagaaa agtctccaag cagagatgac attacttctg aatgctcata aaccacacca 3840 tgaaataaaa gctctttgtt gttttaagat tgtgaagtgt cgttaatggg tccccacaga 3900 tggtccctgc tggactcacc tggaatctct ccacagccat acccactcat cactatcatt 3960 gagacctgca catcttaata gaaatattat aaacatcgaa aatcatgact tacctagaag 4020 ttcgcttgta actaatgaaa ttaaacaaat gtgttgcctt ttgtcatgtg tttctctcct 4080 gtgacatttc aaaatatcac atcttgataa ataatgtgtt tcatcttgaa tagctgaact 4140 aattgctttg gaaacagagt cctagaaaag tgacttcaac agaattgtta ctaaaatttg 4200 cactcacaac atgaaataaa ttttcttcct atggaataat cgtg 4244 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TSHR exon forward primer <400> 3 ctgggcaatg tctttgtcct gcttattc 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> TSHR exon reverse primer <400> 4 tatgtgttgg gggtgtcatg ggattg 26 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DIO2 exon2 forward primer <400> 5 tgtgaattca agtggcaatg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DIO2 exon2 reverse primer <400> 6 ccaatagggc tctgttgaaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DIO2 amplification forward primer <400> 7 tccagtgtgg tgcatgtctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DIO2 amplification reverse primer <400> 8 ctggctcgtg aaaggaggtc 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Gapdh amplification forward primer <400> 9 tgttgccatc aatgacccct t 21 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Gapdh amplification reverse primer <400> 10 ctccacgacg tactcagcg 19

Claims (8)

A polynucleotide consisting of SEQ ID NO: 1 encoding a DIO2 (type 2 iodothyronine deiodinase) protein has 10-100 consecutive sequences containing a single base polymorphism (C.274A> G) in which the 274th base is changed from A to G (SEQ ID NO: 2) encoding a TSHR (Thyroid Stimulating Hormone Receptor) protein, and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 encoding a TSHR (Thyroid Stimulating Hormone Receptor) protein. (Levothyroxine) and L-T3 (liothyronine), which comprise a polynucleotide comprising 10 to 100 contiguous bases or a complementary polynucleotide thereof, Microarray for patient diagnosis.
The microarray for diagnosing hypothyroidism patient according to claim 1, wherein the continuous base is 10 to 70 consecutive bases.
A kit for the diagnosis of hypothyroidism patients requiring combination administration of L-T4 (Levothyroxine) and L-T3 (liothyronine) comprising the microarray of claim 1.
Single nucleotide polymorphism (C.274A> G) and Thyroid Stimulating Hormone Receptor (TSHR) in which the nucleotide located at position 274 is changed from A to G in the polynucleotide consisting of SEQ ID NO: 1 encoding DIO2 (type 2 iodothyronine deiodinase) A L-T4 (SEQ ID NO: 2) encoding a protein comprising a complementary probe or primer set capable of detecting a mutation (C.1349G > A) in which the base G at position 1349 in the polynucleotide consisting of SEQ ID NO: (Levothyroxine) and L-T3 (liothyronine).
1) separating the nucleic acid from the biological sample separated from the subject; And
2) determining L-T4 (Levothyroxine) comprising the SNP base sequence located at the 274th position of the polynucleotide of SEQ ID NO: 1 from the nucleic acid and the 1349th mutant nucleotide sequence of the polynucleotide of SEQ ID NO: 2 L-T3 (liothyronine) is needed for the diagnosis of hypothyroidism.
1) separating the protein from the biological sample separated from the subject; And
2) Thyroid Stimulating Hormone Receptor (TSHR) protein whose arginine (R) as the 450th amino acid from the N-terminal is changed to histidine (H) (C.1349G> A) and threonine The combination of L-T4 (Levothyroxine) and L-T3 (liothyronine), which involves the step of detecting a DIO2 (type 2 iodothyronine deiodinase) protein that has been changed to alanine (A) (C.274A> G) Methods of providing information for the diagnosis of hypopnea.
7. The method according to claim 6, wherein the expression of TSHR (C.1349G > A) protein and DIO2 (C.274A> G) protein are measured using a mutant protein- Providing information necessary for patient diagnosis.
7. The method according to claim 5 or 6, wherein the sample is selected from the group consisting of hair, blood, tissue, cells, serum, plasma, saliva, sputum and urine. Way.

Images