EP1368492A2

EP1368492A2 - Coronary chip

Info

Publication number: EP1368492A2
Application number: EP02702408A
Authority: EP
Inventors: Paul Cullen; Udo Seedorf
Original assignee: OGHAM GmbH
Current assignee: OGHAM GmbH
Priority date: 2001-03-13
Filing date: 2002-03-13
Publication date: 2003-12-10
Also published as: WO2002072882A3; DE10111925A1; WO2002072882A2

Abstract

The invention relates to a method for determining the genetic risk of arteriosclerosis, comprising the steps, selection of reference nucleotide sequences relevant to the risk of arteriosclerosis, including functional characteristic mutation thereof, application of probes of said nucleotide sequences or the complementary sequences thereof on a support, hybridisation of the probes with nucleotide sequences from a patient sample, or with nucleotide sequences synthesised from a patient sample, verification of the hybridisation and evaluation of the hybridisation.

Description

"Heart smart"

The invention relates to a gene chip and a method for determining the genetic predisposition for the development of arteriosclerotic diseases and claims the priority of the German patent application 101 11 925.9, to which reference is made in terms of content.

Cardiovascular diseases are one of the most common causes of death worldwide. In the majority of cases, such diseases are due to calcification of the large and medium-sized artery (arteriosclerosis). Arteriosclerosis of the coronary arteries and carotid artery is of particular importance, as this can lead to a heart attack or stroke. Around 250,000 people currently suffer a heart attack in Germany, which in half of the cases is fatal. The heart attack often leads to considerable consequential damage and disabilities in the surviving patients. The frequency of stroke in Germany is around half of heart attacks; about a quarter of all cases are fatal. The consequential damage is often more devastating than with a heart attack.

Atherosclerosis is a complex clinical picture that goes back to multifactorial causes. Chemical and mechanical damage to the vessels is primarily caused, for example, by persistent nicotine consumption, metabolic disorders such as diabetes melitus or psychological disorders leading to adrenaline or noradrenaline releases. Chosomatic influences discussed as major causes. In addition, persistent high blood pressure and high blood cholesterol play a key role.

In addition, it was recognized more than 100 years ago that heart attacks and strokes increased in some families, so that heredity was considered to play an important role in the development of arteriosclerosis. However, it has only started in the last 10 to 15 years to understand the reasons for the familial accumulation of these diseases on a molecular basis.

Within medical diagnostics, the recording of risk factors for determining the risk of atherosclerosis is of great importance. For this purpose, biochemical analyzes, for example of the cholesterol or fat levels in the blood, are carried out and the patient is asked about lifestyle habits and diseases that occur frequently in the family. If the genetic risk factors are recorded at all, this is done by means of a complete sequencing of the corresponding nucleotide sequence of the relevant gene or by displaying the affected gene segments, for example by means of the allele-specific polymerase chain reaction, restriction length polymorphisms or the single strand conformation polymorphism method (SSCP).

A disadvantage of these methods for recording the genetic disposition is that they generally only record individual sequence changes, at most individual genes. In addition, they are often very labor-intensive and require special knowledge in both implementation and interpretation. Therefore, they are not suitable for examining a large number of people who may be affected. The invention is therefore based on the problem of providing a diagnostic option which allows a quick and meaningful diagnosis of the genetic predisposition to the disease of arteriosclerosis.

This problem is solved with a method according to the main claim and a nucleotide carrier according to the secondary product claim. Advantageous further developments are the subject of corresponding subclaims.

The invention is based on the idea of bringing a suitable selection of nucleotide sequences relevant to the genetic arteriosclerosis risk (or the sequences complementary thereto) as well as their functionally characterized mutations onto a carrier (hereinafter also referred to as a gene chip), these nucleotide sequences with a nucleotide sequence sample of a patient hybridize ducks and evaluate the hybridization multifactorial. The nucleotide sequences represent probes for so-called reference sequences whose indirect or direct functional connection with arteriosclerosis is known or suspected. If a probe hybridizes on the carrier with oligonucleotides from the patient sample, the evidence for the presence of the corresponding reference sequence in the patient is provided.

This simultaneous acquisition and multifactorial analysis of a large number of mutated sequences relevant to the risk of arteriosclerosis is accompanied by the particular advantage that any synergy effects between different mutations can be recognized and represented. It is therefore possible that individual mutations do not pose a significant risk on their own, but if they occur in combination with other mutations - which are in themselves of minor importance - this can result in a considerable genetic risk, which clearly outweighs the risk exceeds the sum of the individual mutations. This particularly applies to mutations that are discussed in connection with arteriosclerosis, without being able to assign a specific function to them at present (so-called candidate mutations).

These advantages are illustrated below using an example:

It has been known for some time that a high blood level of the fat particle lipoprotein (a) increases the risk of heart attack. It is also known that a factor V of the coagulation cascade can partly be present in a changed form. Only the combination of the mutated factor V with a high level of lipoprotein (a) leads to an unexpectedly large increase in the risk of blood clotting in the central vein of the brain and therefore an early, severe stroke. These synergy effects can only be detected by a suitable selection of the nucleotides and a simultaneous detection of the genetic changes.

The method according to the invention and the nucleotide carrier according to the invention thus permit a more precise diagnosis of the risk of arteriosclerosis, in particular when this method is combined with the detection of the conventional risk factors. A more precise diagnosis is an important prerequisite for new treatment methods that will allow specific treatment of genetic defects in the near future, such as gene therapy.

Another advantage of the invention is that all relevant mutations of the selected reference sequences can be recorded in a single operation, the implementation of which requires no special knowledge. This method is also suitable for automation and thus for the rapid processing of large series of samples. Thus, according to the invention, the simultaneous detection of relevant gene changes is possible inexpensively, quickly and reliably. However, the method according to the invention is not only suitable for clinical diagnostics but in particular also as an aid for scientific research. In this way, for example, population studies aiming to clarify the connection between genetic modification and the risk of heart attack or stroke can be facilitated, since a much higher number of study participants can be recorded compared to conventional studies.

Since the nucleotide carrier according to the invention allows the identification of interactions between mutations or mutation combinations and between mutations and conventional risk factors, for example from the family history, the metabolism or the blood cholesterol level, it can contribute to a better understanding of the development of arteriosclerotic phenomena. It is therefore particularly suitable as a research reagent.

The use of the nucleotide carrier according to the invention is based on the hybridization of the nucleotide sequences applied to the carrier with complementary nucleotide sequences from the patient's sample material. Since the selected nucleotide sequences represent probes for reference genes which are functionally relevant for the formation of arteriosclerosis, individual genes or parts thereof can be detected in a preparation of genomic DNA or the transcription product of a gene (mRNA) by means of hybridization. [Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed., Vol. 2, 1989, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 13.96-97; Constanzi C, Gillespie D: Fast blots: immobilization of DNA and RNA from cells. In: Guide to molecular cloning techniques. Edited by Berger SR and Kimmel AR, Academic Press Inc., San Diego: Methods in Enzymology 1987, 152: p582-87; Schena M et al .: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995; 270: p467-470]. The hybridization can be combined with a detection reaction and subsequent identification of a nucleic acid sequence. For this purpose, one strand of nucleic acid can be marked, for example, by dyes, radioactivity or chemiluminescent or fluorescent molecules, while the second is bound to a solid support, for example made of plastic or glass. The nucleotide sequence is preferably marked during the synthesis of the sequence from the patient sample. Several thousand oligonucleotides can be applied to the carrier as probes. The subsequent hybridization on the carrier can advantageously be evaluated using a scanner, so that the evaluation can be largely automated.

In the context of this application, the following terms are used as follows:

The term "hybridization" includes any form of pairing or juxtaposition of nucleotide sequences. The hybridization can take place both under stringent and under relaxed conditions.

The term "nucleotide sequence" refers to any oligomeric or polymeric sequence of ribonucleotides or deoxyribonucleotides or a combination thereof. Ribonucleotides or deoxyribonucleotides with base analogs are also included. Nucleotide sequences can be of synthetic or natural origin and can be traced back to recombinant processes.

The term "atherosclerosis risk" refers to any increase (or decrease) in the likelihood of developing atherosclerosis. The probability is measured by the average probability in the entire population. If the arteriosclerosis risk is due to genetic causes, the term genetic arteriosclerosis risk or genetic predisposition or just disposition is used. "Mutations" are understood to mean all forms of change in the genetic information of a nucleotide sequence in comparison with the wild-type sequence - including a native sequence. These can be substitutions, insertions or deletions, for example. More complex mutations such as inversions and indels are also possible.

The term "reference sequence" refers to certain nucleotide sequences of selected genes, where a gene can include a large number of reference sequences. The reference sequences can be native, i.e. in wild type formation, or be mutated. A connection between the mutations and the risk of arteriosclerosis is known or is suspected, i.e. the mutation is functionally characterized or relevant.

The term "probe" refers to specific oligonucleotide sections of a reference sequence or sequences complementary thereto. The hybridization of an oligonucleotide sample with a probe thus allows the detection of the reference sequence in the original tissue of the sample. The probes are applied to the nucleotide carrier.

In a preferred embodiment, probes for reference sequences of the genes listed in Table 1 below are applied to the carrier, the mutations of which directly or indirectly contribute to an increase in the genetic risk of arteriosclerosis. Alternatively, probes of sequences complementary to the reference sequences can also be used (hereinafter also complementary sequences). The genes are specified both by their code number from the GenBank of the National Center for Biotechnology Information (NCBI) and by the symbol used for them. The table also contains information on the frequency of genetic changes in these genes and the number of known mutations. Table 1

Genes Symbol Frequency of the number of disease traits Gene changes currently in the generally known population Gene changes (as far as known)

Lipolipoprotein genes A-I APOA1 Less than 28 reduction in high-density lipoprotein cholesterol.

1: 1,000

Apolipoprotein B 3500 APOB 1: 600 Like LDL receptor, somewhat milder.

Apolipoprotein B APOB 1: 2 13 Possible protection factor against heart attack and stroke. Apolipoprotein E APOE ApoE2: 10% of 26 At homozygosity for ApoE2: hyperlipidemia, carrier of a

Population; ApoE4- Alleis have an increased risk not only for AρoE4 heart attack: 20% of but also for Alzheimer's disease.

population

Apolipoprotein H APOH 1: 4 2 High triglycerides in women. Possible importance for blood clotting

Adenosine triphosphate- ABCA1 Less than 14 Severe decrease in high-density lipoprotein cholesterol binding cassettes 1: 10,000 level. Possibly increased risk of heart attack. transporter 1

Cholesterol Ester Trans CETP Less than 7 Elevated HDL Cholesterol Levels. Possible protection against Artefer protein 1: 10,000, except in riosclerosis. Japan

Cholesterol 27- CYP27A1 Less than 9 Very high risk of arteriosclerosis.

Hydroxlase (CTX) 1: 10,000

Hepatic Lipase LIPC Less than 1: 5 hypertriglyceridemia; possible increased risk of arterial 10,000 riosclerosis of the coronary arteries.

Lecithinxholesterin LCAT Less than 31 reduction in high-density lipoprotein cholesterol.

Acyltransferase 1: 10,000 corneal opacity and kidney dysfunction may occur.

Low-density Lipopro LDLR 1: 500 36 increase in blood cholesterol. Severe arteriosclerosis tein receptor before the age of 40 in men, before the age of 50 in women.

Lysosomal acid lipase LIPA 1: 750 25 Liver function disorders, high blood cholesterol, high triglycerides, low HDL cholesterol. Possibly increased risk of heart attack.

Lipoprotein Lipase LPL Less than 81 increase in blood triglyceride level. Probably a slight 1: 10,000 increase in myocardial infarction risk. Very high blood triglyceride levels can lead to severe, sometimes life-threatening, inflammation of the pancreas.

Microsomal Triglyze- MTP Unknown Possible protection factor against heart attack and stroke. rid transfer protein

blood pressure

Alpha-adducin ADD1 1:10 1 hypertension.

Beta2 adrenergic recep- ADRB2 1: 5 4 hypertension. goal

Angiotensinogen AGT 1: 3 8 hypertension.

Angiotensin receptor 1 AGTR1 1: 3 1 connection with high blood pressure. bleak of Differentiation CD36 1: 300 3 high blood pressure, fat metabolism disorder, arteriosclerosis.

36

Cytochrome P450 / 11 beta CYP11B1 Unknown, 1:50 in 25 hypertension.

Hydoxylase some populations

Angiotensin-Converting DCP1 1: 4 1 hypertension. Possibly increased risk of heart attack

Enzyme and stroke.

Dopamine receptor DRD2, variable, up to 1:10 4 Possible association with high blood pressure. DRD4

Guanine nucleotide GNB3 1: 2 1 hypertension. ending protein, beta 3

Hydroxysteroid-11-beta-HSD11B2 Unknown 17 Hypertension.

Dehydrogenase 2

Neurofibromatose-Fak- NF1 1: 3500 22 Neurofibromatosis type 1, high blood pressure. gate 1

Glucocorticoid receptor NR3C1 1:20 5 Severe high blood pressure.

Renin REN Unknown 2 Hypertension.

Voltage independent SCNN1B unknown 7 high blood pressure, sodium channel 1 beta Voltage independent SCNNIG unknown 2 high blood pressure. Sodium channel 1 gamma voltage independent SCNNIA unknown 2 high blood pressure, sodium channel 1 alpha

Adhäsionmoleküle

Glycoprotein la GPIBA, 1: 2 10 association with arteriosclerosis of the coronary arteries. GPIBB

Intercellular AdhesiICAM1 1:10 2 Possible association with coronary heart disease on molecule 1

Endothelial LeukoITGA2 Unknown 3 Association with Heart Attack Cyte Adhesion Molecule 1

Integrin-ß3 ITGB3 Unknown 23 Association with heart attack.

E-selectin SELE 1: 5 3 Possibly increased risk of heart attack.

P-selectin SELP 1: 5 1 Possibly protective factor against the development of arteriosclerosis.

Homocysteine metabolism

Cystathion-beta- CBS 1:10 88 Greatly increased risk of heart attack. synthase

Methyl tetrahydrofolate- MTHFR variable, some 12 high homocysteine levels, increased risk of arteriosclerosis homocysteine methyl polymorphisms of the coronary arteries. transferase reductase very common methionine synthase MTR 1: 7 1 Like MTHFR. Methyl tetrahydrofolate- MTRR 1: 3 20 Like MTHFR. reductase

coagulation

Antithrombin 3 AT3 1: 500 116 Increased coagulation.

Coagulation factor II F2 1: 20 17 Increased coagulation. Relation to atherosclerosis still unclear.

Coagulation factor V F5 1:20 12 Assured risk factor for thrombophilia and stroke

(especially together with increased Lp (a)).

Coagulation factor NU F7 Unknown: 1:50 in 40 B bleeding tendency. some populations

Coagulation factor VIII F8 1: 10,000 480 haemophilia A.

Coagulation factor IX F9 1: 30,000 667 hemophilia B.

Coagulation factor X F 10 Less than 29 Slight bleeding tendency.

1: 10.000

Coagulation factor XI F 11 Unknown: 1:10 in 20 Slight bleeding tendency. some populations

Coagulation factor XII F12 Up to 50% 10 Symptom-free when occurring on its own, combination effect unknown.

Coagulation factor XIIIA F13A Unknown, true38 Slight bleeding tendency. apparently less than 1: 10,000

Coagulation factor XIIIB F13B 1: 5 3 Slight bleeding tendency.

Fibrinogen alpha FGA Less than 1 in 9 Statistical relationship between polymorphism and Herzin5,000 farkt.

Fibrinogen beta FGB 1:10 8 Possibly increased risk of heart attack.

Fibrinogen gamma FGG Less than 1 in 13 Asymptomatic on its own, combination effect 5,000 unknown.

Heparin cofactor 2 HCF2 Unknown 3 Severe arteriosclerosis.

Lipoprotein (a) LPA 1: 5 3 Increased levels lead to severe arteriosclerosis with heart attack and stroke.

Plasminogen activator PAH 1: 8 2 Possibly increased risk of heart attack.

Inhibitor 1

Plasminogen PLG 1: 3 14 tendency to clot.

Protein C PROC 1: 30 190 Partially severe tendency to clot.

Protein S PROS Unknown 113 Coagulation tendency.

Tissue Factor Pathway TFP1 1:10 2 Coagulation tendency.

Inhibitor 1

Thrombomodulin THBD 1: 100 7 connection with heart attack described. von Willebrand factor VWF 1:50 117 Most common cause of a clinically important bleeding tendency.

obesity

Leptin LEP 1:10 4 mutation described, which leads to extreme obesity.

Leptin receptor LEPR 1: 3 1 Not related to obesity.

Diabetes mellitus

Glucokinase GCK 1: 100 56 Some mutations lead to diabetes.

Hepatocyte nuclear HNF1 1:30 67 Like glucokinase. factor 1

Hepatocyte nuclear HNF4A unknown like glucokinase. factor 4

Peroxisome prolifera- PPARG 1: 5 diabetes mellitus, lipid disorders. tion activating

Receptor-gamma

Peroxisome prolifera- PPARA unknown hypertriglyceridemia. Lowering HDL cholesterol. tion activating

Receptor alpha

cytokines

Stromelysin MMP3 1: 2 1 Possible association with atherosclerosis of the coronary arteries.

Tumor necrosis factor TNF Some are very 6 possible association with atherosclerosis. alpha common (1: 3)

The individual reference sequences of these genes are the subject of Table 2. The reference sequences can in turn be identified by their code numbers from the GenBank of the National Center for Biotechnology Information (NCBI) (hereinafter also GenBank).

The invention is explained in more detail below with reference to the following exemplary embodiments.

Example 1:

Detection of changes in the lipoprotein lipase gene

Probes of the complementary sequences of the reference sequences listed in Table 3 below are applied as oligonucleotides using standard techniques (DE 19612356; US5837832) to a suitable support, for example glass coated with gold. 10 to 25-mer nucleotide sequences, preferably 15-mers, are used. The sequences are chosen so that they include the functional mutations and the mutated complementary sequence (i.e. complementary sequence to the mutated reference sequence) is approximately in the middle relative to the native complementary sequence (i.e. complementary sequence to the native reference sequence).

The oligonucleotide probes are applied to the support in such a way that there is only one variant of the oligonucleotide in each case on the fields of the support. With regard to the known mutations in the lipoprotein lipase gene (LPL gene), there are 164 fields, with field 1.1, for example, the sequence 5'-CTGGAGCCACACGGC-3 '(complementary to the reference sequence 5'-GCCGTGTGGCTCCAG-3'; Table 2, line 2 1, column 2, underlined sequence underlined) and field 1.2 carries the mutated complementary sequence 5'-CTGGAGICACACGGC-3 '(complementary to the mutated reference sequence 5'-GCCGTGTGACTCCAG-3'; Table 2, line 1, column 3; mutation underlined) , For the assignment of fields 2.1 and 2.2 the Sequence information of row 2 of table 2 used accordingly etc.

Table 3 A) to H) contains information about the nucleic acid sequences, the complementary sequences of which are applied as probes to the gene chip (for the sake of clarity, only the central region of the 10- to 25-mers is shown, the position with reference to the native reference sequence is specified by specifying the codon number). The complementary sequence of the native reference sequence shown in the third column is applied to each reference sequence at a precisely defined location on the support. The codon numbers given relate to the native gene sequence with the GenBank number of NCBINM_000237.

Table 3:

A) Missense or nonsense mutations

Column 1 identifies the codon number. In the last column it is the

B) Splice variants

The location of the native reference sequences (derived from the gene sequence with GenBank numbers M29549, M94221, M15856, M76722, M94222) is shown in columns 1-3. Column 4 specifies the mutation.

C) Small deletions

The deleted nucleotides are underlined.

* Derived from the normal gene sequence with the GenBank numbers M29549, M94221, M15856, M76722, M94222.

D) Small insertions

E) Indels (insertions / deletions)

Codon No. * Deletion insertion 69 AAACTTGTGGccgcCCTGT GG ACAAG

The deleted bases are in lower case.

The mutations are derived from the normal gene sequence with the GenBank numbers M29549, M94221, M15856, M76722, M94222.

F) Larger deletions

The larger deletion consists in the deletion of 2,100 nucleotides including exon 9. The mutation was described at the level of the genomic DNA of Benlian (Journal of Lipid Research 1995, 36, page 356).

G) Complex rearrangements

The rearrangement consists in the insertion of approx. 2,000 nucleotides into the 6th exon or 6th intron. The mutation was determined at the level of the genomic DNA by Devlin et al. (American Journal of Human Genetics 1990, 46, page 112 ff).

H) Regulatory defects

* Relative to the starting point of the transcription of the LPL gene with the GenBank numbers M29549, M94221, M15856, M76722, M94222.

Example 2: Determination of the genetic risk of arteriosclerosis

Analogous to embodiment 1, a gene chip with preferably 15 to 18-mer oligonucleotide probes is produced. The probes are complementary to the known mutated or native reference sequences of the following genes listed in Table 4:

Table 4:

The number of hybridization sites and diagnosable diseases required is shown in Table 5. The known functionally relevant mutations are listed in the appendix.

Table 5: Assessment of the genetic predisposition to the development of a

A total of 6,298 hybridization sites are used in this exemplary embodiment for determining the genetic predisposition for arteriosclerosis on a DNA chip. The common presence of these oligonucleotide probes on a support allows the simultaneous detection of these sequence variations from a patient sample. Therefore, this DNA chip is particularly suitable for use in epidemiological studies that aim to research the relative importance of genetic factors for the risk of heart attack.

The functionally relevant mutations of the genes listed in Table 5 are contained in Table 6. These or their complementary sequences are applied to a nucleotide carrier.

List 3

point mutations

Spleißdefekte

Small deletions

1544 | ACTCC ^Λ TACATgGTGACCGTGG VWF

Other mutations

Claims

claims:

1. A method for determining the genetic arteriosclerosis risk comprising the following steps:

Selection of reference nucleotide sequences relevant to the risk of arteriosclerosis, including their functionally characterized mutations;

Applying probes of these nucleotide sequences or their complementary sequences to a support;

Hybridizing the probes with nucleotide sequences from a patient sample or with nucleotide sequences synthesized from a patient sample;

Detection of hybridization and

Evaluate the hybridization.

2. The method according to claim 1, characterized by a marking of the nucleotide sequence synthesized from a patient sample.

3. The method according to claim 1 or 2, characterized by a multifactorial evaluation of the hybridization.

4. The method according to any one of the preceding claims, characterized by probes made of 10- to 25-mer oligonucleotides.

5. The method according to claim 4, characterized by 15- to 18-mer oligonucleotide probes.

6. The method according to claim 5, characterized by 15-mer oligonucleotide probes.

7. The method according to any one of the preceding claims, characterized by one or more of the reference sequences given in Table 2 or their complementary sequences.

8. The method according to claim 7, characterized by a selection of the functional mutations indicated in Tables 3 and 6.

9. The method according to any one of claims 1 to 7, characterized by oligonucleotide probes which are at least 90% identical or complementary to a selection of the functional mutations given in Tables 3 and 6.

10. Nucleotide carrier with a selection of oligonucleotide probes which are identical or complementary to sections of reference sequences relevant for the genetic arteriosclerosis risk.

11. Nucleotide carrier according to claim 10, characterized in that the reference sequences represent a selection of the reference sequences listed in Table 2.

12. Nucleotide carrier according to claim 11, characterized by a selection of the oligonucleotide probes shown in Tables 3 and 6.

13. Nucleotide carrier according to one of claims 10 to 12, characterized by 10- to 25-mer oligonucleotide probes.

14. Oligonucleotide carrier according to claim 13, characterized by 15- to 18-mer oligonucleotide probes.

15. Nucleotide carrier according to claim 14, characterized by 15-mer oligonucleotide carrier.

16. Nucleotide carrier according to one of claims 10 to 15, characterized in that the carrier consists of glass coated with gold.

17. Use of the oligonucleotide carrier according to one of claims 10 to 16 for the simultaneous diagnosis of at least two genes relevant for the genetic arteriosclerosis risk.