JPS63502240A - Polymorphisms predictive of atherosclerosis and related to lipid metabolism: ApoB, ApoC2, ApoE, ApoA4 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] When the restriction enzyme includes apoB, EcoRV, HpaI, and selected from the group consisting of aI; if apoCII is included, BamHI; selected from the group consisting of BanI + BglI and NcoI; and ap XbaI, Taql, Drag, and Nco if oATV is included Selected from the group consisting of I.

kit.

Specification Measure the atherosclerotic ΦJ pulse, and the tongue proboscis connected to A for fat expulsion: A o B, A ocII, A oE, to ATV Ibaragai Public Corporation The present invention relates to the use of genetic polymorphisms for determining disease status. Especially the original Apolipoproteins are used to diagnose susceptibility to atherosclerosis. The invention relates to the use of polymorphisms of the Cm, Cm, E and AIV genes.

Disaster rice and balls Degree of morbidity and mortality associated with atherosclerosis in developing countries is higher than for any other disease (even cancer). This disease is , manifested in the form of cholesterol deposits in the arterial cell walls. This deposition occurs slowly It is irreversible and begins at a young age. Clinical symptoms appear It can take years to and.

Very serious. Its symptoms include coronary heart disease and stroke. in general, This disease process is caused by these D! Begins long before the onset of sitting symptoms ing.

Not only genetic factors but also environmental factors influence cholesterol deposition and early warning of the onset of deposition, since it has implications for at least mitigating the process. It is desirable to have diagnostic technology that provides Existing technology is It relies on measuring levels of terols or triglycerides, and these Even if the levels of can be measured very precisely.

They do not give the desired high correlation to true sensitivity.

Depends on the detection of atherosclerotic lesions. More reliable prediction methods are highly damaging. Use a method that allows you to This method is very painful and expensive, so it is not commonly used for general examination. It cannot be used for diagnostic purposes, and it is a special group selected based on family medical history. It cannot even be applied to loops. These techniques also. Atheromatous lesions appear Until then, the most effective time for treatment is because it has very little effect and takes a very long time. has passed.

Long-term treatments that are effective but inconvenient and uninteresting (i.e. - complete diet) Lower serum cholesterol by restricting or lowering cholesterol The importance of early discovery is due to the fact that depressants (using depressants) are effective. is much more obvious. If it is not clear that the “loss” is obvious, then 9 There will be resistance to this method. The question is, what kind of treatment should be given? Not that.

The question is to whom the treatment should be applied.

If the results can be effectively related to the disease being evaluated, the advantage of early detection is The technology to provide this genetically is genetic analysis. The characteristics of an individual's genome are basic The concept is that genetic abnormalities that are indicators of later metabolic disorders are is considered an ideal initial diagnostic tool. Genetic testing, existing methods It can be performed repeatedly using fetuses as young as 7 months. past Over the past decade, restriction enzyme and DNA detection techniques have become available. but.

``Restriction fragment polymorphism J (RFLP) can be used for such diagnosis. did. Southern blot hybridization technology is well established to date. (Southern, E., J. Mol. Biol (1975) 98: 503-517), it is now possible to diagnose the 9th disease. became. Such diseases include sickle cell anemia (Kan, Y, W, et al. al, Proc Natl Acad Sci (USA) (1978) Mutual: 5631), β-thalassemia (Antonarakis, S, E.

et al, Proc Natl Acad Sci (USA) (198 3) 79: 137) Type ■ diabetes (Rotwein, P., et al. al, 5science (1981) 213: 1117).

Familial growth hormone deficiency (Phillips, J, A, + III + Ruri 4. Co1d Spring) Labor atory (1983) pp 305-315), phenylketonuria (W oo, S, L, C,, et at, Nature (et at, Lanc et (1984) i: 239+ Grunenbaum, et al , J C11n Invest (1984) 73: 1491) .

All of the above success stories are related to the disease or illness in question. is based on the identification of multiple specific polymorphs. Predicted polymorphism in human genome The number of shapes is calculated based on the assumption that the probability that a given nucleotide is polymorphic is 0.05. According to Study of the globulin gene cluster locus (Jeffreys, A.J.) Ce1l (1979) 18: 1-10; 0ster, t(,.

etal+ 8m J Hum Gen (1984) 36 (suppl) 150S), it is calculated that it should be on the order of 107. Ru.

The present condition is to determine which of these polymorphisms are associated with specific diseases. The objective is to determine which types of molecule are present and provide a method for detecting them.

The present invention aims to develop genes related to lipid metabolism (these genes are associated with atherosclerosis). Apolipoprotein B, CIO, E and Δ are useful in predicting susceptibility to (encoding)) to provide a polymorph located within. Prediction of atherosclerosis Other polymorphisms in the apoAI/C111 gene complex that are useful for Application No. 724.192 (issued on April 17, 1985) and its partial continuation application As disclosed in U.S. Patent Application No. 782.666 (filed September 30, 1985), It is shown.

Ko 1B doctor! Kenichi The present invention provides polymorphic compounds that are useful as predictive indicators of the continued progression of atherosclerosis. Provides an identification method. Most of these polymorphisms are found in genomes that regulate lipid metabolism. Since it is present in the sequence, the pattern of its presence or absence can be interpreted as a trend in the progression of the disease. Related. Furthermore, the presence or absence of these polymorphisms can be used as a form of genetic identification. Rui is used as a fingerprint to confirm individual and family relationships. Useful.

Thus, from a person's point of view, the present invention can improve atherosclerosis in nine individuals. intended as a method of predicting the likelihood of progression or genetically identifying said individual; The method includes one or more of the following: PvuU, 5tul, EcoRVa, EcoRVb, EcoRVc, Presence or absence of HpaI or Dra I polymorphism; "Bam" of apoCII gene Presence of polymorphisms of t(I”, “Band”, “BglI” or Ncol’”) presence or absence; Presence or absence of “HpaI” polymorphism of apoE gene; apoA IV probe Four XbaI in the apoAI/C'llI/AIV gene complex detected in Presence or absence of polymorphism; apoAI/CIII detected by apo 8 day probe /^■ Presence or absence of “’Taql” polymorphism in the gene complex; apoAIV protein “’Dra” in the apoAI/CIII/AIV gene complex detected in the lobe presence or absence of a polymorphism of ``g''; and apoA detected with an apoAIV probe. I/CII[/to ■ Presence or absence of polymorphism of “'NcoI’” in the gene complex.

Furthermore, insulin genetics is a potential predictor of atherosclerosis. Two of the offspring 5° insertion polymorphisms (reported by others) are shown here.

Stated in another way, the present invention examines the sensitivity of nine individuals to atherosclerosis. A method for predicting susceptibility, or by decomposing the human genomic DNA of an individual subject and or more, to genetically identify the above subjects. Intended to provide: 5.5kb PvuU degradation hybridizing to 970bp apoB probe presence or absence of fragments; The presence of a 5.2kb 5tul fragment that hybridizes to the 2kb apoB probe. presence or absence; Presence of 4,8kb EcoRV fragment hybridizing to 3kbapoB probe presence or absence; 11.0 and 7.0 kb Eco hybridizing to 3 kbapoB probe Presence or absence of RV fragments; 3, 6 and 2.7kb EcoR hybridizing to 3kbapoB probe Presence or absence of V fragment; 8, 3 and 6.2kbtla hybridized to 3kb apoB probe Presence or absence of pI fragment; 2, 3-2.6 kb (variable) or Presence or absence of al fragment; Presence or absence of 1.2kb Ban1 degradation fragment, presence or absence of 3.8kb Bgll fragment, Presence or absence of 5.9 kb BamHI degradation fragment, presence or absence of 15.8 Nco1 degradation fragment , and the presence or absence of the 17.8 kb Nco1 degradation fragment (all of which are associated with the apoCn protein). (hybridizes to the lobe); Presence or absence of a 5 kb Hpa1 degradation fragment that hybridizes to the apoE probe; 10k hybridizing to the apoAIV probe (“XbaI-a” polymorph) b Presence or absence of XbaI fragment; apoAIV probe (“Xbal-b”′ polymorphism 2. Presence or absence of Okb XbaI degradation fragment that hybridizes to apoAIV 20kb XbaI that hybridizes to the probe (“Xbal-c” polymorphism) Presence or absence of degraded fragments; hive on apo88day probe “xbal-d” polymorphism Presence or absence of redized 4,8 kb Xba1 fragment; apoA IV protein Presence or absence of a 3 to 6 kb TaqI-digested fragment that hybridizes to the probe; The presence of a 7.4 kb Dra1 degradation fragment that hybridizes to the apoAIV probe. presence or absence; and The 9.5 kb Nco1 degradation fragment hybridizing to the apoA IV probe presence or absence.

Of course, the presence or absence of fragments corresponding to each of the more frequently occurring polymorphs mentioned above is also important. Ta. Relevant to both susceptibility prediction and genetic fingerprinting.

Furthermore, the present invention decomposes genomic DNA from an individual and extracts the insulin gene 5" presence of a 600-1600 bp insert or a 1600-2200 bp insert Predict an individual's susceptibility to atherosclerosis by detecting Concerning how to measure

Therefore, 1 the present invention is based on the patient's genetic fingerprint (this fingerprint). lipid metabolism and transport (which may be associated with diseases of lipid metabolism and transport), and the tasks involved in these functions. The present invention relates to determination by using polymorphisms of genes associated with proteins.

This genetic fingerprint also. Identifying specific individuals and determining ancestry It is useful for The invention also contemplates kits suitable for carrying out the methods of the invention. do.

Two-dish plate Figure 1 shows the DNA sequence of the apoB (0.97kb) probe, and Figure 2 is the DNA sequence of the apoE probe, and Figure 3 is the DNA sequence of the apoE probe. Figure 4a shows the DNA sequence of the two parts of the apoA probe. A sequence, and Figure 4 shows the apoAI/CII[/AIV gene complex. Show your body.

Kinushisato Nakuseise Plate 2 In the description below, the distance of the polymorph from the reference point and the length of the deletion are often Given in bp or kb. If the sequence is known 2, such a measurement is Although it can be very accurate.

where assessed by measuring fragment size on gel or other experimental means. If so, these measurements contain some degree of inaccuracy. This is in the technical field is well understood. Generally, for measurements >4kb, the range of inaccuracy is approximately for shorter lengths, the error is approximately ±10%. this As in, “a deletion of 300 bpll may be slightly larger or slightly smaller. It is also small, and the distance of 4 kb from the apoAI gene is simply It is an approximation.

A. Many 6 methods Predicting individuals suspected of having atherosclerosis.

or some of the polymorphisms associated with a specified genomic region.

Application of the method of the invention to obtaining a genetic fingerprint based on or all using standard methods of DNA extraction, purification, restriction enzyme digestion and size fractionation. ing. Depending on the method of hybridization using the probe and the molecular weight Methods for detecting successfully hybridized substrates that are lined up in the same manner are also within the skill of the art. Well known in the wild. A general approach to finding and detecting important polymorphs are as follows: Somatic cells of the individual to be examined 1 e.g. white blood cells, placental cells, cultured cells. Extract DNA from fibroblasts or, in the case of female individuals, cells from amniotic fluid. Ru. Separate the high molecular weight DNA fraction with 2 eg EcoRI.

Treat with restriction enzymes such as BamHI, MstI+XmnI, etc. High molecular After decomposing the amount of DNA, transfer the two decomposition products to polyacrylamide or agarose gel. The DNA fragments obtained by restriction enzyme digestion can be separated by electrophoresis. However, the position on the gel is determined using a nitrocellulose filter or probe hybridizer. by transfer to other suitable matrices to be used as ization carriers. Copy. D either before or after transfer to the nitrocellulose filter replica. NA fragments are treated with denaturing agents such as sodium hydroxide/salts. denatured single strand The electrophoretic pattern in which the DNA was transferred was labeled (usually by 32P). A single-stranded DNA fragment is used as a probe. Non-radioactive, such as fluorescent molecules Other labels can also be used.

Depending on the probe selected, fragments originating from specific regions on the genome are detected. Ru. For example, in the method of the present invention.

Apolipocumbac B (apoB) or apolipoprotein Cl0 (apoCU ) or a cDNA sequence derived from the apolipoprotein E (apoE) gene sequence. Use the column as a probe. Therefore, on the hybridized filter The only fragments that appear in are those that contain sequences complementary to the specified probe. Ru. That is, if it is not cut within the genome itself, the complementary apo B or apoCI [or cleaved by restriction enzyme digestion derived from the apoE fragment] It's not even something that was given to me. To put it another way.

By using a specific probe, a sequence similar to that corresponding to that probe can be obtained. Changes in the genome are detected.

´　The specific steps used in the general method described in the above section are Understood in the technical field. The procedure for extracting DNA from somatic cells is 2. For example, Kan , Y, W, et al., Proc Natl Acad 5ci (USA) (19 78) 75:5631-5635; Taylor, J.M., et al., Natu re (1984) 251:392-393; and Kan, Y.W., et al. Headed in N En J Med (1977) 297:1080-1084. It is possible. Furthermore, improved methods for rapidly extracting DNA include Law, D, G, et al., Gene (1984) 28:153-158. restriction enzyme Methods for size-based separation of treated DNA fragments are also described in the above references. . Restriction enzyme digestion is generally standard in the art and depends on the production of specific restriction enzymes. Specified by manufacturer. carried out under buffer, ionic strength and temperature conditions. Ru.

Transfer to nitrocellulose or other support and plate with non-specific DNA. Rehybridization and then hybridization with the labeled probe. It is also standard procedure to read the book rather than reading it2.For example, read the above references and and 5Othern, E. (supra). fingerprinted Of course, the parts of the genome that are being researched using the results are It depends on the nature of the web. Probes useful in the present invention include apoB, selected from apoCU and apoE genes.

The production of the probe in B and H If the selected probe has not been resolved from the polymorph by restriction digestion, Complementary to a DNA sequence close enough to the polymorph on the system that cross-over If it is unlikely to be isolated from the polymorph, the resulting fragment pattern Used to investigate polymorphism. Therefore, the region of complementarity of the probe and the polymorphism The limit on the satisfactory distance between is arbitrary.

Generally, hybridization to DNA sequences within 10 kb upstream or downstream of the polymorphism probes that are sized give satisfactory results. Sometimes restriction enzymes The solution pattern is determined by hybridization of the terminal probe onto a fragment unrelated to the polymorphism. tion sites may be placed. The closer the probe is to the polymorph.

The range of restriction enzymes that can be used is increased. Therefore, as used herein, A probe that is “substantially equivalent” to a given probe is one that uses the same restriction enzyme. The same fragment in the DNA digest obtained from one individual for a specific polymorph It is a probe that gives length. For example, Hybridize; slightly shorter probe or slightly longer probe can be used without changing the results; Hybridizing probes may also be substituted.

Atherosclerosis is disclosed in U.S. Patent Application No. 724.192, supra. As shown, apoAI/CI [ In addition to the I gene, lipids are associated with defects in cholesterol metabolism. Genes encoding other proteins involved in metabolism are also useful. Many here Genetic regions of interest regarding shapes are apoB, apoCU, and apoE. and the gene region of the apoA IV gene.

Apolipoprotein B is present in very low-density vertebrate (VLDL) and Cairo protein B. It is the main protein component of microns. Apolipoprotein B is low-density lipoprotein It is the only protein in the proteinaceous protein (LDL), chylomicron and VLDL. is essential for the association and secretion of Apolipoprotein B also acts as a protein for seeding cells. 8 cad Sci, which removes LDL from the circulation by receptor-mediated uptake. USA) (1985) 82:4597-4601. ) four major blood groups of apoB Five serous species have been described (Kane J, P, et al., Proc Natl Ac ad 5ci (USA) (1980) 77:2465-2469). But long two of these are linked to the other by protease activity found in plasma. It seems to be coming from one of the. (Cardin, A, D, et al.

J Biol Chem (1984) 259:8522-8528; Ya mamoto, M. et al., J. BioChem' (1985) 260: 8509-8513. ) One of the early types, apoB-48.

Synthesized in the small intestine and is a component of chylomicron; The other early form, apoB-100, which is attacked directly, binds to the LDL receptor. It is a protein ligand on LDL.

Leads to uptake and metabolism of LDL by the liver. (Deeb, S, S.

et al., Proc Natl Acad 5ci (USA) (1985) 82: 4983-49'86), and nika<, encoded by the apoB gene Apolipoproteins are essential for cholesterol and fat metabolism. It is. Elevated plasma levels of apoB-100 in individuals with early coronary artery disease (Brunzel, J.D., et al. rosis (1984) 4nia 9-83), and familial hyperlipidemia and Individuals with high cholesterol also appear to have elevated levels of this protein. (Brunzel, J. D., et al., supra; Brunzel, J.

D, et al. (1983) 24:147-155), but other It is shown by people. At least in part due to such interests.

cDNA clones for apoB or parts thereof have been prepared (Deeb , S. S. et al., and Lu5is, A. J. et al., both supra; Prott Er.

Another apolipoprotein, apoc, also plays an important role in related metabolic pathways. I am accomplishing it. It is caused by an abundance of circulating triglycerides. It is a 79 amino acid peptide related to lomicron and VLDL (Mykl ebost, O, et al.

J Biol Chem (1984) 7:4401-4404), this It is known to activate acupuncture lipase (LaRosa, J. , C, et al.

Biochem Biohs Commun (1970) 41:57-62 ; Breckenridge, W.

C0 et al., New En J Med (1978) 298=1265-12 72), apocII deficiency.

An association with hypertriglyceridosis and other glycemic abnormalities has been reported.

(Breckenrichlge, W.C., et al., supra; Cox, D.

et al., New En J Med (1978) 299:1421-1424 ; Yamamura, T. et al.

8 Theroscleosis (1979) 34:53-65; Mill er, N, E, et al. Eur JClin Invest (1981) Dan: 69 -76) Prepare a cDNA clone encoding this gene. t, O, supra) and was digested with TaqI to extract apoclll-related probes. Polymorphisms in this gene that can be detected by examining Humphr ies, F, E, et al.

Reported by Mol Biol Med (1983): 463-471 There is. However, as reported by Humphries, this There is an association between TaqI polymorphisms in 9 individuals and factors that predispose individuals to hyperlipidemia. These results are confirmed by our study.

A third related gene sequence encodes human apolipoprotein E. be. ApoE is also a component of chylomicrons and chylomicron debris; This 299 amino acid protein found in VLDL and HDL The sequence is known and cDNA clones have been prepared (McLean J , W. et al., J BiolChem (1984) 25:6498-6504 ), apoE mediates the uptake of polymorphs through specific receptors. Mah. ley, R.W., K11n Wochenschr (1983) 61: 225-232), and appears to bind to the LDL receptor (Innera ritytT, L., Biochemistr (1978) 17:1440 -1447), various forms of apoE protein have been found, and apo An abnormal form of B2.

appears to be associated with genetically determined hyperlipoproteinosis (Mahley , R, et al., Adv Intern Med (1983) 29:385-41 1).

A fourth protein whose coding sequence serves as a substrate for useful probes is human Apolipoprotein AIV. The genetic map shows apoAIV and ap oAI/CII [The gene region is actually.

It shows that they are closely related (Schamaun, O. et al., Hum Genet (1984) 68: 181-184; s, S, ni, Proc NatlAcad Sci USA (1985) 82:6374-6378; Elshourbagy, N.A., et al.

J Biol Chem (1986) 261:1998-2002), and Numerous structural and compositional similarities exist between apoAI and apoAIV. It is attracting attention that it is located between genes. apoAIV is a 376 amino acid protein. The complete amino acid sequence is known (Elshourbagy).

et al. (supra)). apoA IV is similar to apoAI/CII [, Mediate various metabolic steps involved in the metabolism of terol and other lipids It is considered.

This is because the apoAI/CIII gene is similar to the apoAIV gene.

The polymorphism detected using the apoAIV probe is actually apoAI10I Present in the II complex. It is important to note that changes in the DNA sequence can be detected. It is possible. Therefore, the polymorphism detected using this probe is “apoAI/ These proteins are called polymorphisms of the CI [[/AIV gene complex'. The relative positions and reading directions of the areas to be read are shown in FIG.

In summary, apoB, apoCII, apoE and ap6AIV Each of the above gene sequences encoding determines the metabolism of cholesterol and other lipids. It appears to be closely related to the metabolic steps leading to atherosclerosis. suitable for predicting disease. Therefore, it is possible to hybridize to regions of these genes. Two probes designed for this purpose can be used in the method of the present invention.

Probes and restrictions suitable for detecting the 5' insertion polymorphism of the insulin gene A description of the enzyme can be found in Beil, C.1, et al., Nature (1980), 28. 4:26-32; Proc Natl Acad Sci LISA (19 81) 78:5759-5763; Diabetes (1984) 33: Found at 176-183.

The probe was nick transcribed using α(”P)dCTP and α(”P)dGTP. Label with rayon and fragment the probe. In this way, genetic cDND that is complementary only to the xon region and extends beyond the intron region NA probes can be used in the methods of the invention.

C, kit Suitable reagents for detecting suitable polymorphs by applying the method of the present invention include the necessary substances. organized into a convenient kit that provides:

placed in a suitable container, and sometimes in a container or carrier useful in carrying out the analysis. ing. Essential components of the analysis include restriction enzymes associated with the polymorph and appropriate probes. Includes Additionally, hybridization, prehybridization .

Packages containing concentrated reagents used for DNA extraction, etc.

Can be included if necessary. However, in particular, labeled probes, Alternatively, there are two suitable reagents for forming conveniently labeled probes. Useful to facilitate implementation of the law. Instructions for carrying out the method are also included in the kit. It is rare. These instructions include two operations that constitute an analysis - one for the genome involved; Fragment detection and atherosclerosis prediction. How to point out correlations in results is listed.

D, Design a DNA sequence that is more frequently encountered than Yani with atheroma Φ , “obtaining fragments that are more frequently encountered as ‘normal’ may be a counter-response to disease. First of all, it should be noted that there is no special meaning in obtaining seki. higher A higher frequency sequence or pattern is a “polymorph” or a lower frequency sequence or pattern. may be correlated with a disease instead of a pattern. These results are purely a matter of convenience. The terms "normal" and "polymorph" are used.

Polymorphisms in the apoB, apoCU, apoE and apoAIV regions teeth.

May be correlated with a tendency to exhibit signs of atherosclerosis. Such a correlation is and a control population by selecting a sample. Separate patients from controls Ru. One valid criterion is the presence of atherosclerotic plaques detected by angiography. The question is, please. Thus, a collection of samples can be prepared using this technique. There is a difference between those showing plaque and those without atherosclerosis. The DNA sample is then transferred to the patient. and control populations.

obtained from leukocytes or another convenient source, as described here.

A method for detecting related polymorphs is provided. Correlation is a useful statistical method It can also be obtained using the method. Especially for calculating the relative incidence of atherosclerosis. One convenient method is illustrated below. However, what other useful A related method can also be used.

Insertions between 1600 and 2200 bp (“U”) with a frequency of 0.32 in the population shape correlates with atherosclerosis (Owenbach, D.B., et al.

Lancet (1982) 1291-1293; Mandrup-Poul sen, T., Lancet (1984) 250-252) or correlation No (Jowett, N. 1. et al., Lancet (1984) 348) One document is unclear regarding this. The inventors found that “°U” inserts pose an increased risk. and shorter 600-1600 bp “M” insertions. (frequency = 0.04) is moderately correlated with decreased risk of atherosclerosis. I am discovering that.

E, 1 hill ■ The following examples relate to two exemplary probes and include DNA extraction and probe hives. Specified regarding the exact conditions such as redization. These factors are examples It can be seen that it is a proof, but not a restriction. Regarding detection of specific polymorphs The essential feature of the invention lies in the selection of enzymes and probes.

For example, implementation of Pvu II/B for prediction of atherosclerosis In embodiments, Pv+, uII degradation of genomic DNA may be used.

and the genome close to this polymorphic site (i.e., within <5.5 kb in this case) Sequences (in non-repetitive regions) that are complementary to the sequence can be probed. On the other hand, this polymorph Other restriction enzymes can be linked to probes that hybridize particularly close to the target. You may also use raw materials.

Other specific restriction enzymes may be used in fingerprint polymorphism methods. seed Substantially one equivalent probe for each can be designed in relation to this region. specific Restriction enzymes and DNA probes are arbitrarily selected. However, the focus here is As the probe moves close to the polymorph site, as one skilled in the art will know, what should be done is As a result, the efficacy of the probe is enhanced. Otherwise, polymorphs and pro There may be additional cleavage points between the fibers and the fibers. This probe site is also cross-presented. It becomes separated from the polymorphic site during replication.

E.1. Five ways to organize White blood cells were collected from various human organs. Obtained from freshly drawn blood.

High molecular weight genomic DNA is described by Law, D-J, + et al., Gene (198 4) 28: Isolated according to the method of 153-158.

Divide the high molecular weight DNA into two parts and add each part to the supplier (New England Bi). olabs and Bethesda Re5earch Laborato It was completely digested with two different restriction enzymes under the conditions recommended by M. Ries. child The decomposition product was analyzed on a horizontal agarose gel using 30mM Na0HzPO, 36m). 1 Electrophoresis was performed in Tris, 1mM EDTA, pH 7.7. After electrophoresis .

DNA fragments were placed in situ in 0.5M NaOH/1.5M NaCl. Denature for 2 x 10 minutes and neutralize with H ammonium acetate (pH 7,2) for 2 x 10 minutes. - At night, nitrocellulose paper (Schleicher and Schuell ). This filter is 2XSSC (1x ssc is 0.1 Wash with 5M NaCl, 0.015M sodium heptate, pH 7.4) , baked in vacuum at 80°C for 2 hours, then 5XSSPE (1X5SP E is 10mM sodium phosphate, pn 7.4.0.18M NaCl or and 1 mM EDTA) and 5x Denhardt's solution (1x Denhardt's solution Ficoll, polyvinylpyrrolidone and bovine serum albumin were each ．． 2 mg/d), 40% (νol/vol) formamide, and sheared 0.3ml/c containing 250μg/mp of denatured salmon sperm DNA Prehybridize for 5 hours in a plastic bag using a solution of rfl. Ta. In the same bag, add 0.1ml/c+fl of 5X5SPE, IX Denhardt's solution.

40% (vol/vol) formamide, 10% dextran sulfate, and shear Hybridize overnight with 100 μg/rtdl of denatured salmon sperm DNA. , per bag (containing 1 or 2 filters), as described below. The mixture was mixed with the appropriate probe labeled with 32p of 1100n. prehybrid Dization and hybridization were performed at 42°C.

Filters were washed 6 times then 2×SSC at room temperature and 2×SSC at 65°C. and 1× Denhart i solution. 32p labeled probe DNII self hyphenated into JIJ & Yo.

XAR-5 film (Kodak) and C, r o n e x intensifying screen Lean (Dupont) at -70″C for 18 hours to 2 days. Visualization was performed by autoradiography.

Probes related to 4 specific genes are used as shown below = 3 apoB probe; apoB (0,97), apoB (2kb) and apoB (3kb); apocI [probe; apoE probe, and The two parts of the apoATV probe were mixed together.

One of the apoB probes, apoB (0.97kb), has a 30kd tag. Contains a 900bp protein-encoding sequence and a 7obp 5” untranslated region. , a 970 bp EcoRI/EcoRI insert fragment. While I'm at it, The probes are Deeb, S, S, and Lu5is, A, J, (mentioned above). does not overlap with any of the known apoB clones described by. probe and Isolation of the EcoRI fragment used as a probe was carried out by Protter.

A, A, et al.'s Proc Natl Acad Sci (LISAC (currently in print) ) is described. The complete NA sequence of this insert is shown in the library shown in Figure 1. Lee (inserts with an average size of 1 kb were ligated into the EcoR1 site of λgtlO). Huynh, T., et al., DNA cloning method: A. Pr. actual Approach (1984) + Grover, D, + Prepared by the method of ED, +IRL Press, Oxford. Screeni For testing, 9X105 plaques grown in C600 (HFL) cells were .

Manipulated as described in J. J., et al., DNA 3:309 (1984). .

This filter has 3 XNaC1/Cit (I XNaC1/Cit is 1 50mM NaC1/15mM sodium citrate, pH 7.0), 0.1 In %SDS.

Prewashed at 55°C for 2 hours, then 6X NaCl/Ci t.

200 Mg 7 ml denatured salmon sperm DNA, 5X Denhardt's solution.

Prehybridize in 0.05% sodium pyrophosphate for 1 hour at 50°C. let

The first eight amino acids of the previously determined apoB-26 sequence (85p-Glu -Pro-Pro-Gin-Ser-Pro-Trp), considering codon redundancy. We probed 192 concatenations of 23 base oligonucleotides encoding It was used as a buffer. This probe is T4 polynucleotide kinase (PLB iochemicals) and the 5′ end labeled with r-(”P)-8TP This was added to the filter and incubated at 50°C for 14 hours. child The filter was prepared with 5XNaCl/Cit, 0.1% SDS, 0.0 at room temperature. 5% sodium pyrophosphate twice for 15 min and once for 20 min at 50 °C. Washed, dried and autoradiographed with intensifying screen. Ta.

One positive plaque, designated LB25-1, was purified. This cDNA fragment Bidirectional subcloning was performed into M13/mp8 for nucleotide sequencing. For amplification For this purpose, the EcoRI insert was subcloned into pBR322 and pB2 I got 5-1. Therefore, pB25-1 contains some 5” untranslated region, null sequence, and the first 266 amino acids of the mature protein (i.e., apoB (0,97kb) probe).

The remaining two apoB probes include additional apoB-encoding sequences. A linearized and denatured insert of pB25-1 was used as the first probe. I got it. Inserted into λgtlo.

A 2×105 human adult small intestine cDNA library was screened with this insert. -ning. Then, a cDNA named IB7 (this is approximately 1.3k b, and the 3' end of clone pLB25 was extended by approximately 800 bp) was gotten. The isolated and denatured IB7 insert was transferred to pBR322 for amplification. subcloned to.

I made plB7. This purified pIB7 insert was denatured.

It was then used to screen a small intestine library. A certain sun named 110 The sex cDNA fragment contains a 3 kb insert, of which approximately 2.5 kb b, the 3” end of IB7 was extended.

This cDNA insert was subcloned into the EcoR1 site of pBR322. , pBlo was inserted. This insert becomes the second apoB probe, which This was named apoB (3kb).

The linearized and denatured pB10 insert was transformed into another one designated IB-(2)-1. It was used as a probe to further obtain cDNA fragments.

IB-(2)-1 contains an insert of approximately 2 kb, and approximately 1 kb is the IB-10 sequence. Stretch the column in the 3' direction. This EcoRI cDNA insert also pB(2)1 was created by subcloning into the ECoRl site of 322. .

This insert represents the third apoB probe and is similar to apoB (2kb). be named.

This apoCII probe is part of the Myklebost cDNA (mentioned above). This is a 1.03 kb EcoRI/EcoRI insert fragment corresponding to 1. This break The fragment was made with λgt-10 (with an EcoRI linker added and the EcoR1 part of the phage The human fetal liver cDNA constructed by inserting the A 51-base synthetic oligonucleotide containing the coding sequence of leotide 73-122 was was used for screening.

soo,ooo screened, got 2 positive clones, sequenced one Established. It goes through 38 bases of the 3” untranslated region.

Spanning nucleotides 10-432, including amino acid -14 of the signal sequence . The complete sequence of this probe (excluding linker) is shown in FIG.

This apoE probe is a lkb EcoRI/EcoRI fragment; The fragment spans the entire sequence encoding the mature protein.

and corresponds to the sequence published by McLean et al. (supra). this is , (by adding an EcoRI linker and inserting it into the EcoRI site of the phage) obtained from human fetal liver cDNA library prepared with λgt-10 containing the coding sequence from nucleotides 469 to 514 of the published DNA sequence. A synthetic 46 base oligonucleotide was screened. 450,000 fu One of the 10 positive clones obtained from the AGE was sequenced. the The sequence starts from 14 amino acids of the signal sequence.

3゛Nucleons ranging from -14 to +1020, including up to 120 bases of the untranslated region The protein is encoded by the tide. Complete sequence of this probe (excluding linker) is shown in Figure 3.

This apoAIV probe together encodes the majority of the apoATV protein. It is a mixture of two DNA parts. These “apoAIV-5゛°” pro The complete sequence of the probe and “apoAIV-3” probe is shown in Figure 4a.a The poAIV-5'' probe extends beyond the sequence encoding the N-terminus of the protein. It has spread with additional sequences such as those shown here. This is apoAIV- There is a slight overlap with the 5' end of the 3' probe. This apoA The IV-3' probe starts from the codon for amino acid 187 to amino acid 358. (which is 18 amino acids short from the C-terminus of the protein) It is spreading. These probes are used as a mixture and together 2 It is called "poAIV".

This apoAIV profile was first developed by Protter, A. 984) 3: Starting from 1.9 genome clone to λ described in 449-456 and prepared. Decompose 1.9 to λ with EcoRI and extract the apoAIV gene. A 1.2 kb fragment containing a portion was isolated by electrophoresis. The identification of this 1.2 kb fragment was , was confirmed to correspond to the sequence encoding the apoΔ■ protein. child A 1.2 kb fragment of Contains recombinants.

Human small intestine cDNA library in 2gt-10 stored in CsC1 (human jejunum) was used for screening. This 661bp apoAT V-5' profile, and the 513 bp apoAIV-3' profile.

The lobes hybridized to labeled 1,2 kb fragments.

Each of the probes mentioned above is available from the BRL Nick Translation Kit (Bethe About 1μg using sda Re5search Laboratories) 2 to 5 Nick translayer to achieve a constant activity of XX108 cp. Labeling was performed using the cation method. This labeling combines unlabeled dATP and In the presence of dTTP, ("P) dGTP and ("P) dCTP (800 Ci/mmole; Amersham Corporation) was used. It was carried out under the recommended conditions. This probe.

Immediately before hybridization, incubate for 5 minutes in a boiling water bath. and denatured by rapid cooling in ice water.

E, 2.　Multiliter　and measurement to poB, to poCU, to eight poE or apoArV brochures, to those in chapter A. The method was used to determine the number of polymorphisms found in the appropriate genomic regions. these The polymorphs are summarized in Table 1.

(EcoRV, t(decomposition by pal and leaf al, followed by apo The usual pattern of detection by B (3kb) includes fragment diversity. . The results shown in this table distinguish the standard parameters from the individual polymorphs. Only shown. ) This polymorph has been shown to correlate with the risk of atherosclerosis. tested. to determine their correlation.

Control and patient groups were used to determine atherosclerotic plaque formation (angiographically determined). criteria for positive or negative results for With this measurement method, plaque People who show this are classified as “Patients” regardless of whether they have developed heart disease or not. It was. If the result of this test was negative, it was considered a “control”.

None of these people had ever had a heart attack.

Interpreting this result, standard λ-fit analysis can be used to determine the two critical levels. I was able to stay. This significant level represents the likelihood that the correlation is due to chance alone. vinegar. Therefore, the results obtained may be limited by the use of many subjects or by many independent trials. If you do, you will not be prevented. For example, a critical level below 0.05 There is a greater than 95% chance that the observed result is true. i.e. It means that the hypothesis tested is not a futile hypothesis. Therefore, additional or Similar results would be obtained if more subjects were tested. 0.10 The important level is that if more or different samples are tested, This means that 1 in 10 times the results could be different.

(Margin below) Engraving (no changes to the content) Engraving (no changes to the content) This conclusion suggests that people with polymorphisms are more likely to be sick than people without polymorphisms. interpreted in terms of relative risks. These statistically calculated ´°possible "Sex ratio" is by Wolf.

B., according to Ann Hum Genet (1955) 19: 251 Calculated. Apply the relative incidence in the analysis as follows.

PP　X　CN PN　X　CP It was calculated as

here.

pp is the number of patients with the polymorphism; PN is the number of patients without polymorphism; cp is the number of controls with the polymorphism; CN is the number of pairs without polymorphism.

If the value calculated by this ratio is greater than 1.

People with polymorphisms have a high risk of contracting the disease, and if it is less than 1, they are less susceptible to the disease. This indicates that the data is protected.

Applying this analytical method, the reported TaqI/CII polymorphism is.

There seems to be no correlation at all. Ban1/CII polymorphism may exert protective effects. It is clear that Regarding the TaqI/C1l polymorphism.

28 of 41 patients, or 68%, exhibited polymorphism; 12 of 18 controls 67% of people showed polymorphism. From this, the calculated relative occurrence The rate is 1.07, which roughly matches the risk for people without the polymorph. On the other hand, BanI/ CI 3 out of 5 controls, or 60%, showed this abnormality. Therefore, the calculated relative incidence value of 0.8 may indicate a slight protective effect. Recognize. Although the level of importance (0,6) is unfavorable, this proportion There is still a fair chance that the trial will remain in place.

Similarly, as reported by others and The shape (mentioned above) was evaluated. This “U” insert has a relative incidence of 3.0 (high It was determined that there was a clear and dangerous correlation. On the other hand, “M” The inserts were considered adequately protected with a relative incidence of 0.6.

Box notification 2” record pressure point Some of the approximately 10 million polymorphisms that exist in the human genome are unique to each individual. It can be seen that this is a useful index for predicting the risk of romanic arteriosclerosis. these Polymorphisms are fragments obtained by digesting each subject's genomic DNA with specific restriction enzymes. It can be detected by detecting the size of the piece and the specific DNA sequence described here. Ru. This means of early diagnosis of people at risk of atherosclerosis Utility is an early measure of treatment to avoid fatal symptoms of the disease. be.

Procedural amendment (formality) June 13, 1986

Claims (37)

[Claims] 1. A method for predicting the tendency of progression of atherosclerosis in an individual human, the method comprising: apoB, apoCII, apoE, and apoAI/CIII/AIV. A method comprising detecting the presence or absence of a polymorphism in a gene selected from the group consisting of: 2. The polymorphs are PvuII/B, StuI/B, EcoRVa/B; EcoR Vb/B, EcoRVc/B, HpaI/B, DraI/B, BamHI/CI I, BanI/CII, BgII/CII (15,8), NcoI/CII (1 7,8), HpaI/E, XbaI-a/AIV, XbaI-b/AIV, Xb aI-c/AIV, XbaI-d/AIV, TaqI/AIV, DraI/AI V, and the NcoI/AIV polymorph. The method described in Scope No. 1. 3. A method for predicting the tendency of progression of atherosclerosis in an individual human, the method comprising: The method is useful for diagnosis of human genomic DNA degraded by restriction endonucleases. detecting the presence or absence of a DNA fragment of two lengths, the fragment being apoB probe or a substantial equivalent thereof, apoCII probe or a substantial equivalent thereof, a poE probe or substantial equivalent thereof, or apoAIV probe or A method of hybridizing to a substantial equivalent thereof. 4. The restriction endonuclease is PvuII and the probe is apoB. (0.97 kb) or a substantial equivalent thereof, and said DNA fragment is 5 ．． 4. The method according to claim 3, wherein the data size is 5 kb. 5. The restriction endonuclease is StuI, and the probe is apoB ( 2kb) or a substantial equivalent thereof, and said DNA fragment is 5.2kb) The method according to claim 3. 6. The restriction endonuclease is EcoRV and the probe is apoB. (3 kb) or a substantial equivalent thereof, and said DNA fragment is 4.8 kb. The method according to claim 3, which is b. 7. The restriction endonuclease is EcoRV and the probe is apoB. (3 kb) or a substantial equivalent thereof, and the DNA fragment is 11.0 4. The method of claim 3, wherein the method is: kb and 7.0 kb. 8. The restriction endonuclease is EcoRV and the probe is apoB. (3 kb) or a substantial equivalent thereof, and said DNA fragment is 3.6 kb. 4. The method of claim 3, wherein the method is 2.7 kb. 9. The restriction endonuclease is HpaI, and the probe is apoB ( 3 kb) or a substantial equivalent thereof, and said DNA fragment is 8.3 kb) and 6.2 kb. 10. The restriction endonuclease is DraI and the probe is apoB. (3 kb) or a substantial equivalent thereof, and said DNA fragment is 2.3 kb. 4. The method according to claim 3, wherein the data size is between 2.6 kb and 2.6 kb. 11. The restriction endonuclease is BamHI and the probe is apo CII or a substantial equivalent thereof, and the DNA fragment is 5.9 kb. A method according to certain claim 3. 12. The restriction endonuclease is BanI and the probe is apoC. II or a substantial equivalent thereof, and the DNA fragment is 1.2 kb. The method according to claim 3. 13. The restriction endonuclease is BgII and the probe is apoC. II or a substantial equivalent thereof, and the DNA fragment is 3.8 kb. The method according to claim 3. 14. The restriction endonuclease is NcoI and the probe is apoC II or a substantial equivalent thereof, and the DNA fragment is 15.8 kb. A method according to certain claim 3. 15. The restriction endonuclease is NcoI and the probe is apoC II or a substantial equivalent thereof, and the DNA fragment is 17.8 kb. A method according to certain claim 3. 16. The restriction endonuclease is HpaI and the probe is apoE. or a substantial equivalent thereof, and the claimed DNA fragment is 5 kb. The method described in Scope No. 3. 17. The restriction endonuclease is XbaI and the probe is aPoA IV or a substantial equivalent thereof, and said DNA fragment is 10 kb. The method according to claim 3. 18. The restriction endonuclease is XbaI and the probe is apoA IV or a substantial equivalent thereof, and said DNA fragment is 2.0 kb. The method according to claim 3. 19. The restriction endonuclease is XbaI and the probe is apoA IV or a substantial equivalent thereof, and said DNA fragment is 20 kb. The method according to claim 3. 20. The restriction endonuclease is XbaI and the probe is apoA IV or a substantial equivalent thereof, and said DNA fragment is 4.8 kb. The method according to claim 3. 21. The restriction endonuclease is TaqI and the probe is apoI. V or a substantial equivalent thereof, and the DNA fragment is 3.6 kb. The method according to claim 3. 22. The restriction endonuclease is DraI, and the probe is apoA. IV or a substantial equivalent thereof, and said DNA fragment is 7.4 kb. The method according to claim 3. 23. The restriction endonuclease is NcoI and the probe is apoA IV or a substantial equivalent thereof, and said DNA fragment is 9.5 kb. The method according to claim 3. 24. Determining susceptibility to atherosclerosis by analyzing human genomic DNA A kit for determining DNA probes, restriction endonucleases, and kit containing instructions for performing the analysis and interpreting the results. 25. The DNA probe is an apoB (0.97 kb) probe or its substance. and the restriction endonuclease is PvuII. The kit according to item 24. 26. The DM probe is an apoB (2kb) probe or a substantial equivalent thereof. and the restriction endonuclease is StuI. The kit described in Section 4. 27. The DNA probe is an apoB (3kb) probe or substantially the same. and the restriction endonucleases are EcoRV, HpaI, and 25. The kit of claim 24, wherein the kit is selected from the group consisting of DraI and DraI. 28. the DNA probe is an apoCII probe or a substantial equivalent thereof; Yes, and the restriction endonuclease is BamHI, BanI, BgII or and NcoI. 29. the DNA probe is an apoE probe or a substantial equivalent thereof; , and the restriction endonuclease is HpaI. Kit included. 30. the DNA probe is an apoAIV probe or a substantial equivalent thereof; and the restriction endonucleases include XbaI, TaqI, DraI and 25. The kit of claim 24, wherein the kit is selected from the group consisting of: 31. A method for predicting the tendency of progression of atherosclerosis in an individual human, the method comprising: Detecting the presence or absence of a 5' insertion polymorphism of the insulin gene, Method. 32. Claim 31 wherein said polymorph is selected from M polymorph and U polymorph. The method described in. 33. 33. The method of claim 32, wherein the polymorph is a U polymorph. 34. A kit for analyzing human genomic DNA, which includes M and U polymorphisms. A means for detecting a 5' insertion polymorphism of an insulin gene selected from including instructions for conducting the analysis and explaining the results; kit. 35. 1. A method for determining the genetic identity of an individual human, comprising: apoB, apoCII, apoE, and apoAI/CIII/AIV. detecting the presence or absence of one or more polymorphisms in a gene selected from the group includes, Method. 36. The polymorphs are PvuII/B, StuI/B, EcoRVa/B, Eco RVb/B, EcoRVc/B, HpaI/B, DraI/B, BamHI/C II, BanI/CII, BgII/CII (15,8), NcoI/CII ( 17,8), HpaI/E, XbaI-a/AIV, XbaI-b/AIV, X baI-c/AIV, XbaI-d/AIV, TaqI/AIV, DraI/A Claim 35 selected from the group consisting of IV and NcoI/AIV polymorphs. The method described in. 37. A kit for determining the genetic identity of an individual human, comprising: apoB, apoB; At least one species selected from the group consisting of CII, apoE and apoAIV a probe, and at least one restriction enzyme; including instructions for the analysis and interpretation of the results; The restriction enzymes include EcoRV, HpaI, and Dr if apoB is included. selected from the group consisting of aI; if apoCII is included, BamHI, Ba selected from the group consisting of nI, BgII and NcoI; and apoAIV is If included, from the group consisting of XbaI, TaqI, DraI, and NcoI selected, kit.