KR20020023733A - Method for diagnosis of congenital adrenal hyperplasia and oligonucleotide chip - Google Patents

Abstract

PURPOSE: Provided are a diagnosis method of congenital adrenogenital hyperplasia(CAH) by using a specific mutant sequence capable of being used for the selection of metabolism disorder in the newborn and an oligonucleotide chip used therefor. CONSTITUTION: The diagnosis method comprises the steps of: preparing an oligonucleotide, wherein mutant type CAH 21-hydroxylase gene and 7 sets of tandem cDNA of wild type gene (exon 1, intron 2, exon 4) are fixed on a base plate; performing PCR by using a part of 21-hydroxylase genome gene(exon1, intron2, exon4) as a primer; hybridizing amplified genes and 7 sets of tandem cDNA gene fragments fixed on the oligonucleotide chip; and detecting hybridization to diagnose CAH.

Description

Method for diagnosis of congenital adrenal hyperplasia and oligonucleotide chip

The present invention relates to a novel diagnostic method, oligonucleotide chip, for congenital adrenal hyperplasia (abbreviated as 'CAH').

More specifically, the present invention comprises the steps of (1) attaching a specific mutant sequence of CAH to the substrate; (2) amplifying the mutant sequence sites on the chromosome of the diagnostic patient; (3) performing gene hybridization; (4) The present invention relates to a diagnostic method and an oligonucleotide chip capable of easily diagnosing a CAH composed of comparing and searching the results.

Congenital adrenal hyperplasia is a hereditary disease caused by abnormalities in cortisol biosynthesis. Cortisol synthesis requires five enzymes: StAR protein, 17-hydroxylase, 3β-HSD, 21-hydroxylase, and 11β-hydroxylase. More than 90% of patients with the syndrome lack 21-hydroxylase or have impaired function. As a result, patients with adrenal syndrome have various symptoms as a result of decreased biosynthesis of cortisol and aldosterone, which are essential in the adrenal glands.

The 21-hydroxylase gene is located on the chromosome at the chromosome HLA group III gene region and has a complex structure with high variability. The active gene CYP21B is highly homologous to CYP21A, an inactive pseudogene, and these two genes are located in the 3 'region of two genes C4A and C4B, each encoding the fourth element of complement. (repeated tandem unit) C4 / 21OH.

Congenital adrenal syndrome is a relatively common disease that affects one in 10,000 newborn babies. Therefore, the diagnostic method has been developed conventionally, and the conventional method is to measure 17-OH progesterone, a substrate of 21-hydroxylase, in serum. However, unlike these methods, methods for directly diagnosing mutations on chromosomes have recently been developed. Southern blot analysis or polymerase chain reaction is commonly used to diagnose mutations on chromosomes. These diagnostic methods are simpler, cheaper and more sensitive than conventional methods. do. However, this method is also difficult to accurately analyze due to the complex structure of the 21-hydroxylase gene, that is, the presence of a pseudogene, and as a method for overcoming the development of diagnostic methods and diagnostic kits to improve accuracy by searching for mutations at once. This is urgently required.

As a result, the present inventors continued research to effectively diagnose CAH. As a result, three common 21-hydroxyla, P30L (exon 1), splicing mutation (A / C655G, intron 2) and I172N (exon 4) The mutant was used to prepare a plasmid vector comprising the specific mutant sequence, and then used to prepare an oligonucleotide chip, thereby confirming that it is possible to accurately diagnose CAH due to 21-hydroxylase deficiency. Completed.

It is an object of the present invention to provide a method for diagnosing congenital adrenal syndrome using a specific mutation sequence that can be used for neonatal metabolic abnormality screening, and an oligonucleotide chip for diagnosis thereof.

1 is an assembly diagram of seven tandem assembled plasmid vectors used for diagnosing congenital adrenal syndrome of the present invention.

Accordingly, an object of the present invention is an oligonucleotide chip in which mutations in the 21-hydroxylase gene of adrenal syndrome and 7 sets of tandem cDNA fragments of normal genes (exon 1, intron 2, exon 4) are fixed on a solid substrate. To prepare; Amplifying a part of the 21-hydroxylase genomic gene (exon 1, intron 2, exon 4) of the person to be diagnosed by PCR using a primer; Performing hybridization with seven sets of tandem cDNA gene fragments fixed on the amplified gene and oligonucleotide chip; And it provides a method for diagnosing the adrenal gland syndrome comprising the step of searching for the hybridization.

On the other hand, the step of preparing the oligonucleotide chip iii) pCAH-I2N (KCTC 0759BP), pCAH-I2N2 (KCTC 0760BP), pCAH-I2P (KCTC 0761BP), pCAH-E1N (KCTC 0758BP), pCAH-E1P (KCTC 0592BP), pCAH-E4N (KCTC 0762BP), pCAH-E4P (KCTC 0763BP) to prepare 7 sets of assembly vectors, and ii) the tandem cDNA fragments in the 7 sets of assembly vectors in SEQ ID NO: 26 and SEQ ID NO: 27 Amplifying by the PCR method using the two labeled primers, and i) attaching the amplified seven sets of tandem cDNA fragments onto a solid substrate through a coupling reaction.

Meanwhile, the present invention provides an oligonucleotide chip in which a mutation of 21-hydroxylase gene of adrenal syndrome and 7 sets of tandem cDNA fragments of normal genes (exon 1, intron 2, exon 4) are fixed on a solid substrate. ; Ii) labeled primers for PCR amplifying a portion of the 21-hydroxylase genomic gene of the person to be diagnosed (exon 1, intron 2, exon 4); Iii) enzymes and buffers for polymerase chain reaction; Iii) to provide an oligonucleotide chip kit for diagnosing adrenal gland syndrome comprising a buffer solution for hybridization. At this time, the label is characterized in that the ³² P radioisotope label or fluorescence response label.

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

The present invention provides a method for diagnosing whether there is an adrenal syndrome from a subject, and the diagnostic method includes the following steps. Iii) obtaining a sample containing genomic DNA from the sample ii) amplifying a 21-hydroxylase gene site (exon 1, intron 2, exon 4) of normal or abnormal persons with a primer iii) Preparing an oligonucleotide chip in which seven sets of tandem cDNA fragments of normal or abnormal 21-hydroxylase genes are fixed on a solid substrate iii) on the amplified sample gene and on the oligonucleotide chip for reading normality Hybridization with tandem cDNA to confirm the result.

1. Amplification of Sample Genes

In the present invention, P30L (exon 1), splicing mutations (A / C655G, intron 2) and I172N (exon 4), which are specific mutation sites commonly found in 21-hydroxylase gene of CAH patients, are used. P30L is a point mutation of 212 base C to T in the nucleotide sequence of the normal steroid 21-hydroxylase gene of SEQ ID NO: 1, and the 30th amino acid residue, proline (Pro), is expressed as leucine (Leu). 21-hydroxylase is inactivated.

In the second intron, A / C655G, which has a point mutation of A or C, which is base 655 (base 778 in SEQ ID NO: 778) to G, undergoes mutation during splicing after transcription, resulting in a frameshift. (truncated) protein is produced. In addition, I172N, a point mutation appearing in the fourth exon, is expressed with tryptophan (Trp) of arginine (Arg), which is the 172th amino acid residue, to inactivate steroid 21-hydroxylase.

In order to amplify the specific mutant sequence of the 21-hydroxylase gene of the diagnostic patient in the diagnostic method and oligonucleotide chip kit of the present invention, the DNA isolated from the patient is used as a template and the mutant sequence that causes CAH is amplified. Perform polymerase chain reaction with primers.

Specifically, in the present invention, the genomic DNA obtained from the sample to be diagnosed is used as a template, and the first exon and the second intron of the 21-hydroxylase gene using P1 primer (SEQ ID NO: 2) and P2 primer (SEQ ID NO: 3). After amplification of the site, amplify the P30L mutant site of the first exon using E1TS primer (SEQ ID NO: 4), E1TAS primer (SEQ ID NO: 5), I2TS primer (SEQ ID NO: 6), and I2TAS primer (SEQ ID NO: 7). Amplify the A / C655G mutation site of the second intron.

In addition, the I172N mutant site of the fourth exon is a DNA obtained from the patient to be diagnosed as a template, the first PCR is performed using P3 primer (SEQ ID NO: 8) and P4 primer (SEQ ID NO: 9), and then E4TS primer (SEQ ID NO: 10), amplify by performing a second PCR using E4TAS primer (SEQ ID NO: 11).

In the above process, the primer is used by labeling with radioisotopes or fluorescent materials.

2. Oligonucleotide Chip Manufacturing

In the present invention, a DNA fragment comprising a P30L mutant sequence is designed and used to prepare an oligonucleotide chip, and a DNA fragment comprising an A / C655G mutant sequence is designed and used to prepare an oligonucleotide chip, which also includes an I172N mutant sequence. Fragments are designed and used to prepare oligonucleotide chips.

Specifically, in the present invention, a plasmid vector including the specific mutation sequence is prepared to prepare a DNA fragment for preparing an oligonucleotide chip including the specific mutation sequence. In addition, the present invention prepares a DNA fragment designed using the restriction enzyme Sfi I recognition site on both sides of the specific mutant sequence.

Restriction enzyme Sfi Ⅰ has a nucleotide sequence of CAT and GTA, respectively, after the end of the cut-off gene segment, so that the gene fragment is inserted in a unidirection rather than self ligation unlike other restriction enzymes. With this feature, genes containing specific mutation sequences can be repeatedly arranged side by side in a plasmid vector.

In this case, using a phagemid vector that produces single-stranded DNA (ssDNA) as a plasmid vector, a DNA fragment required for an oligonucleotide chip can be easily produced.

In the present invention, a plasmid vector including a DNA fragment is prepared, and the plasmid vector of the present invention includes multiple copies by repeating a specific mutant sequence appearing in CAH, thereby improving the sensitivity of gene search to efficiently diagnose a disease. Oligonucleotide chips can be prepared.

In the present invention, seven plasmid vectors of pCAH-I2N, pCAH-I2N2, pCAH-I2P, pCAH-E1N, pCAH-E1P, pCAH-E4N, pCAH-E4P were prepared for oligonucleotide chip preparation. More specifically, the pCAH-I2N vector has a sense sequence of SEQ ID NO: 12 which is 15 tandem copies and an antisense sequence of SEQ ID NO: 13, and the pCAH-I2N2 vector has a sense sequence of SEQ ID NO: 14 which is 23 tandem copies and an antisense sequence of SEQ ID NO: 15 Wherein the pCAH-I2P vector has a sense sequence of SEQ ID NO: 16 which is 25 tandem copies and an antisense sequence of SEQ ID NO: 17, wherein the pCAH-E1N vector has a sense sequence of SEQ ID NO: 18 and an antisense sequence of SEQ ID NO: 19 Wherein the pCAH-E1P vector has a sense sequence of SEQ ID NO: 20, which is ten tandem copies, and an antisense sequence of SEQ ID NO: 21, and the pCAH-E4N vector has a sense sequence of SEQ ID NO: 22, and an antisense sequence of SEQ ID NO: 23 the carry, the pCAH-E4P vectors has the antisense sequence of 19 tandem copies of SEQ ID NO: 24 and SEQ ID NO: 25 in the sense sequence, and the sequence was used for restriction enzyme recognition site alongside Shi ⅰ Clothing can be arranged.

In addition, the seven plasmid vectors of pCAH-I2N, pCAH-I2N2, pCAH-I2P, pCAH-E1N, pCAH-E1P, pCAH-E4N, and pCAH-E4P are genes in the biotechnology research institute, located at 52, Yue-dong, Yuseong-gu, Daejeon. Deposited international banks in accordance with the Budapest Treaty. The international access numbers of the vectors are as follows.

pCAH-I2N was deposited with KCTC-0759BP, March 23, 2000, pCAH-I2N2 was deposited with KCTC-0760BP, March 23, 2000, and pCAH-I2P, March 23, 2000. Deposited under Accession No. KCTC-0761BP, pCAH-E1N was deposited with Accession No. KCTC-0758BP on March 23, 2000, pCAH-E1P was deposited on March 19, 1999 with Accession No. KCTC-0592BP, pCAH-E4N was deposited on March 23, 2000 with Accession No. KCTC-0762BP, and pCAH-E4P was deposited on March 23, 2000 with Accession No. KCTC-0763BP.

Seven sets of tandem cDNA fragments inserted into the seven deposited vectors were PCR amplified by labeled primers. At this time, primers of SEQ ID NO: 26 and primers of SEQ ID NO: 27 with functional groups were used as primers. Such primers are specific for the deposited vector and used to amplify only tandem cDNA fragments. In addition, the sequences of the primers used for amplifying tandem cDNA fragments of the seven vectors are all identical.

Seven amplified tandem cDNA fragments and the like are coupled to each other by a functional group attached to a solid substrate to be fixed by a nucleophilic functional group such as alcohol, amine or thiol labeled at the ends of the primer. Seven tandem cDNA fragments amplified through such coupling bonds are fixed on a solid substrate, thereby preparing an oligonucleotide chip in which the cDNA fragments are fixed.

3. Hybridization

The genomic DNA fragment of the amplified subject is examined for hybridization with DNA fragments in the oligonucleotide chip. Hybridization of the oligonucleotide chip can be observed using a fluorescent microscope or a CCD camera using a fluorescent material or the like.

Specifically, in the present invention, as a result of hybridization analysis, DNA fragments prepared by amplification from pCAH-I2N, pCAH-I2N2, pCAH-E1N, pCAH-E4N, and hybridization signals were identified. In the case of patient genes, the DNA fragments prepared by amplifying specific mutant sequences from pCAH-I2P, pCAH-E1P, and pCAH-E4P could be observed with hybridized signals.

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

The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1 Amplification through Polymerase Chain Reaction of Sample Genomic DNA

(1-1) Polymerase Chain Reaction and Purification of Products

DNA obtained from the patient to be diagnosed was used as a template, and a polymerase chain reaction was performed using a primer to amplify specific mutation sites of CAH. Polymerase chain reaction was performed with 100 ng of genomic DNA, 10 pmole of each primer, 2.5 mM dNTP mixture and 20 mM tris-hydrochloric acid (pH 8.0), 100 mM potassium chloride, 0.1 mM EDTA, 1 mM DTT, 0.5% twin 20, 0.5% noni The composition of P-40 (Nonidet P-40), 50% glycerol, 1.25 units of polymerase (Takara Shuzo) was added to the volume of the reaction mixture to a volume of 100 μl Perkin-Elmer DNA Dermal Cycler 480 (Perkin-Elner Cetus) Was performed in.

The reaction mixture was denatured at 98 ° C. for 1 minute and then denatured at 94 ° C. for 20 seconds, and the cycle of annealing was repeated 35 times at the temperatures shown in Table 2. Next, the samples were incubated at 72 ° C. for 10 minutes and purified in a JetJet PCR purification kit (GENOMED).

(1-2) Amplification of Exon 1

Amplification of exon 1 is carried out by the first polymerase chain reaction using two primers, SEQ ID NO: 2 and SEQ ID NO: 3. The size of the obtained PCR product was 1339 bp. In a second step, using the two primers of SEQ ID NO: 4 and SEQ ID NO: 5, the PCR product obtained in the first step is obtained by amplifying exon 1 by polymerase chain reaction.

(1-3) Amplification of Exon 2 and Exon 4

Amplification of exon 2 is performed by polymerase chain reaction using two primers of SEQ ID NO: 6 and SEQ ID NO: 7 to the 1339 bp PCR product obtained in the first step of the polymerase chain reaction. The size of the obtained PCR product was 104 bp. For amplification of exon 4, a product obtained by amplifying exon 4 by polymerase chain reaction using two primers of SEQ ID NO: 8 and SEQ ID NO: 9 to the 1339 bp PCR product obtained in the first step of the polymerase chain reaction.

(1-4) Labeling of PCR Products

1 μl of the PCR product was mixed with 5 × of the above reaction buffer, 150 μl Ci [γ- ³² P] -dATP, 20 U T 4 polynucleotide kinase (GibcoBRL), and the reaction mixture was brought to a volume of 50 μl. 30 minutes-1 hour was performed. Unreacted [γ- ³² P] ATP was then filtered from the end labeled PCR product using Sephadex G-50 minicolumn (Phamacia Biotech).

Example 2 Preparation of a Vector for Obtaining a cDNA Fragment

(2-1) Synthesis of Complementary Oligonucleotides

In the present invention, a plasmid vector to be used to prepare a DNA fragment used in an oligonucleotide chip was prepared, and for this purpose, a complementary oligonucleotide was first synthesized.

(2-2) Preparation of plasmid vector

0.7 μg each of the complementary oligonucleotides prepared in (2-1) was mixed with 40 μl of 1 × annealing buffer solution (ph7.5, 25 mM tris hydrochloric acid, 300 mM NaCl, 1 mM EDTA) at 95 ° C. Heat for 3 minutes and at 65 ° C. for 30 minutes, then cool gradually. To obtain complementary phosphorylated oligonucleotide fragments, purification was performed by overnight 4 ° C. electrophoresis on 8% unmodified polyacrylamide gels in DNA rehydration solution (10 mM Tris, 1 mM EDTA) and purified by T4 DNA ligase (Takara Shuzo G) allow self ligation to occur at 16 ° C. for 30 minutes. Next, 1 μg of the fragment is mixed with a cloning vector pBluescript ks (+/−)- Shi 10 ng (Stratagene), mixed in 20 μl of buffer solution, and linked using a DNA ligation kit.

The DNA fragment inserts of the seven vectors are as follows.

That is, the pCAH-I2N vector has a sense sequence of SEQ ID NO: 12, which is 15 tandem copies, and an antisense sequence of SEQ ID NO: 13, and the pCAH-I2N2 vector has a sense sequence of SEQ ID NO: 14, which is 23 tandem copies, and an antisense sequence of SEQ ID NO: 15 Wherein the pCAH-I2P vector has a sense sequence of SEQ ID NO: 16, which is 25 tandem copies, and an antisense sequence of SEQ ID NO: 17, wherein the pCAH-E1N vector has a sense sequence of SEQ ID NO: 18, which is seven tandem copies, and an antisense sequence of SEQ ID NO: 19 The pCAH-E1P vector has a sense sequence of SEQ ID NO: 20 and 10 antisense sequences of SEQ ID NO: 21, and the pCAH-E4N vector has a sense sequence of SEQ ID NO: 22 and an antisense sequence of SEQ ID NO: 23, which are 12 tandem copies The pCAH-E4P vector has a sense sequence of SEQ ID NO: 24 which is 19 tandem copies and an antisense sequence of SEQ ID NO: 25.

The plasmid vector containing the designed fragment was transformed into E. coli Top10F 'strain and then cultured to isolate plasmid DNA.

Also, determine the size of the plasmid vector with a restriction enzyme Eco RⅠ and insertion by cutting with Sfi Ⅰ gene DNA in 1% agarose gel electrophoresis, followed by determining its nucleotide sequence. As a result, the plasmid vectors pCAH-I2N, pCAH-I2N2, pCAH-I2P, pCAH-E1P, pCAH-E1P, and pCAH-E4N, pCAH-E4P and the like were confirmed.

Example 3 Preparation of Oligonucleotide Chip

(3-1) Polymerase Chain Reaction to Obtain cDNA Fragments

Seven kinds of vectors pCAH-I2N, pCAH-I2N2, pCAH-I2P, pCAH-E1N, pCAH-E1P, pCAH-E4N, and pCAH-E4P obtained in Example 2 were used as templates. Polymerase chain reaction was performed using two labeled primers. At this time, two primers labeled with 20 pmole of functional groups, 4 μL of 2.5 mM dNTP, and 1 μL of Taq polymerase (5 unit / μL) were added to 200ng of the plasmid. At this time, add buffer (20 mM of Tris-HCl pH 8.0, 10 mM potassium chloride, 0.1 mM EDTA) to 0.2 ml tube to make final volume 49 μL. This mixed liquid tube is transferred to a thermocycler and denatured at 94 ° C for 5 minutes. At this time, the polymerase chain reaction had 35 summer cycles, the denaturation temperature was 1 minute at 94 ° C, and the annealing temperature was 1 minute at 60 ° C. The PCR product is held at 72 ° C. for 10 minutes and cooled to 4 ° C. Obtain the PCR product labeled with the final marker.

(3-2) Preparation of Oligonucleotide Chip

The 7 amplified cDNA fragments thus obtained are fixed to cDNA fragments on a solid substrate through ester, amide or thioester bonds between a functional group linked to the cDNA fragment and a substance coated on the solid substrate. As described above, the amplified seven cDNA fragments are fixed on a solid substrate to prepare an oligonucleotide chip. In this case, polystyrene, silica gel, polyacrylamide, polyacrylate, polyamide, hydroxyethyl or methacrylate may be used as the solid substrate.

Example 4 Hybridization in Oligonucleotide Chips

(4-1) Preparation of Specimen

The PCR product labeled with the fluorescent material obtained by the method in Example 1 is used as a sample. Dissolve the sample using hybridization buffer (2.5 x SSC) before placing the sample on the oligonucleotide chip. Fluorescent materials can be observed through fluorescent microscopy when cDNA fragments are hybridized on fluid samples and oligonucleotide chips.

(4-2) Hybridization Method

Samples are spotted onto oligonucleotide chips for hybridization and washed again, and finally DNA fragments on the hybridized oligonucleotide chips and DNA fragments of the specimens are detected. These observations can usually be seen by the GSI scan array method. In addition, such hybridizations can be observed through fluorescent microscopy and CCD cameras.

DNA fragments obtained by amplification from pCAH-I2N, pCAH-I2N2, pCAH-E1N, and pCAH-E4N for normal samples are hybridized and amplified from pCAH-I2P, pCAH-E1P, pCAH-E4P for abnormal samples. Hybridization with the obtained DNA fragment is formed.

As described above, the present invention is to prepare an oligonucleotide chip using a specific mutation sequence of the 21-hydroxylase gene, through which the hybridization of the sample with the gene amplification of the sample, by examining the presence of mutations CAH can be effectively diagnosed and treated.

In particular, the diagnostic method and the oligonucleotide chip of the present invention include specific mutant sequences in several copies, and thus have excellent sensitivity when searching, and three specific mutant sequences can be searched at once to enable accurate diagnosis.

Claims (4)

Preparing an oligonucleotide chip in which a mutation of the 21-hydroxylase gene of adrenal syndrome and 7 sets of fragments of tandem cDNA of normal genes (exon 1, intron 2, exon 4) are fixed on a solid substrate; Amplifying a part of the 21-hydroxylase genomic gene (exon 1, intron 2, exon 4) of the person to be diagnosed by PCR using a primer; Performing hybridization with seven sets of tandem cDNA gene fragments fixed on the amplified gene and oligonucleotide chip; And detecting whether the hybridization is performed. The method of claim 1, wherein the preparing of the oligonucleotide chip comprises: iii) pCAH-I2N (KCTC 0759BP), pCAH-I2N2 (KCTC 0760BP), pCAH-I2P (KCTC 0761BP), pCAH-E1N (KCTC 0758BP), pCAH Prepare 7 sets of assembly vectors of -E1P (KCTC 0592BP), pCAH-E4N (KCTC 0762BP), pCAH-E4P (KCTC 0763BP), and ii) sequence the tandem cDNA fragments in the 7 sets of assembly vectors Amplification by a PCR method using two labeled primers of No. 27, i) adrenal group comprising the step of attaching the amplified seven sets of tandem cDNA fragments onto a solid substrate through a coupling reaction Diagnosis Method Iii) oligonucleotide chips in which mutations in the 21-hydroxylase gene of adrenal syndrome and 7 sets of tandem cDNA fragments of normal genes (exon 1, intron 2, exon 4) are fixed on a solid substrate; Ii) labeled primers for PCR amplifying a portion of the 21-hydroxylase genomic gene of the person to be diagnosed (exon 1, intron 2, exon 4); Iii) enzymes and buffers for polymerase chain reaction; Iii) Oligonucleotide chip kit for diagnosing adrenal gland syndrome composed of buffer solution for hybridization 4. The kit of claim 3, wherein the label is a ³² P radioisotope label or a fluorescence lesson label.