KR100526702B1 - Kit for the Diagnosis of Chronic Respiratory and Pancreatic Diseases Associated with CFTR variants in East Asian - Google Patents

Abstract

The present invention relates to a nucleic acid molecule comprising a 782th G base, a nucleic acid molecule comprising a 1540th G base, and a 4188th C base in a CFTR gene having a nucleotide sequence of SEQ ID NO: 1. The present invention also relates to a kit for diagnosing chronic respiratory and pancreatic diseases comprising at least one of the above nucleic acid molecules.

Description

Kit for the Diagnosis of Chronic Respiratory and Pancreatic Diseases Associated with CFTR variants in East Asian}

The present invention relates to a kit for diagnosing chronic respiratory and pancreatic diseases caused by mutations in the CFTR gene of Asians. More specifically, in the CFTR gene having a nucleotide sequence of SEQ ID NO: 1, a nucleic acid molecule comprising the 782th G base, 1540 The present invention relates to a kit for diagnosing chronic respiratory and pancreatic diseases containing at least one of a nucleic acid molecule comprising a first C base and a nucleic acid molecule comprising a 4188th C base as a probe.

The loss of anion transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not only a well-known cystic fibrosis (CF). It is also associated with a wide range of respiratory and pancreatic diseases. To date, more than a thousand CFTR variants have been registered with the Cystic Fibrotic Genetic Analysis Consortium (CFGAC http://www.genet.sickkids.on.ca/). Several CFTR mutations (eg F508del, G542X and N1303K) are associated with severe CF phenotype and show high disease penetrance. However, many other mutants are associated with mono symptomatic diseases of the lung, pancreas or vas deferens and exhibit partial disease penetration.

The main approach used to describe these various phenotypes is to analyze each individual's haplotype background, particularly the intragenic region of CFTR. Various relationships or additional influences between multiple mutant and / or polymorphic sites will determine the final function of the gene product.

On the other hand, if there is any functional CFTR gene mutation in the population, it may be related to epithelial disease. Because CFTR plays an important role in the overall regulation of ion transport in epithelial cells, there are no functional analogs or substituents to replace this protein. Although mutations and polymorphisms in CFTR have been studied extensively in Westerners, their importance in East Asians has been less emphasized due to the unusual appearance of typical CF. However, other diseases known to be associated with CFTR mutations, such as bronchiectasis, chronic pancreatitis, also occur in Asians. Thus, the present inventors investigated the possibility of linking these diseases with the genetic variation of CFTR in Asians, especially Koreans, by using integrated genetic analysis combining molecular and functional studies.

Accordingly, the present invention is to provide a kit for diagnosing chronic respiratory and pancreatic diseases caused by CFTR gene mutation of Asians, in particular, Koreans.

In one aspect, the present invention provides a CFTR gene having a nucleotide sequence of SEQ ID NO: 1, a nucleic acid molecule comprising a 782th G base, a nucleic acid molecule comprising a 1540th G base and a nucleic acid molecule comprising a 4188th C base to provide.

In another aspect, the present invention provides a kit for diagnosing chronic respiratory and pancreatic diseases comprising at least one of the above nucleic acid molecules.

As used herein, the amino acid single letter means the following amino acids in accordance with standard abbreviation provisions in the field of biochemistry:

A: alanine; B: asparagine or aspartic acid; C: cysteine;

D: aspartic acid; E: glutamic acid; F: phenylalanine;

G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine;

M: methionine; N: asparagine; P: proline; Q: glutamine;

R: arginine; S: serine; T: threonine; V: valine;

W: tryptophan; Y: tyrosine; Z: glutamine or glutamic acid.

In both case-control studies and molecular and functional studies according to the invention, it was confirmed that Q1352H is a notable variation. Q1352H is present in a glutamine-rich conservative sequence called 'linker peptide', 'signature motif' or 'motif C' in the second nucleotide binding domain (NBD 2) of CFTR. Structural studies of the NBD domains of the ABC carriers of bacteria confirmed that the linker peptide was an essential motif for the integrity of the folded NBD structure and for the regulation of ATP hydrolysis and was consistent with that identified by the inventors. The linker portion of CFTR NBD 2 consists of the amino acid sequence LSHGHKQ and was somewhat different from the typical linker sequence LSGGQ. However, this sequence is highly conserved in all vertebrates, and in the event that the last glutamine mutates to histidine, this leads to a defect in the expression of the protein, affecting the switching properties of single channel dynamics.

Meanwhile, E217G mutations have already been identified in patients with respiratory CF who did not involve the pancreas of the Polish, and showed a case-related disease linkage in the case control study of the present invention. These results suggest that E217G is a mild mutation and has also been confirmed in the molecular studies of the present invention. E217G mutations reduce membrane protein expression and anion transport activity by 60-70%, and Q1352H mutations associated with the most disease show 70-80% protein expression and functional activity, comparable to E217G mutations, as measured in the M470 haploid background. It was confirmed to reduce until. The haploid analysis of the present invention confirmed that E217G occurs in haploids with high active M470 and Q1352H occurs in haploids with low active V470. Thus, it can be seen that the combination with the M470V position in the Q1352H mutation affects the overall function of the haploid gene product. In the present invention, using the double mutagenesis induced an additional change in M470V in Q1352H, it was found that the negative ion transport activity of CFTR protein is offset. The reason for the complete reduction in functional activity is unclear, but it is assumed that M470V and Q1352H are related to the correlation between the two NBDs of CFTR as M470V and Q1352H are located in NBD 1 and NBD 2 of CFTR, respectively. In the present invention, it was confirmed that cis-correlation of V470 may further exacerbate diseases caused by CFTR gene mutations, in particular Q1352H.

Accordingly, the present invention relates to the following CFTR gene mutations, which are frequently found in Asians and are associated with chronic respiratory and pancreatic diseases, and more particularly, in the CFTR gene having the nucleotide sequence of SEQ ID NO: 1, in (1) 781 to 783 A CFTR gene wherein the glutamic acid codon (GAG) is substituted with a glycine codon (GGG); (2) a CFTR gene in which the methionine codon (ATG) located at 1540-1542 is replaced with valine codon (GTG); And (3) a CFTR gene in which glutamine codons (CAG) located at 4186 to 4188 are substituted with histidine codons (CAC).

Accordingly, the present invention provides the following nucleic acid molecules that can be used as a probe for diagnosing chronic respiratory and pancreatic diseases characterized by using the CFTR gene mutation: In the CFTR gene having the nucleotide sequence of SEQ ID NO: 1, (1) 782 th A nucleic acid molecule comprising a G base, (2) a nucleic acid molecule comprising a 1540th C base, and (3) a nucleic acid molecule comprising a 4188th C base.

More preferably, in the CFTR gene having the nucleotide sequence of SEQ ID NO: 1, (1) a nucleic acid molecule consisting of 20 to 100 consecutive DNAs containing the 782th G base, and (2) 20 comprising the 1540th C base Nucleic acid molecules consisting of from 100 to 100 contiguous DNA and (3) nucleic acid molecules consisting of 20 to 100 contiguous DNA containing the 4188th C base.

Probe nucleic acid molecules of the present invention are all known hybridization techniques for testing the presence or absence of target nucleic acids in biological samples for diagnostic purposes, for example point deposition techniques on filters known as weddot-blot (MANIATIS et al., Molecular Cloning, Cold Spring Harbor, 1982), DNA delivery technology known as Southern blot (SOUTHERN, EM, J. Mol. Biol., 98, 503 (1975)), and RNA delivery technology known as Northern blot.

The probe of the present invention can be manufactured according to a conventional method, and there are two main methods. First, a single stranded probe is obtained. As the most representative example, a deprotection reaction may be performed by synthesizing by an automatic chemical synthesizer by DMT (dimethoxytrityl) off method, and then a probe consisting of a desired number of bases may be synthesized. The probe thus prepared can be attached to a fluorescent material such as fluorescein isothiocyanate (FITC) at one end of the strand to confirm the presence of a specific nucleic acid. Alternatively, single-stranded DNA is used as a template to make a probe having a base sequence complementary thereto. The primer is hybridized to the DNA of the template, and the Klenow enzyme and the fluorescent material are attached from the primer. Base (dNTP) is used to synthesize the probe. Since the fluorescent material is attached to the inside of the DNA, it is possible to synthesize a probe having high sensitivity and specificity. Second, there is a way to get a double stranded probe. Genomic DNA or plasmid DNA can be cut with specific restriction enzymes to obtain probes containing genes or bases of desired portions. Random priming is a method of hybridizing six random hexamers and template DNA, and then synthesizing probes of various lengths to which fluorescent substances are attached. ³² P at the end of the T4 polynucleotide kinase (polynucleotide kinase) by using a fluorescent material attached to the probe can be synthesized, DNA cleavage (DNase) I using a DNA strand (double-stranded) to make a cleavage, Using this DNA as a template, a probe can be synthesized using DNA polymerase I and a base (dNTP) to which a fluorescent substance is attached. This double-stranded probe is used for hybridization after denaturation to make it single-stranded.

Probes of the invention can be advantageously labeled. Any conventional label can be used. Probes can be labeled using radioactive isotopes such as ³² P, ³⁵ S, ¹²⁵ I, ³ H and ¹⁴ C. Radiolabeling can be carried out according to conventional methods such as end labeling the 3 or 5 position with radiolabeled nucleotides, polynucleotide kinases, terminal transferases or ligases. Another method of radiolabeling is to chemically iodide the probe of the present invention, which results in the binding of several ¹²⁵ I atoms onto the probe. As such, when one of the probes of the present invention is labeled as radioactive, radiation is generally detected using autoradiography, liquid scintillation, gamma coefficients, or other conventional methods. In addition, the probes of the present invention may include residues having immune properties (eg, antigens or haptens), residues having specific affinity for some reagents (eg ligands), residues providing detectable enzymatic reactions (eg enzymes, Coenzymes, enzyme substrates or substrates participating in enzymatic reactions) or in combination with residues which provide the physical properties of fluorescence, luminescence or absorption at certain wavelengths. Moreover, the antibody which specifically detects the hybrid formed by a probe and a target can be used. Non-radioactive labels can be provided when chemically synthesizing the probe of the present invention. Adenosine, guanosine, cytidine, thymidine and uracil residues readily bind to other chemical residues and allow detection of hybrids formed between the probe or probe and complementary DNA or RNA fragments.

Probes of the invention can also be used as gene chips. A preferred embodiment of the present invention provides a DNA chip comprising the probe of the present invention. A DNA chip is a high density integration of fragments of several nucleotide sequences on a small substrate and is used to detect DNA of an unknown sample by hybridization between a DNA immobilized on a substrate and an unknown DNA sample complementary thereto. The substrate on which the probe is to be fixed is made of inorganic materials such as glass or silicon, or polymer materials such as acrylic, polyethylene terephtalate (PET), polystyrene, polycarbonate, polypropylene, etc., and the surface of the substrate is flat. Or a hole having a plurality of holes may be used. The probe is fixed either covalently to the substrate at either 5 'or 3'. The immobilization method is a conventional technique, for example using an electrostatic force or by attaching an amine group on the synthesized oligo to an aldehyde coated slide to induce bonding between the two, or on an amine group coated slide or L-lysine It can be done by integrating on this coated slide or on a slide with a nitrocellulose membrane coated. In one embodiment of the present invention, when the probe is synthesized, a base having an amino residue in the 3 'position is inserted to covalently bond to a glass plate coated with an aldehyde residue.

Fixing and arranging different probes on a substrate surface uses methods such as pin microarrays, inkjets, photolithography, electrical arrays, and the like. In one embodiment of the present invention (Yoon et al, J. Microbiol. Biotechnol., 10 (1), 21-) using a microarrayer prepared according to a known method while the probe is dissolved in a buffer solution . 26, 2000). The principle of microarrays is that the microfabricated pins carry DNA from the plate and transfer it to the same location specified by the computer on the substrate. 1 hour to facilitate the reaction between the amine group on the 3 'position of the probe and the aldehyde group coated on the glass plate under the condition that the humidity of 45% to 65% is maintained to fix the probe transferred by the microarray. Adverse reactions are induced and left for at least 6 hours to fix the DNA probes.

If necessary to detect cells derived from a living organism or the organism itself, these cells may be partially or completely lysed by chemical and / or physical methods to facilitate access to the RNA and / or DNA of those cells and Contact. The contact can be carried out on a suitable support such as nitrocellulose, cellulose or nylon filters in a liquid medium or solution. Such contact may occur under optimal conditions, suboptimal conditions or constraint conditions. These conditions reduce the temperature, reactant concentrations, the presence of substances that lower the base pair optimal temperature of the nucleic acid (eg, formamide, dimethylsulfoxide and urea) and those that reduce the reaction volume and / or accelerate hybrid formation (eg, dextran). Sulfates, polyethylene glycols or phenols).

In order to diagnose chronic respiratory and pancreatic diseases caused by CFTR mutations using a DNA chip provided as an aspect of the present invention, a DNA sample capable of contacting the DNA chip from a subject must first be prepared. "DNA sample" means a PCR product obtained by separating genomic DNA from a subject and performing PCR using a desired portion as a template. The "purpose part" means a part of a gene including a CFTR mutation identified in the present invention, which can be easily amplified using an appropriate primer. Furthermore, a fluorescent substance should be added to the PCR reaction solution so that the desired portion is labeled with the fluorescent substance. In the present invention, a fluorescent material known in the art may be used, and preferably, a nucleotide to which the fluorescent material is bound may be easily labeled.

On the other hand, the target DNA that hybridizes with the probe may be prepared by two kinds of methods, depending on which hybridization reaction is used. First, a method used in Southern blot or Northern blot, genomic DNA or plasmid DNA is cut with a suitable restriction enzyme and subjected to electrophoresis on an agarose gel to separate DNA fragments by size. The second method is to amplify and use DNA of a desired portion using a PCR method. At this time, looking at the PCR method used, asymmetric amplification method that can obtain a double strand and a single strand of band simultaneously by asymmetrically adding the most common PCR and primer asymmetrically amplify the sense and antisense primers using the same amount (Asymmetric PCR), multiplex amplification method (multiplex PCR) that can be amplified at the same time by inserting several primers, ligase that amplifies by using a specific four primers and ligase (Ligase) and then fluorescence by enzyme immunoassay Ligase Chain Reaction (LCR) method, as well as hot start PCR, nest PCR, modified oligonucleotide primer PCR, reverse transcriptase PCR, semi-quantitative reverse transcriptase PCR, real time PCR, Rapid Amplification of cDNA Ends (RACE), Competitive PCR, Short Tandem Repeats (STR), Single Strand Polymorphization (Single) Strand Conformation Polymorphism (SSCP), in situ PCR, and DDRT-PCR (Differential Display Reverse Transcriptase PCR).

Hybridization conditions are determined by stringency, i.e. the stringency of the operating conditions. The higher the tightening that hybridization takes place, the more specific the hybridization becomes. Constriction is a function of not only the base composition of the probe / target conjugate but also the degree of mismatch between the two nucleic acids. Constriction may also be a function of hybridization reaction variables such as the concentration and type of ions present in the hybridization solution, the nature and concentration of the denaturant and / or the hybridization temperature. The tightening of the conditions under which the hybridization reaction should be carried out depends in particular on the probe used. All these data are well known and the appropriate conditions can be determined by routine experimentation in the individual case. Generally, depending on the length of the probe used, the temperature of the hybridization reaction is about 20 ° C. to 65 ° C., in particular 35 ° C. to 65 ° C., in a saline solution at a concentration of about 0.8 to 1 M.

Such hybridization conditions can be monitored taking into account several variables, for example, the hybridization temperature, the nature and concentration of the media components, the temperature at the time of washing of the formed hybrid, and the like. Hybridization and wash temperatures are limited to higher values depending on the base sequence, type and length of the probe and the maximum hybridization or wash temperature is about 30 ° C. to 60 ° C. At higher temperatures it competes with duplexing and dissociation or denaturation of the hybrid formed between the probe and the target. Hybridization media are for example about 3xSSC (1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), about 25 mM phosphate buffer pH 7.1 and 20% deion formamide, 0.02% picol, 0.02% bovine serum albumin, 0.02 % Polyvinylpyrrolidone and about 0.1 mg / ml cut and denatured salmon sperm DNA. The wash medium contains, for example, about 3 × SSC, 25 mM phosphate buffer pH 7.1 and 20% deion formamide. Depending on the modification of the probe and medium, the hybridization or washing temperature should be changed according to known associations to achieve the desired specificity (BD HAMES and SJ HIGGINS, (eds.). Nucleic acid hybridzation.A practical approach, IRL Press, Oxford , UK, 1985). In this regard, since DNA: DNA hybrids are generally less stable than RNA: DNA or RNA: RNA hybrids, hybridization conditions must be appropriately adjusted to achieve specific detection, depending on the nature of the hybrid to be detected.

The present invention provides kits for detecting chronic respiratory and pancreatic diseases by detecting CFTR gene mutations. The kit of the present invention comprises (1) a CFTR gene having the nucleotide sequence of SEQ ID NO: 1, (i) a nucleic acid molecule comprising the 782th G base, (ii) a nucleic acid molecule comprising the 1540th C base, and (iii) 4188 At least one of nucleic acid molecules comprising the first C base; (2) a pair of sense and antisense primers for amplifying a nucleic acid molecule comprising the 782th base, a nucleic acid molecule comprising the 1540th base, or a nucleic acid molecule comprising the 4188th base in the subject's CFTR gene; (3) a buffer or component that provides a buffering capacity to induce a hybridization reaction between the probe (1) and the target nucleic acid molecule; (4) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (5) means for detecting the hybrid formed optionally as a result of the hybridization reaction that occurred previously.

Quantification of the hybrid can be accomplished by labeling the target with fluorescent or radioisotopes, as in the probes described above, according to conventional methods. Labeling may be on the primer itself or by using bases labeled with fluorescent and radioisotopes for amplification and transcription.

Hereinafter, the examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

<Example 1>

Sample Preparation

The subjects were 75 Korean patients with unknown cause of disseminated bronchiectasis or chronic pancreatitis. The bronchiectasis group consists of 20 pediatric patients (1 to 18 years old) and 27 adult patients (22 to 74 years old). The diagnosis of disseminated bronchiectasis is reconfirmed by high-resolution computed tomography. It became. Among the patients with bronchiectasis, 15 patients had a history of infancy and 10 patients had asthma symptoms. All registered bronchiectasis patients did not have symptoms and signs (eg, malabsorption and bowel obstruction) associated with typical CF. Twenty eight patients with chronic pancreatitis were also diagnosed using conventional diagnostic methods. Alcoholic subjects who consume 40g or more of ethanol per day were excluded from the examples of the present invention. 117 normal subjects were randomly selected and their health status was confirmed by routine physical, laboratory and radiographic examinations. Blood samples were collected from each subject and DNA was extracted using a purification kit (Qiagen).

<Example 2>

Genetic analysis

a. Mutation / polymorphism pattern analysis of CFTR gene

Mutation / polymorphism patterns of Korean CFTR genes were investigated using two-dimensional gene scanning (TDGS). TDGS was performed on a total of 100 samples from 47 normal bronchiectasis patients and 25 pancreatitis patients. PCR primer sets for human CFTR genes designed for TDGS analysis were purchased from Accelerated Genomics Inc (San Antonio, Texas), and to increase the efficiency of TDGS, primers were fluorescently labeled with HEX and GC-clamp (GC-clamp). ) Was attached to one primer of each pair. Total CFTR coding sequence and lateral intron sequence were amplified by a two-step multiplex PCR reaction. First, 9-plex long range PCR was performed to amplify 9 long fragments containing the coding sequence of CFTR. This product then served as a template for the 4-plex short range PCR reaction to amplify a total of 41 desired fragments, 100 and 500 bp in length. Subsequently, the PCR fragments were subjected to one complete denaturation and regeneration to generate heteroduplex molecules. Mixtures of all CFTR fragments were loaded into an automated 2-D gel electrophoresis instrument (OptiAnalyzer 8008HT; Accelerated Genomics) and separated on a DGGE gel in unison according to the size and melting point of each fragment. Whole DNA fragments were visualized by a fluorescence scanner (Molecular Imager FX, Bio-Rad), and polymorphisms and mutations of heterogeneity were detected with four points representing two homoduplexes and two heteroduplexes rather than one. . The nature and position of the sequence variants were confirmed by DNA nucleotide sequencing. TDGS results are shown in FIG. 1.

As shown in FIG. 1A, samples obtained from normal subjects showed that two heteroduplex bands frequently identified as M470V and 2694T / G (T854) at exon 10 and 14a, respectively. As shown in FIG. 1B, several different heteroduplexes were identified in the sample obtained from the patient, including those identified as Q1352H at exon 22.

After extensive gene scanning and nucleotide sequencing, we found 9 diallelic variations in 5'UTR and several exons (Table 1). Of these, two are novel mutations and the other seven have already been registered for CFGAC.

CFTR genetic variation analyzed in the present invention Variants Identified by TDGS Most common disease-causing mutations worldwide Known Disease-causing Microsatellites -8G / C (5'UTR) ^a R117H (Exon 4) T _5-7,9 (IVS 8) I125T (Exon 4) ^b 621 + 1G ＞ T (Intron 4) E217G (Exon 6a) ^b F508del (Exon 10) 1059C ＞ T (Exon 7, A309) ^a 1717-1G ＞ A (Intron 10) M470V (Exon 10) ^b G542X (Exon 11) I556V (Exon 11) ^b G551D (Exon 11) 2694T / G (Exon 14a, T854) ^b R553X (Exon 11) Q1352H (Exon 22) ^b R1162X (Exon 19) R1453W (Exon 24) ^b W1282X (Exon 20) N1303K (Exon 21)

(Mution names and nucleotide numbers were named according to the Cystic Fibrosis Genetic Analysis Consortium. A is a new genetic variation identified in the present invention, b means a mutation registered with CFGAC.)

On the other hand, CFTR genetic variation name in the specification of the present invention, including Table 1 above is defined as follows:

(1) -8G / C is a base G or C at the 8th position upstream from the point at which translation begins;

(2) 2694T / G has the 2694th base T or G;

(3) I 125 T replaces the 125 th amino acid with I to T;

(4) 1059C> T is more likely that the 1059th base is C than T;

(621) 621 + 1G> T is where 621 is the end of the exon, and 621 + 1 is one base away from the end of the exon, more often G than T; And

(6) 1717-1G> A is the point where 1717 starts exon, and 1717-1 is G more than A before 1 base of exon.

b. Genotyping

To investigate the pathological relationship between CFTR genetic variation and bronchiectasis or chronic pancreatitis, a case-control study was conducted using samples from 192 subjects described in Example 1. In addition to the nine mutations previously found in TDGS, we include the ten most common disease-causing mutations (Dataita obtained from CFGAC) and microsatellite IVS8 Tn, which may be associated with disease. I was. Genotyping of the diallelic position was performed by SNaPshot (Applied Biosystems, Foster City, CA) method following the protocol provided by the manufacturer for automated genetic analyzers (Model 3700, Applied Biosystems) and the Tn number was Confirmed by bidirectional nucleotide sequencing. Of the 10 worldwide disease-causing mutations, only R117H cases were present in normal subjects. The frequency of genotypes found at each location is listed in Table 2.

Proportion of CFTR Gene Mutations in Koreans transition Genotype Group Count Normal person (n = 117) Bronchiectasis (n = 47) Pancreatitis (n = 28) Double allele -8G / C + / ++ / ^-a 10512 443 226 R117H + / ++ /- 1161 470 280 I125T + / ++ /- 1161 461 271 E217G + / ++ /- 1143 43 4 ^b 271 1059C ＞ T (A309) + / ++ /- 1170 470 271 M470V + / ++ /-/- 235242 32816 6148 I556V + / ++ /- 1116 452 280 2694T / G (T854) + / ++ /-/- 415125 16274 8146 Q1352H + / ++ /- 1161 43 4 * 24 4 ** R1453W + / ++ /- 1152 461 280 Microsatellite T _5-7,9 (IVS 8) 5/66/77/77/9 401103 6 * 139 * ^c 1 20260

Genetic variations in several locations were associated with bronchiectasis and / or chronic pancreatitis, most notably Q1352H. The heterozygote ratio of Q1352H was significantly higher in bronchiectasis (P = 0.02) and chronic pancreatitis patients (P = 0.005) than in normal individuals. It has been suggested that IVS8 T ₅ affects RNA splicing and is associated with diseases associated with CF or single symptomatic CF. The table also shows that IVS8 T ₅ in patients with bronchiectasis (P = 0.03). Increased percentage.

c. Haploid analysis

As multiple allele heterogeneity is present in both normal and patients, a haplotype-centric approach was used to provide a better understanding of CFTR mutations associated with disease. Haploids were assembled using a Haplotyper program based on genotype data and Bayesian algorithms obtained from 192 samples. Seventeen positions and microsatellite IVS8 T _n were analyzed, which included 10 dual allelic variants substantially present in Koreans. In the case of IVS8 T _n , T ₅ is recognized as a mutant and all other polymorphisms are entered as wild type because the program can only accept double allele data. After 100 iterations, 15 haploids were assembled and their identification was assigned according to the total sample proportion (Table 3).

Haploid assembly Allele ID end I All la hemp bar four Ah character car Ka group ^a M470V-2694T / G background Normal n (%) Bronchiectasis n (%) Pancreatitis n (%) One G R I E C WT V I T Q R 121 (51.7) 47 (50.0) 24 (42.9) 2-1 2 G R I E C WT M I G Q R 78 (33.3) 25 (26.6) 18 (32.1) 1-2 3 C R I E C WT M I G Q R 11 (4.7) 3 (3.2) 5 (8.9) 1-2 4 G R I E C WT V I T H R 1 (0.4) 4 (4.3) * 4 (7.1) ** 2-1 5 G R I E C 5 V I T Q R 2 (0.9) 5 (5.4) * 1 (1.8) 2-1 6 G R I G C WT M I G Q R 3 (1.3) 4 (4.3) c 1 (1.8) 1-2 7 G R I E C WT V V T Q R 5 (2.1) 2 (2.2) 0 (0.0) 2-1 8 G R I E C WT V I G Q R 4 (1.7) 1 (1.0) 0 (0.0) 2-2 9 G R I E C 5 M I G Q R 2 (0.9) 1 (1.0) 1 (1.8) 1-2 10 G R I E C WT M I G Q W 2 (0.9) 1 (1.0) 0 (0.0) 1-2 11 G R T E C WT V I T Q R 1 (0.4) 1 (1.0) 1 (1.8) 2-1 12 G R I E C WT M I T Q R 2 (0.9) 0 (0.0) 0 (0.0) 1-1 13 C R I E C WT V I G Q R 1 (0.4) 0 (0.0) 0 (0.0) 2-2 14 G H I E C WT V V T Q R 1 (0.4) 0 (0.0) 0 (0.0) 2-1 15 C R I E T WT M I G Q R 0 (0.4) 0 (0.0) 1 (1.8) 1-2 gun 234 (100.0) 94 (100.0) 56 (100.0)

(A: -8G / C, I: R117H, C: I125T, LA: E217G, M: 1059C> T, B: T _5-7,9, SA: M470V, Ah: I556V, J: 2694T / G, Car : Q1352H, car: R1453W)

First we categorized each haploid using two conventional polymorphisms M470V and 2694T / G. Noticeable was the close relationship between the M470V and 2694T / G (T854) locations. All other haploids (corresponding to 97% of normal people) except haploids 8 (2-2), 12 (1-1) and 13 (2-2) are V470-2694T (2-1) or M470-2694G (1- 2) was in the background. Thus, haploids 4, 5, 7, 11 and 14 will be variants of haploid 1 and haploids 3, 6, 9, 10 and 15 will be variants of haploid 2. Haploid 12 was confirmed to have IVS8 T ₉ by haploid assembly using T ₉ instead of T ₅ as a mutation. Thus, haploid 12 has IVS8 T ₉ -M470V-2694T, and 95% of F508dels are known to originate from these haploids. The proportion of alleles of haploid 12 (0.9%) in normal Koreans was much less than that of Kaukasian (7.3%).

Although the ratio of haploid 1 with V470 was the highest in Korean, we used haploid 2 with M470 as the basic haploid for statistical analysis. This is because the sequence of haploid 2 coincided with the CFTR sequence registered in most databases. In addition, when comparing whole cell Cl ^- channel activity, V470 CTFR is known to be about half of M470 CFTR function. Haploids showing an extremely low rate (2%) in all groups (Haploids 8-15) were excluded from statistical analysis.

As expected from genotyping data, haploid 4 comprising Q1352H was closely associated with bronchiectasis and chronic pancreatitis in Koreans ( P = 0.02 and P = 0.008, respectively). The proportion of haploid 6 containing E217G was 5.8 times higher in normal children (1.3%, 3 of 234) in children with bronchiectasis (7.5%, 3 of 40). Haploids 5 and 9 were found to have IVS8 T ₅ , the proportion of which was similar to that of normal persons. Another variation, the proportion of haploid 5 containing V470 ( P = 0.02) increased rapidly in the bronchiectasis group, whereas that of haploid 9 containing M470 did not change.

<Example 3>

Positional Mutations and Expression of CFTR

Molecular and functional studies of CFTR have been conducted to reach specific conclusions about the association with disease and to identify the underlying mechanisms of defective CFTR function. Four of the mutations identified in Koreans were selected for functional studies based on conclusions from the present invention and data reported in CFGAC (Table 4). All mutations were introduced in the same haploid 2 background containing M470 to identify each mutagenesis effect.

Characteristics of CFTR Mutants Selected for Functional Studies name Nucleotide changes Exon domain Evolutionary conservation Related disease E217G 782A> G 6a EC2 b, h, r Pancreatitis (Polish) Pancreatitis (Japanese) I556V 1798A> G 11 NBD1 7 species Chronic Bronchitis (French) Q1352H 4188G> C 22 NBD2 7 kinds CBAVD (Japanese) Pancreas Bronchitis (Japanese) R1453W 4489> T 24 IC10 b, h, m, r, s Pan-bronchiolitis (Japanese)

(Evolutionary preservation compared CFTR genes of bovine (b), mackerel (d), human (h), mouse (m), rabbit (r), sheep (s) and frog (x). The abbreviations used are as follows: CBAVD, congenital weaker epididymis, CF, cystic fibrosis; EC, extracellular domain; IC, intracellular domain; NBD, nucleotide binding domain.)

The use of pNUT-CFTR plasmids for the stable expression of human wild-type CFTR in mammalian cells is already known. The plasmid also contains a gene of dihydrofolate reductase (DHFR), which allows the selection of transformants using methotrexate. Four individual mutations (Table 4) and positional mutations for M470V were performed using the QuickChange Mutation Kit (Stratagene, La Jolla, Calif.). Plasmids for wild-type and mutant CFTR expression were transfected with DHFR-negative CHO-K1 cell line (KCLB 10061; Korea Cell Line Bank, Seoul, Korea) using lipofectamine plus reagent (Life Technologies). CHO-K1 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum penicillin (50 unit / ml) / streptomycin (50 μg / ml). Two days after transfection, methotrexate (5000 μg / ml, Alexis) was added to the culture medium to select the transfected cells. Two or three separate stable cell lines have been developed for each mutant. Primers used for mutagenesis are as follows.

Mutation Sequence Listing Sequence E217G 2 5'-CTC CTC ATG GGG CTA ATC TGG GGG TTG TTA CAG GCG TCT G-3 ' M470V 3 5'-CTG GAG CAG GCA AGA CTT CAC TTC TAA TGG TGA TTA TGG GAG-3 ' I556V 4 5'-AGT GGA GGT CAA CGA GCA AGA GTT TCT TTA GCA AGG TGA AT-3 ' Q1352H 5 5'-CCT AAG CCA TGG CCA CAA GCA CTT GAT GTG CTT GGC TAG-3 ' R1453W 6 5'-GTG AAG CTC TTT CCC CAC TGG AAC TCA AGC AAG TGC AAG TCT-3 '

<Example 4>

Immunoblotting of CFTR and DHFR

CHO-K1 cells transformed with the pNUT-CFTR vector were trypsinized and lysed using conventional lysis buffer containing a protease inhibitor cocktail. 50 μg of protein samples obtained from cells expressing wild-type or mutant CFTR were suspended in SDS sample buffer and separated using SDS-PAGE on 6% gels. The isolated protein was transferred to nitrocellulose membrane and probed with monoclonal antibody M3A7 against the NBD2 domain of CFTR. After treatment with a suitable secondary antibody, protein bands were visualized using an improved chemiluminescence kit (Amersaham Pharmacia). To normalize the level of transfection of the pNUT-CFTR plasmid, the expression level of the cis-gene product DHFR was immunoblow using a smaller amount of lysate (10 μg protein) and anti-DHFR antibody (Research Diagnostics Inc.). Analysis by Ting. Staining intensity of the CFTR and DHFR bands was analyzed using imaging software (MCID version 3.0; Brok University, St. Catharines, Ontario, Canada) and the intensity ratio of CFTR / DHFR was compared in cells transfected with each mutation.

In the CHO cell system, complex-glycosylated 'band C' proteins were identified primarily, rather than primary-translated 'band A' or core-glycosylated 'band B' forms. As shown in FIG. 2A, protein expression of mature glycated CFTR was found to be significantly reduced in E217G and Q1352H mutations. For each transformant, similar expression levels of the cis-gene product dihydrofolate reductase (DHFR) in the expression vector confirmed that these results were not due to low transformation efficiency. When the CFTR band intensities were leveled against that of the cis-gene product DHFR, the expression levels of E217G and Q1352H decreased by 64% and 73%, respectively, compared to the wild type (FIG. 2B).

Example 5

Measurement of Cl ^- Channel Activity

Chloride ion channel activity of wild-type and mutant CFTR was measured in whole cell steric medium and single channel. First, whole cell records were performed on transfected CHO-K1 cells. The pipette solution comprises 140 mM N-methyl D-glucamine chlorite (NMDG-Cl), 5 mM EGTA, 1 mM MgCl ₂ , 1 mM Tris-ATP and 10 mM Hepes (pH 7.2), bath bath solution) 140 mM NMDG-Cl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM glucose and 10 mM Hepes, pH 7.4. All experiments were conducted at room temperature (22-25 ° C.). Pipettes from borosilicate glass It was prepared and had a resistance of 3-5 MΩ after fire polishing. Sealing resistance was typically 3-10 GΩ. After sterilization of the whole cell, CFTR was activated by addition of 5 μM and 100 μM 3-isobutyl-1-methylxanthine (IBMX). The holding potential used in the capacitive test was -30 mV and the patch-clamp results were filtered at 5 kHz. Power was counted and analyzed using an AxoScope 8.1 system and a 1322A AC / DC converter. Average power was leveled as power density (pA / pF).

In total cell measurements, cAMP cocktail (5 μM foscholine and 100 μM IBMX) treatment generated a large current in a linear IV relationship in NMDG-Cl solution (FIGS. 3A and 3B). Treatment with glibenclamide (100 μM) or phenylanthranilic acid (N-phenylanthranilic acid, DPC, 100 μM) inhibited the current by 76 ± 4% and 89 ± 5%. These properties are consistent with previous reports on CFTR Cl ⁻ currents. All mutations showed lower current density (pA / pF) than wild type CFTR. Of these, Q1352H had the largest decrease (71%) and R1463W had the smallest decrease (37%).

Since single channel kinetics did not exactly match protein expression levels (Figure 2) and total cell Cl ⁻ currents (Figure 3), single channel characteristics of wild-type and mutant CFTR resulted in 10-17 MΩ resistance in steric arrangements attached to cells. It was analyzed using a flame-polished pipette to have. The pipette solution contains 140 mM NMDG-Cl, 1.3 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM glucose and 10 mM Hepes, pH 7.4, KCl bath solution contains 140 mM KCl, 1.3 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM glucose and 10 mM Hepes, pH 7.4. Records were performed using Axopatch-200B (Axon Instrument) at room temperature. Voltage and current data were low-pass filtered at 1 kHz and stored for later analysis. Single channel data were filtered numerically at 25 Hz and analyzed using Fetchan and pSTAT v 6.0 software (Axon Instruments).

No spontaneous channel activity was observed before addition of the cAMP cocktail. The cells generated low conduction currents when treated with the cAMP cocktail. The current record for 6 minutes at -80 mV voltage was analyzed for the possibility of opening (Po) (FIG. 3D), and the results thus obtained are summarized in a bar graph (FIG. 3F). Compared with the wild type, Po decreased significantly by 34% at I556V, 55% at Q1532H and 78% at R1453W. To determine the single channel induction size, all point histograms were made at each voltage from -120 mV to +120 mV (in 40 mV increments) and applied to a Gaussian distribution. One example at +80 mV is shown in Figure 3E. The mean conduction of wild type CFTR was 7.6 pS and no change was observed in mutant CFTR. Thus, the reduced current density in total cell Cl ⁻ current at E217G is caused by decreased expression of cell membrane proteins, and I556V and R1453V are caused by reduced Po. In the case of Q1352H, both the expression of the cell membrane protein and the Po are decreased.

<Example 6>

Cl ^- / HCO ₃ ^- Measurement of the exchange

CFTR is known to play an important role in HCO ₃ ⁻ transport of epithelial cells by regulating Cl ⁻ / HCO ₃ ⁻ exchange. Reduced HCO ₃ ⁻ secretion by defective CFTR-dependent HCO ₃ ⁻ transport has been suggested to be an important pathological mechanism in respiratory and pancreatic diseases induced by mutant CFTR. Accordingly, Cl ^- / HCO ₃ ^- exchange activity of HCO3 ^- could be measured in the wild-type and mutant CFTR transformed cells by measuring the pHi increase due to the removal of the (Fig. 4) ^- Cl from the perfusion solution buffered to.

Cl ⁻ / HCO ₃ ⁻ exchange activity was measured by recording the intracellular pH for changes. Briefly, the glass cover slips with cells attached were washed once with Hepes buffer solution A and assembled to form the bottom of the chamber. Hepes buffer solution A comprises 140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 10 mM glucose and 10 mM Hepes, pH 7.4. Cells were incubated for 10 minutes in Solution A with 2.5 μm BCECF-AM and loaded with fluorescent pH probe BCECF. After dye loading, cells were filled with HCO ₃ ⁻ buffer solution and BCECF fluorescence was recorded at excitation wavelengths 490 and 440 nm at a resolution of 2 / s using a recording setup (Delta Ram; PTI Inc.). The HCO ₃ ^- buffer is 120mM NaCl, 5mM KCl, 1mM MgCl 2, 1mM CaCl 2, 10mM glucose, 5mM HEPES _{(pH 7.4), 25mM NaHCO 3} (pH 7.4) Cl - without solution Cl ^- gluconate Prepared by replacing with. The 490/440 ratio was calibrated by filling the cells with a solution containing 145 mM KCl, 10 mM Hepes and 5 μΜ nigericin.

The basal Cl ⁻ / HCO ₃ ⁻ exchange activity in the transformed cells was 0.083 ± .05 ΔpH units / minute, which was not significantly changed by forskolin treatment. However, in cells transformed with wild type CFTR, basal activity increased to 0.141 ± .021 and forskolin treatment significantly increased Cl ⁻ / HCO ₃ ⁻ exchange activity to 0.677 ± .063 (FIG. 4A). Changes activated by cAMP in each mutation were analyzed to individualize CFTR-dependent Cl ⁻ / HCO ₃ ⁻ exchange (FIG. 4B). Compared with the wild type, E217G and Q1352H were found to significantly decrease by 65% and 77%, respectively, in the Cl ⁻ / HCO ₃ ⁻ exchange activated by forskolin.

<Example 7>

Expression of CFTR has a Q1352H and M470, Cl ^- current and Cl ^- / HCO ₃ ^- Measurement of the exchange activity

The Q1352H mutation showed the highest disease association in genotyping data. Since Q1352H appeared only in the V470 background in the subject population, we caused M470V changes in wild-type and Q1352H-CFTR clones and conducted a functional study. As shown in FIG. 5A, additional M470V changes did not alter protein expression levels in either wild type or Q1352H-CFTR. However, additional M470V changes in Q1352H reduced cAMP-activated Cl ⁻ currents (FIGS. 5C and 5D) and cAMP-activated Cl ⁻ / HCO ₃ ⁻ exchanges (FIG. 5E and 5F) by 99% and 95%, respectively. .

<Example 8>

Preparation of DNA Chip for CFTR Mutation Detection

In the CFTR gene having a nucleotide sequence of SEQ ID NO: 1, a nucleic acid molecule having a 755th to 840th sequence (SEQ ID NO: 7), a nucleic acid molecule having a 1487 to 1587th sequence (SEQ ID NO: 8), and a 4153 to 4231 base Each nucleic acid molecule having a sequence (SEQ ID NO: 9) was synthesized. The nucleic acid molecules thus obtained were used as templates, and the three nucleic acid molecules were PCR amplified using the primers shown in Table 6. At this time, PCR was mixed with 20pmol primer, 2.5mM dNTP mixture, 100ng plasmid DNA, 5μl 10 x PCR buffer (Solgent Co.), 2.5 unit Taq DNA polymerase so that the final volume is 50μl and then The mixture was denatured at 94 ° C. for 10 minutes, denatured at 94 ° C. for 1 minute, recombined at 55 ° C. for 45 seconds, and stretched for 34 seconds at 72 ° C. for 34 times. The reaction was terminated after expanding for 10 minutes at 72 ° C.

Primer used for probe amplification CFTR variation SEQ ID NO: E217G sense  10  5 '-TGGCACTCCTCATGGGGCTAAT-3' Antisense  11  5 '-AAAAAGGGCAAGGACTATCAGGAA-3 M470V sense  12  5 '-AGTTGTTGGCGGTTGCTGGAT-3' Antisense  13  5 '-ACTGTGCTTAATTTTACCCTCTGA-3' Q1352H sense  14  5 '-GATGGGGGCTGTGTCCT-3' Antisense  15  5 '-AGATCTTCGCCTTACTGAG-3'

The amplified DNA fragments were purified using a QuiaQuick PCR purification kit (Qiagen), and then electrophoresed on a 2% agarose gel to identify PCR products and subjected to sequencing to determine whether the desired nucleic acid molecules (ie, probes) were amplified. It was confirmed.

The PCR products were dried, dissolved in 20 μl of 50% DMSO solution and integrated onto a Corning Gaps slide for DNA chips to make chips, crosslinked with 300mJ UV irradiation, and then dried at room temperature. Stored at.

Example 9

DNA Sample Preparation and Use of DNA Chips

The DNA chip according to the present invention was tested for CFTR mutations to test whether it is suitable as a kit for diagnosing chronic respiratory and pancreatic diseases. Genomic DNA was isolated from pancreatitis patients identified as normal and containing CFTR mutations (E217H, Q1352H, and M470V), and then subjected to PCR using the primers described in Table 6.

At this time, the PCR reaction was performed with 20 pmol primer, 2.5 mM dNTP mixture, 100ng genomic DNA, 5μl 10 × PCR buffer (Solgent Co.), 2.5 unit (Solgent Co.) Taq DNA polymer so that the final volume was 50μl. Laze, 25 nM 1 μl of Cyanine-5-dUTP phosphor was mixed, and the mixture was denatured at 94 ° C. for 10 minutes, 94 ° C. for 1 minute, recombined at 55 ° C. for 45 seconds, and stretched at 72 ° C. for 45 seconds for 35 times. Repeated. The reaction was terminated after expanding for 10 minutes at 72 ° C.

PCR products labeled with Cy-5 were purified with a QuiaQuick PCR purification kit and concentrated to a final concentration of 25 μl to prepare DNA samples. This DNA sample was hybridized to the prepared DNA chip. At this time, hybridization was mixed with 3 x SSC, 0.2% SDS, 20μg salmon sperm DNA so that the final volume of 40μl and then the mixture was reacted at 50 ℃ for 2 hours.

  After hybridization, the chips were washed with 2 x SSC for 2 minutes, 0.2 x SSC and 0.1% SDS (sodium dodecyl sulfate) for 5 minutes, 0.2 x SSC for 5 minutes x 2 times and dried by centrifugation (700 rpm, 10 min). Next, the hybridization results were confirmed by scanning using an Axon 4000B scanner (Axon Instument).

As a result of the DNA chip test, it was confirmed that DNA samples isolated from normal persons did not hybridize with any probe, while DNA samples isolated from pancreatitis patients specifically hybridized with all the probes of the present invention.

Using the diagnostic kit according to the present invention will be able to accurately diagnose chronic respiratory and pancreatic diseases caused by CFTR mutation in Asians.

Figure 1 shows the entire CFTR coding sequence amplified by two-step PCR and DGGE results of introns, A is a sample obtained from a normal subject, a heteroduplex band at exons 10 and 14a, B is from a pancreatitis patient The sample obtained shows heterobiaxial bands at exons 14a and 22. C shows the result that the genetic diversity at exon 22 was identified as Q1352H (4188G> C) by bidirectional nucleotide sequencing.

FIG. 2A shows immunoblotting results for wild type CFTR, mutant CFTR (E217G / Q1352H / I556V / R1453W) and cis-transformation marker DHFR, and FIG. 2B shows CFTR bands from these immunoblotting results And staining intensity of the DHFR bands and the CFTR / DHFR intensity ratio is shown as a bar graph.

A, B and C in FIG. 3 show Cl ⁻ currents measured in whole cell conformation of wild type CFTR, mutant CFTR (E217G / Q1352H / I556V / R1453W), and D, E and F of FIG. 3 are wild type CFTR , Cl ⁻ current measured in a single channel attached to cells of mutant CFTR (E217G / Q1352H / I556V / R1453W).

4 shows Cl ⁻ / HCO ₃ ⁻ exchange activity of wild type or Q1352H CFTR by measuring the initial pHi after removing Cl ⁻ from HCO ₃ ⁻ buffered perfusate in CHO cells transformed with wild type CFTR or Q1352H CFTR. will be.

5 shows protein expression and functional activity when M470V mutations were introduced into wild-type and Q1352H clones, A and B show the expression rate of CFTR protein from the clone, and C and D are activated by c-AMP the Cl ^- currents, E and F, Cl ^- shows the exchange activity ^- / HCO _3. It can be seen that Cl ⁻ current and Cl ⁻ / HCO ₃ ⁻ exchange activity is significantly reduced when additional M470V mutations are introduced into the Q1352H clone.

<110> Yon Sei University <120> Kit for the Diagnosis of Chronic Respiratory and Pancreatic Diseases Associated with CFTR variants in East Asian <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 6129 <212> DNA <213> Homo sapiens <400> 1 aattggaagc aaatgacatc acagcaggtc agagaaaaag ggttgagcgg caggcaccca 60 gagtagtagg tctttggcat taggagcttg agcccagacg gccctagcag ggaccccagc 120 gcccgagaga ccatgcagag gtcgcctctg gaaaaggcca gcgttgtctc caaacttttt 180 ttcagctgga ccagaccaat tttgaggaaa ggatacagac agcgcctgga attgtcagac 240 atataccaaa tcccttctgt tgattctgct gacaatctat ctgaaaaatt ggaaagagaa 300 tgggatagag agctggcttc aaagaaaaat cctaaactca ttaatgccct tcggcgatgt 360 tttttctgga gatttatgtt ctatggaatc tttttatatt taggggaagt caccaaagca 420 gtacagcctc tcttactggg aagaatcata gcttcctatg acccggataa caaggaggaa 480 cgctctatcg cgatttatct aggcataggc ttatgccttc tctttattgt gaggacactg 540 ctcctacacc cagccatttt tggccttcat cacattggaa tgcagatgag aatagctatg 600 tttagtttga tttataagaa gactttaaag ctgtcaagcc gtgttctaga taaaataagt 660 attggacaac ttgttagtct cctttccaac aacctgaaca aatttgatga aggacttgca 720 ttggcacatt tcgtgtggat cgctcctttg caagtggcac tcctcatggg gctaatctgg 780 gggttgttac aggcgtctgc cttctgtgga cttggtttcc tgatagtcct tgcccttttt 840 caggctgggc tagggagaat gatgatgaag tacagagatc agagagctgg gaagatcagt 900 gaaagacttg tgattacctc agaaatgatt gaaaatatcc aatctgttaa ggcatactgc 960 tgggaagaag caatggaaaa aatgattgaa aacttaagac aaacagaact gaaactgact 1020 cggaaggcag cctatgtgag atacttcaat agctcagcct tcttcttctc agggttcttt 1080 gtggtgtttt tatctgtgct tccctatgca ctaatcaaag gaatcatcct ccggaaaata 1140 ttcaccacca tctcattctg cattgttctg cgcatggcgg tcactcggca atttccctgg 1200 gctgtacaaa catggtatga ctctcttgga gcaataaaca aaatacagga tttcttacaa 1260 aagcaagaat ataagacatt ggaatataac ttaacgacta cagaagtagt gatggagaat 1320 gtaacagcct tctgggagga gggatttggg gaattatttg agaaagcaaa acaaaacaat 1380 aacaatagaa aaacttctaa tggtgatgac agcctcttct tcagtaattt ctcacttctt 1440 ggtactcctg tcctgaaaga tattaatttc aagatagaaa gaggacagtt gttggcggtt 1500 gctggatcca ctggagcagg caagacttca cttctaatgg tgattatggg agaactggag 1560 ccttcagagg gtaaaattaa gcacagtgga agaatttcat tctgttctca gttttcctgg 1620 attatgcctg gcaccattaa agaaaatatc atctttggtg tttcctatga tgaatataga 1680 tacagaagcg tcatcaaagc atgccaacta gaagaggaca tctccaagtt tgcagagaaa 1740 gacaatatag ttcttggaga aggtggaatc acactgagtg gaggtcaacg agcaagaatt 1800 tctttagcaa gagcagtata caaagatgct gatttgtatt tattagactc tccttttgga 1860 tacctagatg ttttaacaga aaaagaaata tttgaaagct gtgtctgtaa actgatggct 1920 aacaaaacta ggattttggt cacttctaaa atggaacatt taaagaaagc tgacaaaata 1980 ttaattttga atgaaggtag cagctatttt tatgggacat tttcagaact ccaaaatcta 2040 cagccagact ttagctcaaa actcatggga tgtgattctt tcgaccaatt tagtgcagaa 2100 agaagaaatt caatcctaac tgagacctta caccgtttct cattagaagg agatgctcct 2160 gtctcctgga cagaaacaaa aaaacaatct tttaaacaga ctggagagtt tggggaaaaa 2220 aggaagaatt ctattctcaa tccaatcaac tctatacgaa aattttccat tgtgcaaaag 2280 actcccttac aaatgaatgg catcgaagag gattctgatg agcctttaga gagaaggctg 2340 tccttagtac cagattctga gcagggagag gcgatactgc ctcgcatcag cgtgatcagc 2400 actggcccca cgcttcaggc acgaaggagg cagtctgtcc tgaacctgat gacacactca 2460 gttaaccaag gtcagaacat tcaccgaaag acaacagcat ccacacgaaa agtgtcactg 2520 gcccctcagg caaacttgac tgaactggat atatattcaa gaaggttatc tcaagaaact 2580 ggcttggaaa taagtgaaga aattaacgaa gaagacttaa aggagtgcct ttttgatgat 2640 atggagagca taccagcagt gactacatgg aacacatacc ttcgatatat tactgtccac 2700 aagagcttaa tttttgtgct aatttggtgc ttagtaattt ttctggcaga ggtggctgct 2760 tctttggttg tgctgtggct ccttggaaac actcctcttc aagacaaagg gaatagtact 2820 catagtagaa ataacagcta tgcagtgatt atcaccagca ccagttcgta ttatgtgttt 2880 tacatttacg tgggagtagc cgacactttg cttgctatgg gattcttcag aggtctacca 2940 ctggtgcata ctctaatcac agtgtcgaaa attttacacc acaaaatgtt acattctgtt 3000 cttcaagcac ctatgtcaac cctcaacacg ttgaaagcag gtgggattct taatagattc 3060 tccaaagata tagcaatttt ggatgacctt ctgcctctta ccatatttga cttcatccag 3120 ttgttattaa ttgtgattgg agctatagca gttgtcgcag ttttacaacc ctacatcttt 3180 gttgcaacag tgccagtgat agtggctttt attatgttga gagcatattt cctccaaacc 3240 tcacagcaac tcaaacaact ggaatctgaa ggcaggagtc caattttcac tcatcttgtt 3300 acaagcttaa aaggactatg gacacttcgt gccttcggac ggcagcctta ctttgaaact 3360 ctgttccaca aagctctgaa tttacatact gccaactggt tcttgtacct gtcaacactg 3420 cgctggttcc aaatgagaat agaaatgatt tttgtcatct tcttcattgc tgttaccttc 3480 atttccattt taacaacagg agaaggagaa ggaagagttg gtattatcct gactttagcc 3540 atgaatatca tgagtacatt gcagtgggct gtaaactcca gcatagatgt ggatagcttg 3600 atgcgatctg tgagccgagt ctttaagttc attgacatgc caacagaagg taaacctacc 3660 aagtcaacca aaccatacaa gaatggccaa ctctcgaaag ttatgattat tgagaattca 3720 cacgtgaaga aagatgacat ctggccctca gggggccaaa tgactgtcaa agatctcaca 3780 gcaaaataca cagaaggtgg aaatgccata ttagagaaca tttccttctc aataagtcct 3840 ggccagaggg tgggcctctt gggaagaact ggatcaggga agagtacttt gttatcagct 3900 tttttgagac tactgaacac tgaaggagaa atccagatcg atggtgtgtc ttgggattca 3960 ataactttgc aacagtggag gaaagccttt ggagtgatac cacagaaagt atttattttt 4020 tctggaacat ttagaaaaaa cttggatccc tatgaacagt ggagtgatca agaaatatgg 4080 aaagttgcag atgaggttgg gctcagatct gtgatagaac agtttcctgg gaagcttgac 4140 tttgtccttg tggatggggg ctgtgtccta agccatggcc acaagcactt gatgtgcttg 4200 gctagatctg ttctcagtaa ggcgaagatc ttgctgcttg atgaacccag tgctcatttg 4260 gatccagtaa cataccaaat aattagaaga actctaaaac aagcatttgc tgattgcaca 4320 gtaattctct gtgaacacag gatagaagca atgctggaat gccaacaatt tttggtcata 4380 gaagagaaca aagtgcggca gtacgattcc atccagaaac tgctgaacga gaggagcctc 4440 ttccggcaag ccatcagccc ctccgacagg gtgaagctct ttccccaccg gaactcaagc 4500 aagtgcaagt ctaagcccca gattgctgct ctgaaagagg agacagaaga agaggtgcaa 4560 gatacaaggc tttagagagc agcataaatg ttgacatggg acatttgctc atggaattgg 4620 agctcgtggg acagtcacct catggaattg gagctcgtgg aacagttacc tctgcctcag 4680 aaaacaagga tgaattaagt ttttttttaa aaaagaaaca tttggtaagg ggaattgagg 4740 acactgatat gggtcttgat aaatggcttc ctggcaatag tcaaattgtg tgaaaggtac 4800 ttcaaatcct tgaagattta ccacttgtgt tttgcaagcc agattttcct gaaaaccctt 4860 gccatgtgct agtaattgga aaggcagctc taaatgtcaa tcagcctagt tgatcagctt 4920 attgtctagt gaaactcgtt aatttgtagt gttggagaag aactgaaatc atacttctta 4980 gggttatgat taagtaatga taactggaaa cttcagcggt ttatataagc ttgtattcct 5040 ttttctctcc tctccccatg atgtttagaa acacaactat attgtttgct aagcattcca 5100 actatctcat ttccaagcaa gtattagaat accacaggaa ccacaagact gcacatcaaa 5160 atatgcccca ttcaacatct agtgagcagt caggaaagag aacttccaga tcctggaaat 5220 cagggttagt attgtccagg tctaccaaaa atctcaatat ttcagataat cacaatacat 5280 cccttacctg ggaaagggct gttataatct ttcacagggg acaggatggt tcccttgatg 5340 aagaagttga tatgcctttt cccaactcca gaaagtgaca agctcacaga cctttgaact 5400 agagtttagc tggaaaagta tgttagtgca aattgtcaca ggacagccct tctttccaca 5460 gaagctccag gtagagggtg tgtaagtaga taggccatgg gcactgtggg tagacacaca 5520 tgaagtccaa gcatttagat gtataggttg atggtggtat gttttcaggc tagatgtatg 5580 tacttcatgc tgtctacact aagagagaat gagagacaca ctgaagaagc accaatcatg 5640 aattagtttt atatgcttct gttttataat tttgtgaagc aaaatttttt ctctaggaaa 5700 tatttatttt aataatgttt caaacatata ttacaatgct gtattttaaa agaatgatta 5760 tgaattacat ttgtataaaa taatttttat atttgaaata ttgacttttt atggcactag 5820 tatttttatg aaatattatg ttaaaactgg gacaggggag aacctagggt gatattaacc 5880 aggggccatg aatcaccttt tggtctggag ggaagccttg gggctgatcg agttgttgcc 5940 cacagctgta tgattcccag ccagacacag cctcttagat gcagttctga agaagatggt 6000 accaccagtc tgactgtttc catcaagggt acactgcctt ctcaactcca aactgactct 6060 taagaagact gcattatatt tattactgta agaaaatatc acttgtcaat aaaatccata 6120 catttgtgt 6129 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctcctcatgg ggctaatctg ggggttgtta caggcgtctg 40 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctggagcagg caagacttca cttctaatgg tgattatggg ag 42 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agtggaggtc aacgagcaag agtttcttta gcaaggtgaa t 41 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cctaagccat ggccacaagc acttgatgtg cttggctag 39 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtgaagctct ttccccactg gaactcaagc aagtgcaagt ct 42 <210> 7 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 tggcactcct catggggcta atctgggggt tgttacaggc gtctgccttc tgtggacttg 60 gtttcctgat agtccttgcc cttttt 86 <210> 8 <211> 101 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 8 agttgttggc ggttgctgga tccactggag caggcaagac ttcacttcta atggtgatta 60 tgggagaact ggagccttca gagggtaaaa ttaagcacag t 101 <210> 9 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 9 gatgggggct gtgtcctaag ccatggccac aagcacttga tgtgcttggc tagatctgtt 60 ctcagtaagg cgaagatct 79 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 10 tggcactcct catggggcta at 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaaagggca aggactatca ggaa 24 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agttgttggc ggttgctgga t 21 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 actgtgctta attttaccct ctga 24 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gatgggggct gtgtcct 17 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 agatcttcgc cttactgag 19

Claims (10)

A nucleic acid molecule comprising 20 to 100 contiguous DNA comprising the 782th G base of the CFTR gene having the nucleotide sequence of SEQ ID NO: 1. delete The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule has a nucleotide sequence of SEQ ID NO. A nucleic acid molecule comprising 20 to 100 contiguous DNA comprising the 1540th G base of the CFTR gene having the nucleotide sequence of SEQ ID NO: 1. delete The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule has a nucleotide sequence of SEQ ID NO: 8. Nucleic acid molecule, characterized in that consisting of 20 to 100 consecutive DNA containing the 4188th C base of the CFTR gene having a nucleotide sequence of SEQ ID NO: 1. delete 8. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule has a nucleotide sequence of SEQ ID NO. 10. A kit for diagnosing chronic respiratory and pancreatic diseases, comprising at least one nucleic acid molecule according to any one of claims 1, 3, 4, 6, 7, and 9.