WO1989012697A1

WO1989012697A1 - Method for detecting abnormal genes

Info

Publication number: WO1989012697A1
Application number: PCT/US1989/002731
Authority: WO
Inventors: Akitada Ichinose
Original assignee: The Board Of Regents Of The University Of Washingt
Priority date: 1988-06-22
Filing date: 1989-06-21
Publication date: 1989-12-28

Abstract

Methods for detecting the presence of selected mutations, such as the Thr-601 mutation and the Phe-355 mutation, in the plasminogen of a patient are disclosed. The methods include exposing amplified genomic DNA to a restriction endonuclease capable of differentially cleaving mutant and wild-type plasminogen DNA sequences, and analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation. Diagnostic kits for the rapid detection of the selected mutation are also disclosed.

Description

METHOD FOR DETECTING ABNORMAL GENES

Technical Field

The present invention is related generally to the detection of abnormal genes. More specifically, the invention provides methods for detecting the presence of abnormal plasminogen genes, such as a gene encoding Thr-601 plasminogen or a gene encoding Phe-355 plasminogen.

Background of the Invention

In order to understand the mechanisms and genetics of human diseases, it is important to identify DNA and protein markers that indicate the presence of genetic defects in populations and families. For example, deficiencies in protein C, protein S, antithrombin III, heparin co-factor II, tissue-type plasminogen activator and plasminogen have been identified as the cause or at least part of the cause of a predisposition for thrombosis in some patients with hereditary thrombophilia (for review, see Bauer and Rosenberg, Blood 70:343-350, 1987, and Mannucci and Tripodi, Thromb. Haemostas. 57: 247-251, 1987). Plasminogen is a single-chain proenzyme that is converted to an active two-chain form (consisting of an A and a B chain connected by two disulfide bonds), called plasmin, by activators such as tissue-type plasminogen activator, urokinase, and streptokinase. Plasmin digests fibrin clots to form soluble fibrin degradation products. In addition, plasmin is thought to play an important role in various biological reactions, such as inflammation, tissue development and remodeling, processing other molecules, etc. The primary structure of plasminogen (790 amino acid residues) was established by Sottrup-Jensen et al. (Prog. Chem. Fibrinol. Thrombol. 3:191-209, 1978). This amino acid sequence has been confirmed by cDNA sequencing (Malinowski et al., Biochemistry 23: 4243-4250, 1984 and Forsgren et al., FEBS Lett. 213:254-260, 1987), which indicated the presence of an additional Ile residue at position 65. Accordingly, plasminogen contains 791 amino acids (See Figure 1). The A chain of the molecule consists of the activation peptide (77 amino acid residues) and five disulfide bond-folded structures called "kringles" (about 90 residues each). The B chain contains the activation site (between Arg-561 and Val-562), the active site His-603 residue region, the active site Asp-646 residue region, the region which is linked to the heavy chain by a disulfide bond, the active site Ser-741 residue region, and the C-terminus (amino acid numbers used herein refer to the sequence shown in Figure 1). The first kringle structure (K1) in the A chain of plasminogen is responsible for its binding to fibrin (Thorsen et al., Biochim. Biophys. Acta. 668: 377-387, 1981). The B chain of plasminogen carries all three active sites essential for catalytic function as a serine protease.

There are at least several genes in the human genome that are homologous to that of plasminogen, such as apolipoprotein(a) (McLean et al., Nature 330:132-137, 1987). Apolipoprotein(a) contains 37 copies of plasminogen kringle 4 and one copy of plasminogen kringle 5. It also contains a serine protease domain that is highly homologous with the B chain of plasminogen.

Several cases of a molecular abnormality of plasminogen in association with a complication of thrombosis have been reported (Aoki et al., J. Clin. Invest. 61: 1186- 1195, 1978; Kazama et al., Thromb. Res. 21:517-522, 1981; Wohl et al., Thromb. Haemostas. 48: 146-152, 1982; Soria et al., Thromb. Res. 32:229-238, 1982 and Scharrar et al., Thromb. Hemostas. 55: 396-401, 1986). These abnormalities have been found most frequently in Japan, but have also been reported in other populations. By an analysis of the plasminogen molecules from these patients, it has been shown that an amino acid substitution of Thr for Ala-601 in the B chain results in the generation of an inactive plasmin molecule (Sakata and Aoki, J. Biol. Chem. 255:5442- 5447, 1980; Miyata et al., Proc. Natl. Acad. Sci. USA 79:6132-6136, 1982; Miyata et al., J. Biochem. 96:227-287, 1984). However, the nature of the underlying abnormality at the DNA level has not heretofore been determined, and other plasminogen disorders have not been characterized.

Since plasminogen is the key enzyme in the fibrinolytic system, responsible for removing fibrin clots from circulation, individuals with abnormal plasminogen or a plasminogen deficiency develop thrombosis. Given the gene frequency of approximately 0.02 among Japanese, the expected number of homozygotes with the Thr-601 plasminogen variant is calculated to be about 50,000 in Japan (population of approximately 125 million). A few homozygotes have been found; however, the homozygous condition is expected to be lethal in most cases. In heterozygotes, the reduced plasminogen activity in plasma seems to be insufficient to prevent thrombosis, which may develop after trauma and is manifested as deep vein thrombosis, thrombophlebitis or pulmonary embolism.

Conventional biological assays for plasminogen activity and antigen concentration do not accurately identify the molecular basis of thrombosis, because plasminogen can be decreased in several acquired disease states, such as liver dysfunction and disseminated intravascular coagulation, or by thrombolytic therapy using plasminogen activators. Because proper therapy is dictated by the nature of the underlying condition, it is important to make a definitive diagnosis in the case of a genetic molecular abnormality. An additional complication in diagnosing plasminogen-related disorders arises from the high degree of homology between plasminogen and apolipoprotein(a). This homology makes it difficult to distinguish between DNA sequences encoding the two proteins. Previously described methods of identifying the presence of the Thr-601 plasminogen mutation are not well suited to clinical use. Miyata et al. (Proc. Natl. Acad. Sci. USA 79:6132-6136, 1982) used proteolytic digestion of plasminogen and amino acid sequence analysis of the resultant peptides to characterize the mutation. Aoki et al. (Biochemical Genetics 22: 871-881, 1984) utilized electrofocusing, zymography and immunofixation of neuraminidase-treated plasminogen. The entire procedure required four or more days to perform.

There is therefore a need in the art for improved methods of detecting the presence of mutations in the plasminogen gene. Such methods should be technically simple and rapid enough to permit clinical use. The present invention provides such methods for genetic diagnosis at the DNA level and has the additional advantage of not being influenced by the presence of other disease conditions.

Disclosure of the Invention

Briefly stated, the present invention is directed toward methods for detecting the presence of a mutation in the plasminogen gene of a patient. In one aspect of the present invention, the method comprises (a) amplifying a portion of genomic DNA from a patient, the portion including a predetermined exon comprising the site of a selected mutation and at least 14 base pairs of each of two intron sequences flanking the exon; (b) exposing the amplified DNA to a restriction endonuclease capable of differentially cleaving DNA having the selected mutation and wild-type plasminogen DNA, under conditions suitable for activity of the endonuclease; and (c) analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation. Within preferred embodiments, the selected mutation is the Phe-355 mutation or the Thr-601 mutation. The method may also include, prior to the step of amplifying, isolating genomic DNA from the patient.

Within a related aspect of the present invention, a method of detecting the presence of a mutation in the plasminogen gene of a patient is disclosed, wherein the method generally comprises (a) denaturing genomic DNA from the patient; (b) annealing the denatured genomic DNA to a pair of oligonucleotide primers, wherein the first primer is complementary to a first sequence of at least about fifteen consecutive nucleotides of a first intron on the coding strand of the genomic DNA, and wherein the second primer is complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, the introns flanking the exon comprising the site of a selected mutation; (c) extending the annealed primers to produce double-stranded DNA fragments, the fragments including the site of the selected mutation; (d) denaturing the double-stranded DNA fragments; (e) annealing the denatured DNA fragments to the pair of oligonucleotide primers and extending the annealed primers to produce selectively amplified DNA; (f) exposing the selectively amplified DNA to a restriction endonuclease capable of differentially cleaving DNA having the selected mutation and wild-type plasminogen DNA, under conditions suitable for activity of the endonuclease; and (g) analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation, wherein the selected mutation is the Phe-355 mutation or the Thr-601 mutation. Within a preferred embodiment, the primers are extended using Taq DNA polymerase.

Within another aspect of the present invention, a diagnostic kit for the rapid detection of the Thr-601 mutation in the plasminogen gene of a patient is disclosed. The kit includes, within suitable compartments: a pair of oligonucleotide primers, the first primer being complementary to a first sequence of at least about fifteen consecu tive nucleotides of an intron on the coding strand of genomic DNA from a patient, the second primer being complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, the introns flanking the exon coding for amino acid residue 601 of plasminogen; Taq DNA polymerase; control DNA; a restriction endonuclease capable of differentially cleaving Ala-601 plasminogen DNA and Thr-601 plasminogen DNA; and suitable buffers. Within yet another aspect of the present invention, a diagnostic kit for the rapid detection of the Phe-355 mutation in the plasminogen gene of a patient is provided. The kit comprises, contained within suitable compartments, (a) a pair of oligonucleotide primers, the first primer being complementary to a first sequence of at least about fifteen consecutive nucleotides of an intron on the coding strand of genomic DNA from a patient, the second primer being complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, the introns flanking the exon coding for amino acid 355 of plasminogen; (b) Taq DNA polymerase; (c) control DNA; (d) a restriction endonuclease capable of differentially cleaving Val-355 plasminogen DNA and Phe-355 plasminogen DNA; and (e) suitable buffers.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.

Brief Description of the Drawings

Figure 1 illustrates the cDNA sequence and amino acid sequence of plasminogen. The positions of certain restriction enzyme recognition sites are shown. Numbers in the left margin refer to nucleotide positions. Numbers above the sequence refer to amino acid positions.

Figure 2 illustrates portions of the sequence of the normal human plasminogen gene. N indicates an undeter mined nucleotide. Arrows indicate exon-intron boundaries. Exon sequences are underlined and labeled with Roman numerals. The 5' end of exon I and the 3' end of exon XIX were not determined; the 3' end of exon XIX is shown at the proposed polyadenylation signal. The partial gene sequence is presented in 10 sections, labeled a through j, showing: a, exon I and adjacent intron sequences; b, exons II and III and adjacent intron sequences; c, exon IV and adjacent intron sequences; d, exon V and adjacent intron sequences; e, exon VI and adjacent intron sequences; f, 10,000 base pairs comprising exons VII, VIII, IX and X; g, 10,000 base pairs comprising exons XI, XII and XIII; h, 10,000 base pairs comprising exons XIV, XV, XVI and XVII; i, intron sequence (4473 bp); and j, exons XVIII and XIX with adjacent intron sequences. Nucleotides in each of sections a through j are independently numbered as designated in the right margin, beginning with 1.

Figure 3 illustrates a portion of the genomic DNA sequence encoding plasminogen and the sequences of two sets of oligonucleotide primers (designated A39, 1A, 10A and 11A) used to selectively amplify a portion of the genomic DNA. The locations of certain restriction enzyme recognition sites are indicated.

Figure 4 shows the results of a Fnu 4HI digest of selectively amplified genomic DNAs from three unrelated patients with abnormal plasminogen and a normal individual. The molecular weight marker is a 123-bp ladder obtained from Bethesda Research Laboratories. AbI, II and III refer to samples from abnormal patients I, II and III, respectively.

Best Mode for Carrying Out the Invention

Prior to setting forth the invention, it may be useful to define certain terms used herein. Selectively amplifying: The process of increasing the copy number of a preselected DNA sequence or fragment relative to the copy number of other sequences or fragments in a sample.

Differentially cleaving: Cleaving a first sequence or set of sequences but not cleaving a second sequence or set of sequences. Restriction endonucleases differentially cleave DNA sequences due to their ability to specifically recognize short stretches of paired bases, frequently palindromic sequences of four to six base pairs. Cleavage may occur within the recognition sequence or at some specific distance away from the recognition sequence. Site of the selected mutation: The position in a gene at which a mutation is known to occur, regardless of whether that particular allele carries the mutant or wild- type sequence at the site.

As noted above, reduced plasminogen activity can lead to thrombotic episodes. Also as noted above, such a reduction in activity can result from a variety of causes, including genetic abnormalities. Practical methods of clinical screening for genetic abnormalities in plasminogen have heretofore been unavailable.

The present invention provides methods useful in diagnosing cases of thrombosis, in genetic screening and in prenatal diagnosis. The methods are simple, rapid, and do not require the use of radioactive isotopes, so are particularly useful in many clinical laboratories that lack in the special facilities necessary for handling radioisotopes.

The present invention is related, in part, to the elucidation of the human plasminogen gene sequence, portions of which are shown in Figures 2 and 3. Knowledge of this sequence has permitted the design of oligonucleotide primers that may be used to selectively amplify those portions of the gene encoding amino acid residue 601 or amino acid residue 355. In a similar manner, other abnormal plasminogen gene sequences may be analyzed, allowing those skilled in the art to selectively amplify exons comprising sites of other selected mutations. The methods of the present invention are applied to genomic DNA samples from a patient. In one embodiment, the genomic DNA is first isolated, using conventional procedures. A convenient source of isolated genomic DNA is leukocytes, which may be readily obtained from a small (e.g., 10 ml) blood sample. Other cell types may also be used. DNA may be isolated from leukocytes using the technique of Bell et al. (Proc. Natl. Acad. Sci. USA 78:5759-5763, 1981). Briefly, blood is collected in the presence of an anticoagulant, the cells are lysed, and the nuclei are collected. The nuclei are then treated with sodium dodecyl sulfate and proteinase K and the DNA is extracted from the mixture with phenol/chloroform/isoamyl alcohol. The DNA is then precipitated and resuspended in a suitable buffer, such as 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Alternatively, by using the method disclosed disclosed by Kogan et al. (New Eng. J. Med. 317: 985-990, 1987), the methods of the present invention may be applied directly to tissue samples, without the need to isolate the DNA. For example, chorionic villus samples can be screened directly by disrupting the tissue by vortexing in a solution of 0.1M NaOH, 2M NaCl, 0.5% SDS. The sample is then boiled for two minutes, centrifuged, and an aliquot is taken for amplification. This facilitates the application of these methods to prenatal diagnosis of the plasminogen abnormality.

Genomic DNA (either isolated or in the form of a suitable tissue sample) is then selectively amplified to provide a high copy number of the desired portion of the plasminogen gene (e.g., the portion encoding amino acid residue 601 or the portion encoding amino acid residue 355). Preferably, a sequence of approximately 200-1,000, most preferably about 300-400, base pairs is selectively amplified. In a preferred embodiment, the exon encoding amino acid 601 and portions of the intron sequences flanking this exon are selectively amplified. Similarly, the exon encoding amino acid 355 and portions of the flanking introns may be selectively amplified. A preferred method of amplification is the polymerase chain reaction, described by Mullis (U.S. Patent Nos. 4,683,202 and 4,683,195). Briefly, the genomic DNA is denatured to separate the coding and noncoding strands. Denaturation is preferably accomplished by heat treatment of the DNA, generally treatment at about 80°C-105°C for about one to ten minutes, although enzymatic denaturation may also be used. Most preferably, the DNA is heated at about 93°C for one minute. The denatured DNA is then combined with a molar excess of a pair of oligonucleotide primers under conditions which allow the DNA strands to anneal to the primers (e.g., 60°C for one to three minutes, preferably about two minutes). Preferably, each primer is used at a concentration of about 1μM for amplification of one microgram of genomic DNA. Suitable results may be obtained with 5μg of primer per pig of target DNA. One of the primers is complementary to a sequence on the coding strand and the second primer is complementary to a sequence on the noncoding strand, the sequences flanking the region to be amplified. "Sequences flanking the region to be amplified" include exon sequences, sequences of introns immediately adjacent to the exon to be amplified and sequences of other introns, so long as the amplified region includes the site of the selected mutation. The flanking sequences should be selected so as to provide an amplified portion of the gene within the size limits noted above. Although 100% complementarity is not required, a high degree of complementarity of primer and genomic DNA is advantageous in that it results in high specificity and efficiency of amplification. For use within the present invention, the primers must be sufficiently complementary to hybridize with their respective strands on the genomic DNA. The annealed primers are enzymatically extended using a DNA polymerase and all four deoxyribonucleotide triphosphates (dNTP's). Suitable polymerases include E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase, and T4 DNA polymerase. Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988) is particularly preferred. The reaction mixture is incubated under conditions of time and temperature suitable for the activity of the polymerase. When using the Taq DNA polymerase the mixture is incubated at about 70°C ± 10°C for approximately three minutes. As will be appreciated by one skilled in the art, the exact time and temperature will be determined by the melting point of the annealed DNA. The resulting extension products are separated from the original DNA strands, preferably by heat denaturation. The annealing, extension and separation steps are then repeated, preferably about 25 to 30 times, until the desired degree of amplification is obtained. At that time, the final separation step is omitted, and double-stranded DNA is isolated. In general, it is preferred to add the primers and dNTP's at the beginning of the amplification reaction in sufficient quantity to allow full amplification to occur without the need to add additional reagents during the course of the reaction series. The use of Taq DNA polymerase facilitates such a process, as this heat-stable enzyme is not inactivated by the heat denaturation steps and the reaction need not be interrupted for the addition of more polymerase.

As noted above, oligonucleotide primers for use in the polymerase chain reaction are constructed to be complementary to sequences flanking an exon comprising the site of a selected mutation, such as the exon containing the codon for amino acid 601 or the exon containing the codon for amino acid 355. A first primer is designed to be complementary to a sequence on the coding strand, and a second primer is complementary to a sequence on the noncoding strand of the DNA. Preferably, the primers will be complementary to intron sequences because intron sequences will exhibit the least amount of intergene homology. The primers are preferably at least about 15-20 bases in length, more preferably at least about 25 bases in length. Primers shorter than about 20 bases will often have reduced specificity, and may anneal to and amplify unwanted sequences. Primers are preferably less than 50 bases in length, more preferably less than about 30 bases in length. Longer primers may self-anneal or their use may lead to reduced specificity. Within the present invention, alternative methods of DNA amplification may also be used. For example, a genomic library may be prepared by digesting genomic DNA from a patient and cloning the resultant DNA fragments into a suitable vector (e.g., plasmid, cosmid or bacteriophage). The library is then amplified by conventional methods, and plasminogen-encoding clones are screened for the presence of the mutation.

The amplified DNA is then incubated with a restriction endonuclease which is capable of differentially cleaving normal and abnormal plasminogen DNA. Suitable restriction endonucleases for identification of the Thr-601 mutation include Fnu 4HI and Bbv I. Endonucleases suitable for identification of the Phe-355 mutation include Ava II, Bam Nxl, Cau I (Bingham and Darbyshire, Gene 18:87-91, 1982; Molemans et al., Gene 18:93-96, 1982), Hgi BI, Hgi CII, Hgi EI and Sau 96I. However, the invention is not limited to the use of particular enzymes, but is intended to include the use of other suitable enzymes which may from time to time become available. Restriction endonucleases are commercially available from, for example, New England Biolabs (Beverly, Mass.), Bethesda Research Laboratories (Gaithersburg, Md.) and other suppliers. The amplified DNA is incubated with the endonuclease under conditions of time, temperature and buffer composition suitable for the activity of the endonuclease. Such conditions are generally specified by the supplier.

Following exposure to the restriction endonuclease, the DNA sample is analyzed to detect the presence or absence of cleavage fragments diagnostic for the selected mutation, for example by electrophoretic separation of DNA fragments. In a preferred embodiment, the DNA is electrophoresed on an agarose gel containing ethidium bromide. Endonuclease Fnu 4HI cleaves the normal plasminogen sequence at the codon for Ala-601. The presence of the Thr-601 mutation prevents this cleavage, resulting in no change in fragment size following exposure to the enzyme. Priming in the introns flanking the codon for amino acid 601 as disclosed in more detail below resulted in amplification of a 340 bp fragment. The normal sequence could be cleaved by Fnu 4HI to yield fragments of about 240 bp and 100 bp. Also, as discussed in more detail below, the mutation of Val-355 to Phe can be detected by amplifying a 390 bp fragment, digesting the amplified DNA with Ava II and analyzing the digested DNA. The Phe-355 mutation results in the presence of a 360 bp fragment, which is not present in the Ava II digest of wild-type DNA. The methods described herein are well suited to clinical use. In particular, the combination of the polymerase chain reaction and restriction analysis can be used to diagnose the specific plasminogen abnormality at the DNA level in a rapid and straightforward manner. Partial purification of genomic DNA from leukocytes takes several hours, and amplification by the polymerase chain reaction takes about three hours . Restr iction d igestion of the amplif ied DNA and its analysis on agarose gels require about one hour or less each. Therefore, the entire diagnostic procedure can be performed in a single day.

As briefly described above, suitable kits for diagnosing these plasminogen mutations contain oligonucleotide primers, Taq DNA polymerase, an appropriate restriction enzyme, buffers, and normal (control) DNA in appropriate packaging.

The following examples are offered by way of illustration, and not by way of limitation.

EXPERIMENTAL

Taq DNA polymerase was obtained from New England Biolabs and The Perkin Elmer Corporation (Norwalk, Conn.). Restriction endonucleases and T4 DNA ligase were purchased from Bethesda Research Laboratories (Gaithersburg, Md.) or New England Biolabs. The Klenow fragment of Escherichia coli DNA polymerase, bacterial alkaline phosphatase, ATP, deoxynucleotides, dideoxynucleotides, M13mp18, and M13mp19 were supplied by Bethesda Research Laboratories. dATP[α- 3⁵S] was provided by Amersham (Chicago, 111.).

Oligonucleotides were synthesized using a nucleotide synthesizer (Applied Biosysterns, Foster City, Calif.) and kindly supplied by Drs. Patrick S.H. Chou, Yim Foon Lee and Jeff Harris.

Example 1 Leukocyte genomic DNA samples were obtained from three unrelated Japanese patients with abnormal plasminogen (named abnormal I, II and III, respectively), a daughter of abnormal III (abnormal III-2) and three unrelated normal American white individuals. Abnormals I, II and III-2 had a history of thrombosis, but abnormal III did not. The plasma of abnormal I had a trace of plasminogen activity in spite of a normal plasminogen antigen concentration, and the plasma from the mother and a sister of abnormal I showed a 50% reduction in enzymatic activity of plasminogen. Accordingly, abnormal I is a homozygote of a nonfunctional plasminogen variant. Abnormal II is a heterozygote of a plasminogen variant, since the plasminogen in the plasma of the patient and his two daughters has about half of the specific activity (activity per antigen) of normal plasminogen. Abnormal III is a homozygote of the plasminogen variant named PLG B (Nishimukai et al., Hum. Hered. 36:137-142, 1986) as determined by isoelectric focusing. Abnormal III-2 is a heterozygote of PLG B with a normal plasminogen concentration and half of normal specific activity.

Genomic DNA samples were prepared from the leukocytes of the patients with abnormal plasminogen and from normal individuals by the method of Bell et al. (ibid.).

Typically, 10-40 ml of blood is collected in citrate buffer. Ten ml of blood is added to 90 ml of 0.32 M sucrose, 10 mM Tris pH7.5, 5mM MgCl₂, 1% Triton X-100, and the mixture is incubated at 4°C to lyse the cells. Nuclei are collected by centrifugation at 1,000 x g for 10 minutes and resuspended in 4.5 ml of 0.075 M NaCl, 0.024 M EDTA, pH 8.0. The nuclei are treated with SDS and proteinase K, and the DNA is extracted with chloroform/phenol/isoamyl alcohol, precipitated with ethanol and resuspended in the appropriate buffer. Nucleotide primers A39 and 1A (Figure 3) for the putative introns N and O flanking the exon coding for the amino acid residue-601 of plasminogen (exon XV) were synthesized for the polymerase chain reaction. These regions were selected because they lie outside the putative exon 15, and upon selective amplification they produce a fragment of a length suitable for analysis by restriction digestion and DNA sequencing. Both the 5'- and 3'-ends were modified to generate convenient restriction sites (Hind III) for cloning directly into the M13 sequencing vector. One μg of genomic DNA was amplified in a 100 μl reaction mixture containing 50 mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl₂, each primer (A39 and 1A, Figure 3) at 1 μM, each dNTP at 200 μM, gelatin at 200 μg/ml, and 2.5 units of Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988). The sample was placed in a small Eppendorf tube and overlaid with 100 μl of mineral oil to prevent evaporation. The sample was heated at 93°C for one minute to denature the DNA, cooled to 60°C for two minutes to anneal the primers, and incubated at 70°C for three minutes to extend the annealed primers. The procedure was repeated for a total of 25-30 cycles of amplification. At the end of the last cycle, the sample was incubated at 70°C for 7 minutes to ensure the completion of the final extension step. After precipitation with ethanol and resuspension in 100 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), 5 μl was applied to a 1.5% agarose gel for submerged electrophoresis, and stained with ethidium bromide. A discrete band of about 340 bp was obtained for each sample, as predicted from the sequence of the gene for normal plasminogen.

The samples from abnormals I, II, III, III-2 and normal individuals were digested with three units of Fnu 4HI endonuclease for one hour or with six units of enzyme for four hours at 37°C. Five microliters of each sample was then applied to a 1.5% agarose gel containing ethidium bromide. The 340 bp fragment of normal DNA was cleaved into two fragments (about 240 and 100 bp), while that of the DNA from abnormal III remained unchanged (Figure 4). The Fnu 4HI digests of the 340 bp fragments from abnormals II and III-2 each showed a mixed pattern of normal DNA and the DNA from abnormal III. In contrast, the DNA from abnormal I was cleaved completely. Prolonged digestion of the samples for four hours with six units of enzyme gave exactly the same results (Figure 4). The amplification and digestion of the genomic DNAs from abnormals I, II, III and III-2 was performed eight, two, three and two times, respectively, and the results obtained were the same in each experiment for each sample. Fnu 4HI recognizes only th GCNGC sequence, suggesting that one or more of these fou nucleotides in the DNAs of abnormals III, III-2, and II i replaced by other nucleotides. Alternatively, a short stretch of nucleotides could be deleted or inserted in the abnormal DNA.

To characterize the mutation(s) at the DNA level, the amplified fragments were sequenced. Since both the 5'- and 3'-end primers were designed to produce double-stranded fragments flanked by Hind III recognition sequences, the amplified 340 bp fragments from normal and abnormal individuals were digested with Hind III and ligated into M13 sequencing vectors cut with Hind III. In order to obtain the DNA sequence coding for the specific region around amino acid residue 601, the amplified DNAs were also digested with Hinc II and Pst I endonucleases. The digested samples were electrophoresed on a 1.5% agarose gel, electroeluted, and dialyzed against 0. IX TBE (1X TBE is 89 mM Tris-borate, 89 mM boric acid, 20 mM EDTA) overnight. The dialyzed samples were extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE, and finally subcloned into M13mp18 or mp19 in order to obtain discrete overlapping sequences. The DNA sequences of the inserts were then obtained using the dideoxynucleotide method (Sanger et al. Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) with dATP [α-³⁵S] and buffer gradient gels (Biggin et al. Proc. Natl. Acad. Sci. USA 80:3963-3965, 1983) .

The DNA sequences obtained from the three normal individuals included 343 bp. These sequences were the same as expected for the normal gene except for the presence of Hind III sites at both the 5'- and 3' ends. The sequence of the Hinc Il-Pst I fragments from the normal DNAs included 205 bp, and was also the same as the established sequence of the normal gene for plasminogen. The actual sequence of the region coding for amino acid 601 (Ala) included ACTGCTGC in the normal gene. On the other hand, the DNA sequence analysis of both Hind III and Hinc Il-Pst I fragments of abnormal III revealed that the gene of abnormal III contained the sequence ACTACTGC. This corresponds to a single base change resulting in the substitution of Thr (ACT) for Ala (GCT). Twenty-three templates .from the amplified samples of abnormal III were sequenced and all of them showed the same abnormal sequence (G to A change). No other alterations of nucleotides were found by DNA sequence analysis. When twelve templates for abnormal II were sequenced, one-half of them showed the same sequence as the normal gene except for a point mutation (T to C) 5 nucleotides prior to the Fnu 4HI site, and the other half had the same abnormal sequence as abnormal III. These results confirmed that abnormal III is a homozygote of a plasminogen variant and that abnormal II is a heterozygote of the same variant allele. The exon XV DNA sequence of abnormal I was the same as that of the normal gene, indicating that the abnormality in this molecule is in another region.

A second set of primers (designated 10A and 11A in Figure 3), flanked by Eco RI recognition sequences and four additional nucleotides, was used to confirm the results. A band of 360 bp was obtained for each sample as predicted.

Example 2

Plasminogen gene exon X DNA of abnormal I was amplified essentially as described above using primers K4a- 5' (5' GTC AGA ATT CTC AGA GGC TAC CGT ACT 3'; coding strand primer) and K4a-3' (5' CTA CGA ATT CTG GCT CTA ACA CAA ATT TCC 3'; noncoding strand primer). The amplified DNA was digested with Eco RI, and the resulting ~390 bp fragment was cloned into an M13 phage vector and sequenced. Sequence analysis revealed the presence of the sequence GTGTTCCAG in six of the templates, as compared to the wild- type sequence GTGGTCCAG. This T for G substitution results in the substitution of a phenylalanine residue for the normal valine residue at amino acid position 355, located several residues upstream of Kringle 4 in the A chain (Figure 1). DNA samples from normal and abnormal individuals were digested with five units of Ava II endonuclease for one hour at 37°C. The 390 bp DNA fragment from the normal individuals was cleaved into three fragments of approximately 230 bp, 130 bp and 30 bp. DNA samples from abnormal I and two daughters (abnormals I-2 and I-3) and a nephew (abnormal I-4) of abnormal I showed a mixture of 360 bp, 230 bp, 130 bp and 30 bp fragments. These results indicated that the abnormal patients were heterozygous for the Phe-355 mutation. Thus, this mutation can be diagnosed by the presence of a 360 bp Ava II fragment when DNA is selectively amplified using primers K4a-5' and K4a-3'. In a second series of experiments, DNA from abnormals I, I-2, I-3 and I-4 was amplified using pr imers K4a-5' and K4a-32 (5' AAA TGA ATT CCT AGG AAG TTG GCT TGA AGC 3'; noncoding strand primer). Digestion of the resulting ~370 bp fragment with Ava II confirmed the loss of an Ava II site in the abnormal DNA, and also confirmed the diagnosis of abnormals I, I-2, I-3 and I-4 as heterozygotes of the Phe-355 mutation.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

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FOR THE PURPOSES OF INFORMA TION ONLY

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DK Denmark MC Monaco

ES Spain MG Madagascar

METHOD FOR DETECTING ABNORMAL GENES

Technical Field

Background of the Invention

In order to understand the mechanisms and genetics of human diseases, it is important to identify DNA and protein markers that indicate the presence of genetic defects in populations and families. For example, deficiencies in protein C, protein S, antithrombin III, heparin co-factor II, tissue-type plasminogen activator and plasminogen have been identified as the cause or at least part of the cause of a predisposition for thrombosis in some patients with hereditary thrombophilia (for review, see Bauer and Rosenberg, Blood 70:343-350, 1987, and Mannucci and Tripodi, Thromb. Haemostas. 57: 247-251, 1987). Plasminogen is a single-chain proenzyme that is converted to an active two-chain form (consisting of an A and a B chain connected by two disulfide bonds), called plasmin, by activators such as tissue-type plasminogen activator, urokinase, and streptokinase. Plasmin digests fibrin clots to form soluble fibrin degradation products. In addition, plasmin is thought to play an important role in various biological reactions, such as inflammation, tissue development and remodeling, processing other molecules, etc. The primary structure of plasminogen (790 amino acid residues) was established by Sottrup-Jensen et al. (Prog. Chem. Fibrinol. Thrombol. 3:191-209, 1978). This amino acid sequence has been confirmed by cDNA sequencing (Malinowski et al., Biochemistry 23: 4243-4250, 1984 and Forsgren et al., FEBS Lett. 213:254-260, 1987), which indicated the presence of an additional Ile residue at position 65. Accordingly, plasminogen contains 791 amino acids (See Figure 1). The A chain of the molecule consists of the activation peptide (77 amino acid residues) and five disulfide bond-folded structures called "kringles" (about 90 residues each). The B chain contains the activation site (between Arg-561 and Val-562), the active site His-603 residue region, the active site Asp-646 residue region, the region which is linked to the heavy chain by a disulfide bond, the active site Ser-741 residue region, and the C-terminus (amino acid numbers used herein refer to the sequence shown in Figure 1). The first kringle structure (K1) in the A chain of plasminogen is responsible for its binding to fibrin (Thorsen et al., Biochim. Biophys. Acta. 668:377-387, 1981). The B chain of plasminogen carries all three active sites essential for catalytic function as a serine protease.

Several cases of a molecular abnormality of plasminogen in association with a complication of thrombosis have been reported (Aoki et al., J. Clin. Invest. 61: 1186-1195, 1978; Kazama et al., Thromb. Res. 21:517-522, 1981; Wohl et al., Thromb. Haemostas. 48:146-152, 1982; Soria et al., Thromb. Res. 32:229-238, 1982 and Scharrar et al., Thromb. Hemostas. 55:396-401, 1986). These abnormalities have been found most frequently in Japan, but have also been reported in other populations. By an analysis of the plasminogen molecules from these patients, it has been shown that an amino acid substitution of Thr for Ala-601 in the B chain results in the generation of an inactive plasmin molecule (Sakata and Aoki, J. Biol. Chem. 255:5442- 5447, 1980; Miyata et al., Proc. Natl. Acad. Sci. USA 79:6132-6136, 1982; Miyata et al., J. Biochem. 96:227-287, 1984). However, the nature of the underlying abnormality at the DNA level has not heretofore been determined, and other plasminogen disorders have not been characterized.

Conventional biological assays for plasminogen activity and antigen concentration do not accurately identify the molecular basis of thrombosis, because plasminogen can be decreased in several acquired disease states, such as liver dysfunction and disseminated intravascular coagulation, or by thrombolytic therapy using plasminogen activators. Because proper therapy is dictated by the nature of the underlying condition, it is important to make a definitive diagnosis in the case of a genetic molecular abnormality. An additional complication in diagnosing plasminogen-related disorders arises from the high degree of homology between plasminogen and apolipoprotein(a). This homology makes it difficult to distinguish between DNA sequences encoding the two proteins. Previously described methods of identifying the presence of Lhe Thr-601 plasminogen mutation are not well suited to clinical use. Miyata et al. (Proc. Natl. Acad. Sci. USA 79:6132-6136, 1982) used proteolytic digestion of plasminogen and amino acid sequence analysis of the resultant peptides to characterize the mutation. Aoki et al. (Biochemical Genetics 22:871-881, 1984) utilized electrofocusing, zymography and immunofixation of neuraminidase-treated plasminogen. The entire procedure required four or more days to perform.

Disclosure of the Invention

Brief Description of the Drawings

Best Mode for Carrying Out the Invention

As noted above, reduced plasminogen activity can lead to thrombotic episodes. Also as noted above, such a reduction in activity.can result from a variety of causes, including genetic abnormalities. Practical methods of clinical screening for genetic abnormalities in plasminogen have heretofore been unavailable.

Genomic DNA (either isolated or in the form of a suitable tissue sample) is then selectively amplified to provide a high copy number of the desired portion of the plasminogen gene (e.g., the portion encoding amino acid residue 601 or the portion encoding amino acid residue 355). Preferably, a sequence of approximately 200-1,000, most preferably about 300-400, base pairs is selectively amplified. In a preferred embodiment, the exon encoding amino acid 601 and portions of the intron sequences flanking this exon are selectively amplified. Similarly, the exon encoding amino acid 355 and portions of the flanking introns may be selectively amplified. A preferred method of amplification is the polymerase chain reaction, described by Mullis (U.S. Patent Nos. 4,683,202 and 4,683,195). Briefly, the genomic DNA is denatured to separate the coding and noncoding strands. Denaturation is preferably accomplished by heat treatment of the DNA, generally treatment at about 80°C-105°C for about one to ten minutes, although enzymatic denaturation may also be used. Most preferably, the DNA is heated at about 93°C for one minute. The denatured DNA is then combined with a molar excess of a pair of oligonucleotide primers under conditions which allow the DNA strands to anneal to the primers (e.g., 60°C for one to three minutes, preferably about two minutes). Preferably, each primer is used at a concentration of about 1μM for amplification of one microgram of genomic DNA. Suitable results may be obtained with 5μg of primer per μg of target DNA. One of the primers is complementary to a sequence on the coding strand and the second primer is complementary to a sequence on the noncoding strand, the sequences flanking the region to be amplified. "Sequences flanking the region to be amplified" include exon sequences, sequences of introns immediately adjacent to the exon to be amplified and sequences of other introns, so long as the amplified region includes the site of the selected mutation. The flanking sequences should be selected so as to provide an amplified portion of the gene within the size limits noted above. Although 100% complementarity is not required, a high degree of complementarity of primer and genomic DNA is advantageous in that it results in high specificity and efficiency of amplification. For use within the present invention, the primers must be sufficiently complementary to hybridize with their respective strands on the genomic DNA. The annealed primers are enzymatically extended using a DNA polymerase and all four deoxyribonucleotide triphosphates (dNTP's). Suitable polymerases include E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase, and T4 DNA polymerase. Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988) is particularly preferred. The reaction mixture is incubated under conditions of time and temperature suitable for the activity of the polymerase. When using the Taq DNA polymerase the mixture is incubated at about 70°C ± 10°C for approximately three minutes. As will be appreciated by one skilled in the art, the exact time and temperature will be determined by the melting point of the annealed DNA. The resulting extension products are separated from the original DNA strands, preferably by heat denaturation. The annealing, extension and separation steps are then repeated, preferably about 25 to 30 times, until the desired degree of amplification is obtained. At that time, the final separation step is omitted, and double-stranded DNA is isolated. In general, it is preferred to add the primers and dNTP's at the beginning of the amplification reaction in sufficient quantity to allow full amplification to occur without the need to add additional reagents during the course of the reaction series. The use of Taq DNA polymerase facilitates such a process, as this heat-stable enzyme is not inactivated by the heat denaturation steps and the reaction need not be interrupted for the addition of more polymerase.

The amplified DNA is then incubated with a restriction endonuclease which is capable of differentially cleaving normal and abnormal plasminogen DNA. Suitable restriction endonucleases for identification of the Thr-601 mutation include Fnu 4HI and Bbv I. Endonucleases suitable for identification of the Phe-355 mutation include Ava II, Bam Nxl, Cau I (Bingham and Darbyshire, Gene 18:87-91, 1982; Molemans et al., Gene 18: 93-96, 1982), Hgi BI, Hgi CII, Hgi El and Sau 961. However, the invention is not limited to the use of particular enzymes, but is intended to include the use of other suitable enzymes which may from time to time become available. Restriction endonucleases are commercially available from, for example, New England Biolabs (Beverly, Mass.), Bethesda Research Laboratories (Gaithersburg, Md.) and other suppliers. The amplified DNA is incubated with the endonuclease under conditions of time, temperature and buffer composition suitable for the activity of the endonuclease. Such conditions are generally specified by the supplier.

Following exposure to the restriction endonuclease, the DNA sample is analyzed to detect the presence or absence of cleavage fragments diagnostic for the selected mutation, for example by electrophoretic separation of DNA fragments. In a preferred embodiment, the DNA is electrophoresed on an agarose gel containing ethidium bromide. Endonuclease Fnu 4HI cleaves the normal plasminogen sequence at the codon for Ala-601. The presence of the Thr-601 mutation prevents this cleavage, resulting in no change in fragment size following exposure to the enzyme. Priming in the introns flanking the codon for amino acid 601 as disclosed in more detail below resulted in amplification of a ~340 bp fragment. The normal sequence could be cleaved by Fnu 4HI to yield fragments of about 240 bp and 100 bp. Also, as discussed in more detail below, the mutation of Val-355 to Phe can be detected by amplifying a ~390 bp fragment, digesting the amplified DNA with Ava II and analyzing the digested DNA. The Phe-355 mutation results in the presence of a 360 bp fragment, which is not present in the Ava II digest of wild-type DNA. The methods described herein are well suited to clinical use. In particular, the combination of the polymerase chain reaction and restriction analysis can be used to diagnose the specific plasminogen abnormality at the DNA level in a rapid and straightforward manner. Partial purification of genomic DNA from leukocytes takes several hours, and amplification by the polymerase chain reaction takes about three hours. Restriction digestion of the amplified DNA and its analysis on agarose gels require about one hour or less each. Therefore, the entire diagnostic procedure can be performed in a single day.

EXPERIMENTAL

Taq DNA polymerase was obtained from New England Biolabs and The Perkin Elmer Corporation (Norwalk, Conn.). Restriction endonucleases and T4 DNA ligase were purchased from Bethesda Research Laboratories (Gaithersburg, Md.) or New England Biolabs. The Klenow fragment of Escherichia coli DNA polymerase, bacterial alkaline phosphatase, ATP, deoxynucleotides, dideoxynucleotides, M13mpl8, and M13mp19 were supplied by Bethesda Research Laboratories. dATP[α- 3⁵S] was provided by Amersham (Chicago, 111.).

Oligonucleotides were synthesized using a nucleotide synthesizer (Applied Biosystems, Foster City, Calif.) and kindly supplied by Drs. Patrick S.H. Chou, Yim Foon Lee and Jeff Harris.

Typically, 10-40 ml of blood is collected in citrate buffer. Ten ml of blood is added to 90 ml of 0.32 M sucrose, 10 mM Tris pH7.5, 5mM MgCl₂, 1% Triton X-100, and the mixture is incubated at 4°C to lyse the cells. Nuclei are collected by centrifugation at 1,000 x g for 10 minutes and resuspended in 4.5 ml of 0.075 M NaCl, 0.024 M EDTA, pH 8.0. The nuclei are treated with SDS and proteinase K, and the DNA is extracted with chloroform/phenol/isoamyl alcohol, precipitated with ethanol and resuspended in the appropriate buffer. Nucleotide primers A39 and 1A (Figure 3) for the putative introns N and O flanking the exon coding for the amino acid residue-601 of plasminogen (exon XV) were synthesized for the polymerase chain reaction. These regions were selected because they lie outside the putative exon 15, and upon selective amplification they produce a fragment of a length suitable for analysis by restriction digestion and DNA sequencing. Both the 5'- and 3'-ends were modified to generate convenient restriction sites (Hind III) for cloning directly into the M13 sequencing vector. One μg of genomic DNA was amplified in a 100 μl reaction mixture containing 50 mM KC1, 10 mM Tris (pH 8.4), 2.5 mM MgCl2, each primer (A39 and 1A, Figure 3) at 1 μM, each dNTP at 200 μM, gelatin at 200 μg/ml, and 2.5 units of Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988). The sample was placed in a small Eppendorf tube and overlaid with 100 μl of mineral oil to prevent evaporation. The sample was heated at 93°C for one minute to denature the DNA, cooled to 60°C for two minutes to anneal the primers, and incubated at 70°C for three minutes to extend the annealed primers. The procedure was repeated for a total of 25-30 cycles of amplification. At the end of the last cycle, the sample was incubated at 70°C for 7 minutes to ensure the completion of the final extension step. After precipitation with ethanol and resuspension in 100 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), 5 μl was applied to a 1.5% agarose gel for submerged electrophoresis, and stained with ethidium bromide. A discrete band of about 340 bp was obtained for each sample, as predicted from the sequence of the gene for normal plasminogen.

The samples from abnormals I, II, III, III-2 and normal individuals were digested with three units of Fnu 4HI endonuclease for one hour or with six units of enzyme for four hours at 37°C. Five microliters of each sample was then applied to a 1.5% agarose gel containing ethidium bromide. The 340 bp fragment of normal DNA was cleaved into two fragments (about 240 and 100 bp), while that of the DNA from abnormal III remained unchanged (Figure 4). The Fnu 4HI digests of the 340 bp fragments from abnormals II and III-2 each showed a mixed pattern of normal DNA and the DNA from abnormal III. In contrast, the DNA from abnormal I was cleaved completely. Prolonged digestion of the samples for four hours with six units of enzyme gave exactly the same results (Figure 4). The amplification and digestion of the genomic DNAs from abnormals I, II, III and III-2 was performed eight, two, three and two times, respectively, and the results obtained were the same in each experiment for each sample. Fnu 4HI recognizes only the GCNGC sequence, suggesting that one or more of these four nucleotides in the DNAs of abnormals III, III-2, and II is replaced by other nucleotides. Alternatively, a short stretch of nucleotides could be deleted or inserted in the abnormal DNA.

To characterize the mutation(s) at the DNA level, the amplified fragments were sequenced. Since both the 5'- and 3'-end primers were designed to produce double-stranded fragments flanked by Hind III recognition sequences, the amplified 340 bp fragments from normal and abnormal individuals were digested with Hind III and ligated into M13 sequencing vectors cut with Hind III. In order to obtain the DNA sequence coding for the specific region around amino acid residue 601, the amplified DNAs were also digested with Hinc II and Pst I endonucleases. The digested samples were electrophoresed on a 1.5% agarose gel, electroeluted, and dialyzed against 0.1X TBE (IX TBE is 89 mM Tris-borate, 89 mM boric acid, 20 mM EDTA) overnight. The dialyzed samples were extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE, and finally subcloned into M13mρl8 or mp.19 in order to obtain discrete overlapping sequences. The DNA sequences of the inserts were then obtained using the dideoxynucleotide method (Sanger et al. Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) with dATP [α-³⁵S] and buffer gradient gels (Biggin et al. Proc. Natl. Acad. Sci,. USA 80:3963- 3965, 1983).

The DNA sequences obtained from the three normal individuals included 343 bp. These sequences were the same as expected for the normal gene except for the presence of Hind III sites at both the 5'- and 3' ends. The sequence of the Hinc Il-Pst I fragments from the normal DNAs included 205 bp, and was also the same as the established sequence of the normal gene for plasminogen. The actual sequence of the region coding for amino acid 601 (Ala) included ACTGCTGC in the normal gene. On the other hand, the DNA sequence analysis of both Hind III and Hinc Il-Pst I fragments of abnormal III revealed that the gene of abnormal III contained the sequence ACTACTGC. This corresponds to a single base change resulting in the substitution of Thr (ACT) for Ala (GCT). Twenty-three templates from the amplified samples of abnormal III were sequenced and all of them showed the same abnormal sequence (G to A change). No other alterations of nucleotides were found by DNA sequence analysis. When twelve templates for abnormal II were sequenced, one-half of them showed the same sequence as the normal gene except for a point mutation (T to C) 5 nucleotides prior to the Fnu 4HI site, and the other half had the same abnormal sequence as abnormal III. These results confirmed that abnormal III is a homozygote of a plasminogen variant and that abnormal II is a heterozygote of the same variant allele. The exon XV DNA sequence of abnormal I was the same as that of the normal gene, indicating that the abnormality in this molecule is in another region.

Example 2

Plasminogen gene exon X DNA of abnormal I was amplified essentially as described above using primers K4a- 5' (5' GTC AGA ATT CTC AGA GGC TAC CGT ACT 3'; coding strand primer) and K4a-3' (5' CTA CGA ATT CTG GCT CTA ACA CAA ATT TCC 3'; noncoding strand primer). The amplified DNA was digested with Eco RI, and the resulting ~390 bp fragment was cloned into an M13 phage vector and sequenced. Sequence analysis revealed the presence of the sequence GTGTTCCAG in six of the templates, as compared to the wild- type sequence GTGGTCCAG. This T for G substitution results in the substitution of a phenylalanine residue for the normal valine residue at amino acid position 355, located several residues upstream of Kringle 4 in the A chain (Figure 1). DNA samples from normal and abnormal individuals were digested with five units of Ava II endonuclease for one hour at 37°C The 390 bp DNA fragment from the normal individuals was cleaved into three fragments of approximately 230 bp, 130 bp and 30 bp. DNA samples from abnormal I and two daughters (abnormals 1-2 and 1-3) and a nephew (abnormal 1-4) of abnormal I showed a mixture of 360 bp, 230 bp, 130 bp and 30 bp fragments. These results indicated that the abnormal patients were heterozygous for the Phe-355 mutation. Thus, this mutation can be diagnosed by the presence of a 360 bp Ava II fragment when DNA is selectively amplified using primers K4a-5' and K4a-3'. In a second series of experiments, DNA from abnormals I, I-2, I-3 and I-4 was amplified using primers K4a-5' and K4a-32 (5' AAA TGA ATT CCT AGG AAG TTG GCT TGA AGC 3'; noncoding strand primer). Digestion of the resulting "370 bp fragment with Ava II confirmed the loss of an Ava II site in the abnormal DNA, and also confirmed the diagnosis of abnormals I, I-2, I-3 and I-4 as heterozygotes of the Phe-355 mutation.

PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification ⁴ _: (11) International Publication Number: WO 89/12697

C12Q 1/68 // COm 21/04 Al (43) International Publication Date: 28 December 1989 (28.12.89

(21) International Application Number : PCT/US89/02731 (81) Designated States : AT (European patent), BE (European patent), CH (European patent), DE (European patent),

(22) International Filing Date : 21 June 1989 (21.06.89) DK, FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent).

(30) Priority data:

210,116 22 June 1988 (22.06.88) US

Published

With international search report.

(71) Applicant: THE BOARD OF REGENTS OF THE UNIBefore the expiration of the time limit for amending th

VERSITY OF WASHINGTON [US/US]; 3755 Universclaims and to be republished in the event of the receipt Oj ity May N.E., Seattle, WA 98195 (US). amendments.

(72) Inventor: ICHINOSE, Akitada ; 6846 40th Avenue N.E.,

Seattle, WA 98115 (US).

(74) Agents: MAKI, David, J. et al.; Seed and Berry, 6300 Colombia Center, Seattle, WA 98104-7092 (US).

(54) Title: METHOD FOR DETECTING ABNORMAL GENES

Amplified FnutHI digest Fnu4H I d i gest ONA ( I hp ) ( 4 hrs )

\

57) Abstract

Methods for detecting the presence of selected mutations, such as the Thr-601 mutation and the Phe-355 mutation, in the plasminogen of a patient are disclosed. The methods include exposing amplified genomic DNA to a restriction endonuclease capable of differentially cleaving mutant and wild-type plasminogen DNA sequences, and analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation. Diagnostic kits for the rapid detection of the elected mutation are also disclosed.

FOR THE PURPOSES OF INFORMATION ONLY

AT Austria FI Finland ML Mali

AU Australia FR France MR Mauritania

BB Barbados GA Gabon MW Malawi

BE Belgium GB United Kingdom NL Netherlands V^

BF Burkina Fasso HU Hungary NO Norway

BG Bulgaria rr Italy RO Romania

BJ Benin JP Japan SD Sudan

BR Brazil KP Democratic People's Republic SE Sweden

CF Central African Republic of Korea SN Senegal f

CG Congo KR Republic of Korea SU Soviet Union

CH Switzerland U Liechtenstein TD Chad

CM Cameroon LK Sri Lanka TG Togo

DE Germany, Federal Republic of LU Luxembourg US United States of America

DK Denmark MC Monaco

ES Spain MG Madagascar

METHOD FOR DETECTING ABNORMAL GENES

Technical Field

Background of the Invention

In order to understand the mechanisms and genetics of human diseases, it is important to identify DNA and protein markers that indicate the presence of genetic defects in populations and families. For example, deficiencies in protein C, protein S, antithrombin III, heparin co-factor II, tissue-type plasminogen activator and plasminogen have been identified as the cause or at least part of the cause of a predisposition for thrombosis in some patients with hereditary thrombophilia (for review, see Bauer and Rosenberg, Blood 70: 343-350, 1937, an d Mannucci and Tripodi, Thromb. Haemostas. 57:247-251, 1987). Plasminogen is a single-chain proenzyme that is converted to an active two-chain form (consisting of an A and a B chain connected by two disulfide bonds), called plasmin, by activators such as tissue-type plasminogen activator, urokinase, and streptokinase. Plasmin digests fibrin clots to form soluble fibrin degradation products. In addition, plasmin is thought to play an important role in various biological reactions, such as inflammation, tissue development and remodeling, processing other molecules, etc. The primary structure of plasminogen (790 amino acid residues) was established by Sottrup-Jensen et al. (Prog. Chem. Fibrinol. Thrombol. 3:191-209, 1978). This amino acid sequence has been confirmed by cDNA sequencing (Malinowski et al., Biochemistry 23: 4243-4250, 1984 and Forsgren et al., FEBS Lett. 213:254-260, 1987), which indicated the presence of an additional Ile residue at position 65. Accordingly, plasminogen contains 791 amino acids (See Figure 1). The A chain of the molecule consists of the activation peptide (77 amino acid residues) and five disulfide bond-folded structures called "kringles" (about 90 residues each). The B chain contains the activation site (between Arg-561 and Val-562), the active site His-603 residue region, the active site Asp-646 residue region, the region which is linked to the heavy chain by a disulfide bond, the active site Ser-741 residue region, and the C-terminus (amino acid numbers used herein refer to the sequence shown in Figure 1). The first kringle structure (K1) in the A chain of plasminogen is responsible for its binding to fibrin (Thorsen et al., Biochim. Biophys. Acta. 668:377-387, 1981). The B chain of plasminogen carries all three active sites essential for catalytic function as a serine protease.

Several cases of a molecular abnormality of plasminogen in association with a complication of thrombosis have been reported (Aoki et al., J. Clin. Invest. 61: 1186-1195, 1978; Kazama et al., Thromb. Res. 21:517-522, 1981; Wohl et al., Thromb. Haemostas. 48: 146-152, 1982; Soria et al., Thromb. Res. 32:229-238, 1982 and Scharrar et al., Thromb. Hemostas. 55:396-401, 1986). These abnormalities have been found most frequently in Japan, but have also been reported in other populations. By an analysis of the plasminogen molecules from these patients, it has been shown that an amino acid substitution of Thr for Ala-601 in the B chain results in the generation of an inactive plasmin molecule (Sakata and Aoki , J. Biol. Chem. 255: 5442- 5447, 1980; Miyata et al., Proc. Natl. Acad. Sci. USA 79:6132-6136, 1982; Miyata et al., J. Biochem. 96:227-287, 1984). However, the nature of the underlying abnormality at the DNA level has not heretofore been determined, and other plasminogen disorders have not been characterized.

Conventional biological assays for plasminogen activity and antigen concentration do not accurately identify the molecular basis of thrombosis, because plasminogen can be decreased in several acquired disease states, such as liver dysfunction and disseminated intravascular coagulation, or by thrombolytic therapy using plasminogen activators. Because proper therapy is dictated by the nature of the underlying condition, it is important to make a definitive diagnosis in the case of a genetic molecular abnormality. An additional complication in diagnosing plasminogen-related disorders arises from the high degree of homology between plasminogen and apolipoprotein(a). This homology makes it difficult to distinguish between DNA sequences encoding the two proteins. Previously described methods of identifying the presence of Lhe Thr-601 plasminogen mutation are not well suited to clinical use. Miyata et al. (Proc. Natl. Acad. Sci. USA 79: 6132-6136, 1982) used proteolytic digestion of plasminogen and amino acid sequence analysis of the resultant peptides to characterize the mutation. Aoki et al. (Biochemical Genetics 22:871-881, 1984) utilized electrofocusing, zymography and immunofixa tion of neuraminidase-treated plasminogen. The entire procedure required four or more days to perform.

Disclosure of the Invention

Briefly stated, the present invention is directed toward methods for detecting the presence of a mutation in the plasminogen gene of a patient. In one aspect of the present invention, the method comprises (a) amplifying a portion of genomic DNA from a patient, the portion including a predetermined exon comprising the site of a selected mutation and at least 14 base pairs of each of two intron sequences flanking the exon; (b) exposing the amplified DNA to a restriction endonuclease capable of differentially cleaving DNA having the selected mutation and wild-type plasminogen DNA, under conditions suitable for activity of the endonuclease; and (c) analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation. Within preferred embodiments, the selected mutation is Lhe Phe-355 mutation or the Thr-601 mutation. The method may also include, prior to the step of amplifying, isolating genomic DNA from the patient.

Brief Description of the Drawings

Best Mode for Carrying Out the Invention

Differentially cleaving: Cleaving a first sequence or set of sequences but not cleaving a second sequence or set of sequences. Restriction endonucleases differentially cleave DNA sequences due to their ability to specifically recognize short stretches of paired bases, frequently palindromic sequences of four to six base pairs. Cleavage may occur within the recognition sequence or at some specific distance away from the recognition sequence. Site of the selected mutation: The position in a gene at which a mutation is known to occur, regardless of whether that particular allele carries the mutant or wild-type sequence at the site.

The present invention is related, in part, to the elucidation of the human plasminogen gene sequence, portions of which are shown in Figures 2 and 3. Knowledge of this sequence has permitted the design of oligonucleotide primers that may be used to selectively amplify those portions of the gene encoding amino acid residue 601 or amino acid residue 355 . In a similar manner , other abnormal plasminogen gene sequences may be analyzed, allowing those skilled in the art to selectively amplify exons comprising sites of other selected mutations. The methods of the present invention are applied to genomic DNA samples from a patient. In one embodiment, the genomic DNA is first isolated, using conventional procedures. A convenient source of isolated genomic DNA is leukocytes, which may be readily obtained from a small (e.g., 10 ml) blood sample. Other cell types may also be used. DNA may be isolated from leukocytes using the technique of Bell et al. (Proc. Natl. Acad. Sci. USA 78:5759- 5763, 1981). Briefly, blood is collected in the presence of an anticoagulant, the cells are lysed, and the nuclei are collected. The nuclei are then treated with sodium dodecyl sulfate and proteinase K and the DNA is extracted from the mixture with phenol/chloroform/isoamyl alcohol. The DNA is then precipitated and resuspended in a suitable buffer, such as 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Alternatively, by using the method disclosed disclosed by Kogan et al. (New Eng. J. Med. 317: 985-990, 1987), the methods of the present invention may be applied directly to tissue samples, without the need to isolate the DNA. For example, chorionic villus samples can be screened directly by disrupting the tissue by vortexing in a solution of 0.1M NaOH, 2M NaCl, 0.5% SDS. The sample is then boiled for two minutes, centrifuged, and an aliquot is taken for amplification. This facilitates the application of these methods to prenatal diagnosis of the plasminogen abnormality.

Genomic DNA (either isolated or in the form of a suitable tissue sample) is then selectively amplified to provide a high copy number of the desired portion of the plasminogen gene (e.g., the portion encoding amino acid residue 601 or the portion encoding amino acid residue 355). Preferably, a sequence of approximately 200-1,000, most preferably about 300-400, base pairs is selectively amplified. In a preferred embodiment, the exon encoding amino acid 601 and portions of the intron sequences flanking this exon are selectively amplified. Similarly, the exon encoding amino acid 355 and portions of the flanking introns may be selectively amplified. A preferred method of amplification is the polymerase chain reaction, described by Mullis (U.S. Patent Nos. 4,683,202 and 4,683,195). Briefly, the genomic DNA is denatured to separate the coding and noncoding strands. Denaturation is preferably accomplished by heat treatment of the DNA, generally treatment at about 80°C-105°C for about one to ten minutes, although enzymatic denaturation may also be used. Most preferably, the DNA is heated at about 93°C for one minute. The denatured DNA is then combined with a molar excess of a pair of oligonucleotide primers under conditions which allow the DNA strands to anneal to the primers (e.g., 60°C for one to three minutes, preferably about two minutes). Preferably, each primer is used at a concentration of about 1μM for amplification of one microgram of genomic DNA. Suitable results may be obtained with 5ug of primer per μg of target DNA. One of the primers is complementary to a sequence on the coding strand and the second primer is complementary to a sequence on the noncoding strand, the sequences flanking the region to be amplified. "Sequences flanking the region to be amplified" include exon sequences, sequences of introns immediately adjacent to the exon to be amplified and sequences of other introns, so long as the amplified region includes the site of the selected mutation. The flanking sequences should be selected so as to provide an amplified portion of the gene within the size limits noted above. Although 100% complementarity is not required, a high degree of complementarity of primer and genomic DNA is advantageous in that it results in high specificity and efficiency of amplification. For use within the present invention, the primers must be sufficiently complementary to hybridize with their respective strands on the genomic DNA. The annealed primers are enzymatically extended using a DNA polymerase and all four deoxyribonucleotide triphosphates (dNTP's). Suitable polymerases include E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase, and T4 DNA polymerase. Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988) is particularly preferred. The reaction mixture is incubated under conditions of time and temperature suitable for Lhe activity of the polymerase. When using the Taq DNA polymerase the mixture is incubated at about 70°C ± 10°C for approximately three minutes. As will be appreciated by one skilled in the art, the exact time and temperature will be determined by the melting point of the annealed DNA. The resulting extension products are separated from the original DNA strands, preferably by heat denaturation. The annealing, extension and separation steps are then repeated, preferably about 25 to 30 times, until the desired degree of amplification is obtained. At that time, the final separation step is omitted, and double-stranded DNA is isolated. In general, it is preferred to add the primers and dNTP's at the beginning of the amplification reaction in sufficient quantity to allow full amplification to occur without the need to add additional reagents during the course of the reaction series. The use of Taq DNA polymerase facilitates such a process, as this heat-stable enzyme is not inactivated by the heat denaturation steps and the reaction need not be interrupted for the addition of more polymerase.

The amplified DNA is then incubated with a restriction endonuclease which is capable of differentially cleaving normal and abnormal plasminogen DNA. Suitable restriction endonucleases for identification of the Thr-601 mutation include Fnu 4HI and Bbv I. Endonucleases suitable for identification of the Phe-355 mutation include Ava II, Bam Nxl, Cau I (Bingham and Darbyshire, Gene 18:87-91, 1982; Molemans et al., Gene 18:93-96, 1982), Hgi BI, Hgi CII, Hgi EI and Sau 961. However, the invention is not limited to the use of particular enzymes, but is intended to include the use of other suitable enzymes which may from time to time become available. Restriction endonucleases are commercially available from, for example, New England Biolabs (Beverly, Mass.), Bethesda Research Laboratories (Gaithersburg, Md.) and other suppliers. The amplified DNA is incubated with the endonuclease under conditions of time, temperature and buffer composition suitable for the activity of the endonuclease. Such conditions are generally specified by the supplier.

EXPERIMENTAL

Taq DNA polymerase was obtained from New England Biolabs and The Perkin Elmer Corporation (Norwalk, Conn.). Restriction endonucleases and T4 DNA ligase were purchased from Bethesda Research Laboratories (Gaithersburg, Md.) or New England Biolabs. The Klenow fragment of Escherichia coli DNA polymerase, bacterial alkaline phosphaLase, ATP, deoxynucleotides, dideoxynucleotides, M13mp18, and M13mp19 were supplied by Bethesda Research Laboratories. dATP[α-³⁵S] was provided by Amersham (Chicago, 111.).

Oligonucleotides were synthesized using a nucleotide synthesizer (Applied Biosystems, Foster City, Calif.) and kindly supplied by Drs. Patrick S.H. Chou , Yim Foon Lee and Jeff Harris.

Genomic DNA samples were prepared from the leukocytes of the patients with abnormal plasminogen and from normal individuals by the method of Bell et al. (ibid.). Typically, 10-40 ml of blood is collected in citrate buffer. Ten ml of blood is added to 90 ml of 0.32 M sucrose, 10 mM Tris pH7.5, 5mM MgCl₂, 1% Triton X-100, and the mixture is incubated at 4°C to lyse the cells. Nuclei are collected by centrifugation at 1,000 x g for 10 minutes and resuspended in 4.5 ml of 0.075 M NaCl, 0.024 M EDTA, pH 8.0. The nuclei are treated with SDS and proteinase K, and the DNA is extracted with chloroform/phenol/isoamyl alcohol, precipitated with ethanol and resuspended in the appropriate buffer. Nucleotide primers A39 and 1A (Figure 3) for the putative introns N and 0 flanking the exon coding for the amino acid residue-601 of plasminogen (exon XV) were synthesized for the polymerase chain reaction. These regions were selected because they lie outside the putative exon 15, and upon selective amplification they produce a fragment of a length suitable for analysis by restriction digestion and DNA sequencing. Both the 5'- and 3'-ends were modified to generate convenient restriction sites (Hind III) for cloning directly into the M13 sequencing vector. One μg of genomic DNA was amplified in a 100 μl reaction mixture containing 50 mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl₂, each primer (A39 and 1A, Figure 3) at 1 μM, each dNTP at 200 μM, gelatin at 200 μg/ml, and 2.5 units of Taq DNA polymerase (Saiki et al., Science 239:487-491, 1988). The sample was placed in a small Eppendorf tube and overlaid with 100 μl of mineral oil to prevent evaporation. The sample was heated at 93°C for one minute to denature the DNA, cooled to 60°C for two minutes to anneal the primers, and incubated at 70°C for three minutes to extend the annealed primers. The procedure was repeated for a total of 25-30 cycles of amplification. At the end of the last cycle, the sample was incubated at 70°C for 7 minutes to ensure the completion of the final extension step. After precipitation with ethanol and resuspension in 100 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), 5 μl was applied to a 1.5% agarose gel for submerged electrophoresis, and stained with ethidium bromide. A discrete band of about 340 bp was obtained for each sample, as predicted from the sequence of the gene for normal plasminogen.

The samples from abnormals I, II, III, III-2 and normal individuals were digested with three units of Fnu 4HI endonuclease for one hour or with six units of enzyme for four hours at 37°C. Five microliters of each sample was then applied to a 1.5% agarose gel containing ethidium bromide. The 340 bp fragment of normal DNA was cleaved into two fragments (about 240 and 100 bp), while that of the DNA from abnormal III remained unchanged (Figure 4) . The Fnu 4HI digests of the 340 bp fragments from abnormals II and III-2 each showed a mixed pattern of normal DNA and the DNA from abnormal III. In contrast, the DNA from abnormal I was cleaved completely. Prolonged digestion of the samples for four hours with six units of enzyme gave exactly the same results (Figure 4). The amplification and digestion of the genomic DNAs from abnormals I, II, III and III-2 was performed eight, two, three and two times, respectively, and the results obtained were the same in each experiment for each sample. Fnu 4HI recognizes only the GCNGC sequence, suggesting that one or more of these four nucleotides in the DNAs of abnormals III, III-2, and II is replaced by other nucleotides. Alternatively, a short stretch of nucleotides could be deleted or inserted in the abnormal DNA.

To characterize the mutation(s) at the DNA level, the amplified fragments were sequenced. Since both the 5'- and 3'-end primers were designed to produce double-stranded fragments flanked by Hind III recognition sequences, the amplified 340 bp fragments from normal and abnormal individuals were digested with Hind III and ligated into M13 sequencing vectors cut with Hind III. In order to obtain the DNA sequence coding for the specific region around amino acid residue 601, the amplified DNAs were also digested with Hinc II and Pst I endonucleases. The digested samples were electrophoresed on a 1.5% agarose gel, electroeluted, and dialyzed against 0. IX TBE (IX TBE is 89 mM Tris-borate, 89 mM boric acid, 20 mM EDTA) overnight. The dialyzed samples were extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE, and finally subcloned into M13mpl8 or mpl9 in order to obtain discrete overlapping sequences. The DNA sequences of the inserts were then obtained using the dideoxynucleotide method (Sanger et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977) with dATP [α-³⁵S] and buffer gradient gels (Biggin et al. Proc. Natl. Acad. Sci. USA 80:3963- 3965, 1983).

The DNA sequences obtained from the three normal individuals included 343 bp. These sequences were the same as expected for the normal gene except for the presence of Hind III sites at both the 5'- and 3'ends. The sequence of the Hinc Il-Pst I fragments from the normal DNAs included 205 bp, and was also the same as the established sequence of the normal gene for plasminogen. The actual sequence of the region coding for amino acid 601 (Ala) included ACTGCTGC in the normal gene. On the other hand, the DNA sequence analysis of both Hind III and Hinc Il-Pst I fragments of abnormal III revealed that the gene of abnormal III contained the sequence ACTACTGC. This corresponds to a single base change resulting in the substitution of Thr (ACT) for Ala (GCT) . Twenty-three templates from the amplified samples of abnormal III were sequenced and all of them showed the same abnormal sequence (G to A change). No other alterations of nucleotides were found by DNA sequence analysis. When twelve templates for abnormal II were sequenced, one-half of them showed the same sequence as the normal gene except for a point mutation (T to C) 5 nucleotides prior to the Fnu 4HI site, and the other half had the same abnormal sequence as abnormal III. These results confirmed that abnormal III is a homozygote of a plasminogen variant and that abnormal II is a heterozygote of the same variant allele. The exon XV DNA sequence of abnormal I was the same as that of the normal gene, indicating that the abnormality in this molecule is in another region.

Example 2

Plasminogen gene exon X DNA of abnormal I was amplified essentially as described above using primers K4a-5' (5' GTC AGA ATT CTC AGA GGC TAC CGT ACT 3'; coding strand primer) and K4a-3' (5' CTA CGA ATT CTG GCT CTA ACA CAA ATT TCC 3"; noncoding strand primer). The amplified DNA was digested with Eco RI, and the resulting ~390 bp fragment was cloned into an M13 phage vector and sequenced. Sequence analysis revealed the presence of the sequence GTGTTCCAG in six of the templates, as compared to the wild-type sequence GTGGTCCAG. This T for G substitution results in the substitution of a phenylalanine residue for the normal valine residue at amino acid position 355, located several residues upstream of Kringle 4 in the A chain (Figure 1). DNA samples from normal and abnormal individuals were digested with five units of Ava II endonuclease for one hour at 37°C. The 390 bp DNA fragment from the normal individuals was cleaved into three fragments of approximately 230 bp, 130 bp and 30 bp. DNA samples from abnormal I and two daughters (abnormals I-2 and I-3) and a nephew (abnormal I-4) of abnormal I showed a mixture of 360 bp, 230 bp, 130 bp and 30 bp fragments. These results indicated that the abnormal patients were heterozygous for the Phe-355 mutation. Thus, this mutation can be diagnosed by the presence of a 360 bp Ava II fragment when DNA is selectively amplified using primers K4a-5' and K4a-3'. In a second series of experiments, DNA from abnormals I, I-2, I-3 and I-4 was amplified using primers K4a-5' and K4a-32 (5' AAA TGA ATT CCT AGG AAG TTG GCT TGA AGC 3'; noncoding strand primer). Digestion of the resulting ~370 bp fragment with Ava II confirmed the loss of an Ava II site in the abnormal DNA, and also confirmed the diagnosis of abnormals I, I-2, I-3 and I-4 as heterozygotes of the Phe-355 mutation.

Claims

Cla ims

1. A method of detecting the presence of a mutation in the plasminogen gene of a patient, comprising: amplifying a portion of genomic DNA from the patient, said portion including a predetermined exon comprising the site of a selected mutation and at least 14 base pairs of each of two intron sequences flanking said predetermined exon; exposing said amplified DNA to a restriction endonuclease capable of differentially cleaving DNA having the selected mutation and wild-type plasminogen DNA under conditions suitable for activity of the endonuclease; and analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation.

2. The method of claim 1 wherein the selected mutation is the Phe-355 mutation or the Thr-601 mutation.

3. A method of detecting the presence of a mutation in the plasminogen gene of a patient, comprising: a. denaturing genomic DNA from the patient; b. annealing the denatured genomic DNA to a pair of oligonucleotide primers, wherein the first primer is complementary to a first sequence of at least about fifteen consecutive nucleotides of a first intron on the coding strand of the genomic DNA, and wherein the second primer is complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, wherein said introns flank the exon comprising the site of a selected mutation; c. extending the annealed primers to produce double- stranded DNA fragments, said fragments including the site of the selected mutation; d. denaturing the double-stranded DNA fragments; e. annealing the denatured DNA fragments to the pair of oligonucleotide primers and extending the annealed primers to produce selectively amplified DNA; f. exposing said selectively amplified DNA to a restriction endonuclease capable of differentially cleaving DNA having the selected mutation and wild-type plasminogen DNA, under conditions suitable for activity of the endonuclease; and g. analyzing the exposed DNA to detect the presence or absence of cleavage fragments diagnostic for the selected mutation, wherein the selected mutation is the Phe-355 mutation or the Thr-601 mutation.

4. The method of claim 3 wherein the primers are extended using Taq DNA polymerase.

5. The method of claim 3 wherein each of said first and second primers is from about twenty to about thirty nucleotides in length, inclusive.

6. The method of claim 3 wherein said selected mutation is the Thr-601 mutation and said first primer includes the sequence CAA TTT AAC TAA AAT TTG AAC TAA AT or TGT ACA ATG GAG CAG AAC AAA.

7. The method of claim 3 wherein said selected mutation is the Thr-601 mutation and said second primer includes the sequence TCA TGT CTA CTA AAA CAC CCG GAC TTA or TCT CCT TTC TGT GTC ATG TCT A.

8. The method of claim 3 wherein said selected mutation is the Phe-355 mutation and said first primer includes the sequence GTC AGA ATT CTC AGA GGC TAC CGT ACT.

9. The method of claim 3 wherein said selected mutation is the Phe-355 mutation and said second primer includes the sequence CTA CGA ATT CTG GCT CTA ACA CAA ATT TCC or AAA TGA ATT CCT AGG AAG TTG GCT TGA AGC.

10. The method of claim 3, further comprising the step of isolating genomic DNA from the patient prior to the step of denaturing the genomic DNA.

11. The method of claim 3 wherein the endonuclease differentiates between G and A in the first position of the codon for amino acid 601 of plasminogen.

12. The method of claim 11 wherein the restriction endonuclease is selected from the group consisting of Fnu 4HI and Bbv I.

13. The method of claim 3 wherein said endonuclease differentiates between G and T in the first position of the codon for amino acid 355 of plasminogen.

14. The method of claim 13 wherein the restriction endonuclease is selected from the group consisting of Ava II and Sau 96I.

15. The method of claim 3 wherein the steps of denaturing comprise heat treatment of the DNA.

16. The method of claim 3 wherein approximately 300-400 bp of genomic DNA is amplified.

17. The method of claim 3 wherein steps d and e are repeated in sequence from about twenty-three to about twenty-eight times prior to step f.

18. A diagnostic kit for the rapid detection of the Thr-601 mutation in the plasminogen gene of a patient, comprising in suitable compartments within the kit: a pair of oligonucleotide primers, the first primer being complementary to a first sequence of at least about fifteen consecutive nucleotides of an intron on the coding strand of genomic DNA from a patient, the second primer being complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, the introns flanking the exon coding for amino acid residue 601 of plasminogen;

Taq DNA polymerase; control DNA; a restriction endonuclease capable of differentially cleaving Ala-601 plasminogen DNA and Thr-601 plasminogen DNA; and suitable buffers.

19. A diagnostic kit for the rapid detection of the Phe-355 mutation in the plasminogen gene of a patient, comprising in suitable compartments within the kit: a pair of oligonucleotide primers, the first primer being complementary to a first sequence of at least about fifteen consecutive nucleotides of an intron on the coding strand of genomic DNA from a patient, the second primer being complementary to a second sequence of at least about fifteen consecutive nucleotides of a second intron on the noncoding strand of the genomic DNA, the introns flanking the exon coding for amino acid 355 of plasminogen;

Taq DNA polymerase; control DNA; a restriction endonuclease capable of differentially cleaving Val-355 plasminogen DNA and Phe-355 plasminogen DNA; and suitable buffers.