DE20220351U1 - Nucleotide support, useful for assessing genetic risk of arteriosclerosis, comprises probes directed against nine risk-associated genes or their variants - Google Patents

Abstract

Nucleotide support (A) for determining the genetic risk of arteriosclerosis comprises oligonucleotide probes (I) for investigating nine specified genes (II) in a patient sample, and each (I) is at least partly identical with, or complementary to, a segment of (II).

Description

The invention relates to a gene chip for determining the genetic predisposition for the development of arteriosclerotic diseases.

Diseases of the cardiovascular system are one of the most common causes of death worldwide. In the majority of cases, such diseases are caused by calcification of the large and medium-sized arteries (arteriosclerosis). Arteriosclerosis of the coronary arteries and carotid arteries is of particular importance, as it can lead to heart attacks or strokes. In Germany, around 250,000 people suffer a heart attack each year, with half of the cases being fatal. Heart attacks often lead to significant consequential damage and disabilities in the surviving patients. In Germany, strokes occur in around half of the cases of heart attacks; around a quarter of all cases are fatal. The consequential damage is often more devastating than with a heart attack.

Arteriosclerosis is a complex disease that has multifactorial causes. It is primarily caused by chemical and mechanical damage to the vessels, for example through prolonged nicotine consumption, metabolic disorders such as diabetes mellitus or psychological disorders that lead to the release of adrenaline or noradrenaline.

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Cholestatic influences are discussed as major causes. In addition, persistent high blood pressure and high cholesterol levels in the blood play a significant role.

In addition, it was recognized over 100 years ago that heart attacks and strokes occurred more frequently within certain families, so that heredity was considered to play an important role in the development of arteriosclerosis. However, it was only in the last 10 to 15 years that we began to understand the molecular reasons for the familial clustering of these diseases.

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

The disadvantage of these methods for determining genetic predisposition is that they usually only detect individual sequence changes, or at most individual genes. In addition, they are often very labor-intensive and require specialist knowledge both in their implementation and in their interpretation. Therefore, they are not suitable for examining a large number of potentially affected people.

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The invention is therefore based on the problem of providing a diagnostic option that allows a rapid and meaningful diagnosis of the genetic predisposition to arteriosclerosis.

This problem is solved with a nucleotide carrier according to the main 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 risk of arteriosclerosis (or the sequences complementary to them) and their functionally characterized mutations onto a carrier (hereinafter also a gene chip), hybridizing these nucleotide sequences with a nucleotide sequence sample from a patient and evaluating the hybridization in a multifactorial manner. 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 on the carrier hybridizes with oligonucleotides from the patient sample, proof of the presence of the corresponding reference sequence in the patient is provided.

This simultaneous recording and multifactorial analysis of a large number of mutated sequences relevant to the risk of arteriosclerosis has the particular advantage that any synergistic effects between different mutations can be identified and presented. It is possible that individual mutations do not represent a significant risk on their own, but if they occur in combination with other mutations - which are again of little significance on their own - this can result in a considerable genetic risk that significantly exceeds the risk from the sum of the individual mutations. This is particularly true for mutations that are associated with arteriosclerosis.

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202 20 351.4 17 July 2003

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discussed, although no concrete function can currently be assigned to them (so-called candidate mutations).

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

A further advantage of the invention is that a large number of relevant mutations of the selected reference sequences can be detected in just one operation, which does not require any special knowledge. The nucleotide carriers are also suitable for automation and thus for the rapid processing of extensive sample series. The invention thus makes it possible to simultaneously detect relevant genetic changes inexpensively, quickly and reliably.

The nucleotide carriers according to the invention are not only suitable for clinical diagnostics, but also in particular as an aid for scientific research. For example, they can facilitate population studies that aim to determine the connection between genetic changes and the risk of heart attack or stroke, since a much larger number of study participants can be included in comparison to conventional studies.

Since the nucleotide carrier according to the invention enables the identification of interactions between mutations or mutation combinations as well as between mutations and conventional risk factors, for example from

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family history, metabolism or blood cholesterol levels, it can help to better understand the development of arteriosclerotic

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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 sample material from the patient. Since the selected nucleotide sequences represent probes for reference sequences of certain genes that are functionally relevant for the development of arteriosclerosis, individual genes or parts thereof in a preparation of genomic DNA or the transcription product of a gene (mRNA) can be detected by means of the 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, GiIIespie 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].

Hybridization can be combined with a detection reaction and subsequent identification of a nucleic acid sequence. For this purpose, one nucleic acid strand can be marked, for example, with dyes, radioactivity or chemiluminescent or fluorescent molecules, while the second is bound to a solid carrier, for example made of plastic or glass. The nucleotide sequence is preferably marked during 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 annealing of nucleotide sequences. Hybridization can occur under both stringent and relaxed conditions.

The term "nucleotide sequence" refers to any oligomeric or polymeric sequence of ribonucleotides or deoxyribonucleotides or a combination thereof. It also includes ribonucleotides or deoxyribonucleotides with base analogues. Nucleotide sequences can be of synthetic or natural origin or can be derived from recombinant processes.

The term "arteriosclerosis risk" refers to any increase (or decrease) in the probability of developing arteriosclerosis. The probability is measured against 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 changes to the genetic information of a nucleotide sequence compared to the wild-type sequence (also known as the native sequence). These can be, for example, substitutions, insertions or deletions. More complex mutations such as inversions and indels are also possible.

The term "reference sequence" refers to specific nucleotide sequences of selected genes, whereby a gene can include a large number of reference sequences. The reference sequences can be native, i.e. in the wild-type form, or mutated. A connection between the mutants

functions and the risk of arteriosclerosis is known or 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 to it. Hybridization of an oligonucleotide sample with a probe thus allows the detection of the reference sequence in the tissue of origin of the sample. The probes are applied to the nucleotide carrier.

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 be used (hereinafter also referred to as 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

Gene Gene-
symbol Frequency of genetic changes
changes in general
Population (as far as
known) Clinical picture Lipid metabolism genes Apolipoprotein B 3500 APOB 1:600 (binding defective apolipoprotein B)
elevated LDL cholesterol levels Apolipoprotein E APOE ApoE2: 10% of the population
ApoE4: 20% of
Population in case of homozygosity for ApoE2: hyperlipidemia,
Carriers of an ApoE4 allele have an increased
Risk not only for heart attack but also
for Alzheimer's disease. Cholesterol ester trans-
ferprotein CETP less than 1:10,000, except
in Japan increased HDL cholesterol levels; possible
Protection against arteriosclerosis. Cholesterol 7-alpha
Hydroxylase CYP7A1 rare allele in about 40%
the population High lipid levels. Possibly associated
treatment with coronary sclerosis Lipoprotein lipase LPL rare allele at about 8%
the population Increase in blood triglyceride levels.
slight increase in the risk of heart attack.
Very high blood triglyceride levels can lead to
a severe, sometimes life-threatening
Inflammation of the pancreas.

Homocysteine metabolism MTHFR variable, some polymorph-
pismen very common high homocysteine levels, increased risk
for atherosclerosis of the coronary arteries. Methyltetrahydrofolate-
Homocysteine Methyltrans-
ferase Reductase Coagulation PAIl rare allele at about 12%
the population Possibly increased risk of heart
farct. Plasminogen activator
Inhibitor-1 ITGB3 rare allele in 15% of
Population altered aggregation behaviour of the thrombo-
bocytes; possible correlation with increased
Arteriosclerosis risk Glycoprotein IHa Other PONl rare allele in about 40%
the population Association with a reduction in anti-
oxidative effect of HDL and a
increased risk of arteriosclerosis Paraoxonase 1

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

Table 2

No. 1. symbol gene GenBank sequence numbers APOB Apolipoprotein B J02610,, J02630, J02775, J04838, J05157, K03175, M10374, M12413, 2 . M12480,M12681 APOE Apolipoprotein E K00396, M10065, M12529, NM_000041, XOOl 99, X92000, 3. Z70760 CETP Cholesterol ester trans- AF027656, J02898, M30185, egg protein M32992, M32993, M32994, M32995, 4 . M32996, M32997, M32998 CYP7A1 Cholesterol 7-alpha L13460, M89803, M93133, 5. Hydroxylase NM 000780, X56088 LPL Lipoprotein lipase AF050163, M15856, M29549, M76722, M94221, M94222, NM_000237, X14390, Xl5736, 6. X54516 MTHFR 5,10-Methylene-Tetrahy- AF105977, AF105978, AF105979, drofolate reductase AF105980, AF105981, AF105982, AF105983, AF105984, AF105985, 7. AF105986, AF105987, U09806 PAIl Plasminogen activator- J03764, J03836, M14083, M16006, Inhibitor-1 Ml8082, M22313, M22314, M22315, M22316, M22317, M22318, M22319, M22320, M22321, M33136, M91557, X04429, X04729, X04731, X04744

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8th. ITGB3 Glycoprotein Ilia J02703, J05427, L28832, M20311,
M25108, M32673, M32686,
M35999, M57494, NM000212 9. PONl Paraoxonase AC004022, D84371, M63012,
M63013, M63014, NM000446,
S64615, S64696,
U53784,U55885

In the following, the invention is explained in more detail using two embodiments.

Example 1:

Probes of the complementary sequences of the reference sequences listed in Table 2 are applied as oligonucleotides to a suitable support, for example gold-coated glass, using standard techniques (DE 19612356; US5837832). 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 carrier in such a way that only one variant of the oligonucleotide is present on each field of the carrier.

Table 3 contains information on the nucleic acid sequences whose complementary sequences 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 respect to the native reference sequence

is specified by stating the codon number). For each reference sequence, the complementary sequence of the native reference sequence shown in the third column is applied to a precisely defined position on the carrier. The codon numbers given refer to the native gene sequence.

The number of required hybridization sites and diagnosable diseases can be derived from Table 1. The known functionally relevant mutations are listed in Table 3.

In this embodiment, a total of 410 hybridization sites are used on a DNA chip to determine the genetic predisposition to arteriosclerosis. The joint presence of these oligonucleotide probes on a carrier allows the simultaneous detection of these sequence variations from a patient sample. This DNA chip is therefore 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 1 are contained in Table 3. These or their complementary sequences are applied to a nucleotide carrier. For the sake of clarity, the mutations are listed in different "types".

Table 3:

A) Point mutations

No. Codon Sequence mutation amino acid gene 1 . 1306 tCGA-TGA Arg term ApoB 2 . 1424 GGT-GTT Gly-Val ApoB 3. 1450 aCAG-TAG Gln term ApoB 4. 1887 AAT-AGT Asn-Ser ApoB (Jl 1896 CAT-CGT His-Arg ApoB 6. 2058 tCGA-TGA Arg term ApoB

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7. 2153 aCAA-TAA Gln term ApoB 8th. 2252 cCAG-TAG Gln term ApoB 9. 2486 cCGA-TGA Arg term ApoB 10. 2495 cCGA-TGA Arg term ApoB 11. 2712 CCA-CTA Pro-Leu ApoB 12 . 3371 GCT-GTT Ala-Val ApoB 13 . 3405 cGAA-CAA Glu-Gln ApoB 14 . 3500 CGG-CAG Arg-GIn ApoB 15. 3500 aCGG-TGG Arg-Trp ApoB 16. 3531 aCGC-TGC Arg-Cys ApoB 17. 3750 TCA-TAA Ser-Term ApoB 18. 3894 cGTA-ATA Val-Ile ApoB 19. 13 cGAG-AAG Glu-Lys ApoE 20 . 20 TGGc-TGA Trp term ApoE 21. 28 CTG-CCG Leu-Pro ApoE 22 . 112 gTGC-CGC Cys-Arg ApoE 23 . 127 GGC-GAC Gly-Asp ApoE 24 . 136 gCGC-TGC Arg-Cys ApoE 25. 136 CGC-CAC Arg-His ApoE 26. 136 gCGC-AGC Arg-Ser ApoE 27. 142 gCGC-TGC Arg-Cys ApoE 28 . 142 CGC-CTC Arg-Leu ApoE

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29. 145 gCGT-TGT Arg-Cys ApoE 30. 145 CGT-CCT Arg-Pro ApoE 31. 146 tAAG-CAG Lys-Gln ApoE 32 . 146 tAAG-GAG Lys-Glu ApoE 33 . 158 gCGC-TGC Arg-Cys ApoE 34. 187 aCAG-GAG Gln-Glu ApoE 35. 210 TGG TAG Trp term ApoE 36. 228 cCGC-TGC Arg-Cys ApoE 37. 57 TAT-DAY Tyr-Term CETP 38. 181 gGGA-TGA Gly-Term CETP 39. 442 GAC-GGC Asp-GIy CETP 40. 33 CTG-CCG Leu-Pro ITGB3 41. 62 cCGA-TGA Arg term ITGB3 42. 117 TTG-TGG Leu-Trp ITGB3 43. 119 gGAC-TAC Asp-Tyr ITGB3 44 . 162 TCA-TTA Ser-Leu ITGB3 45. 214 CGG-CAG Arg Gin ITGB3 46. 214 aCGG-TGG Arg-Trp ITGB3 47. 280 CAT-CCT His-Pro ITGB3 48. 374 TGC-TAC Cys-Tyr ITGB3 49. 407 tCCC-GCC Pro-Ala ITGB3

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50. 542 gTGC-CGC Cys-Arg ITGB3 51. 560 TGT-TTT Cys-Phe ITGB3 52. 579 cGGC-AGC Gly-Ser ITGB3 53 . 616 gGAG TAG Glu term ITGB3 54 . 636 cCGT-TGT Arg-Cys ITGB3 55. 752 gTCT-CCT Ser-Pro ITGB3 56. 9 cGAC-AAC Asp-Asn LPL 57. 43 AAT-AGT Asn-Ser LPL 58. 61 TATg-TAA Tyr-Term LPL 59. 64 TGG TAG Trp term LPL 60. 69 tGTG-CTG VaI-Leu LPL 61. 73 TACa-TAA Tyr-Term LPL 62. 75 AGAg-AGC Arg-Ser LPL 63 . 86 cTGG-CGG Trp-Arg LPL 64. 86 cTGG-GGG Trp-Gly LPL 65. 98 cGCG-ACG Ala-Thr LPL 66. -14 TGGc-TGA Trp term LPL 67. 101 cACC-GCC Thr-Ala LPL 68. 106 aCAG-TAG Gln term LPL 69. 136 CAT-CGT His-Arg LPL 70. 139 tGGC-AGC Gly-Ser LPL

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71. 142 GGA-GAA Gly-Glu LPL 72 . 154 tGGC-AGC Gly-Ser LPL 73. 156 CGAT-AAT Asp-Asn LPL 74. 156 GAT-GGT Asp-Gly LPL 75. 156 cGAT-CAT Asp-His LPL 76. 158 aGCT-ACT Ala-Thr LPL 77. 163 GAG-GGG Glu-Gly LPL 78. 172 TCT-TGT Ser-Cys LPL 79. 176 tGCA-ACA Ala-Thr LPL 80. 180 GACg-GAG Asp-Glu LPL 81. 183 CACa-CAG His-Gln LPL 82. 188 aGGG-AGG Gly-Arg LPL 83. 188 GGG-GAG Gly-Glu LPL 84 . 193 aAGC-CGC Ser-Arg LPL 85. 194 ATT-ACT Ile-Thr LPL 86. 195 GGA-GAA Gly-Glu LPL 87. 204 GACa-GAG Asp-Glu LPL 88. 205 ATT-AGT Ile-Ser LPL 89. 207 CCG-CTG Pro-Leu LPL 90. 216 aTGT-AGT Cys-Ser LPL 91. 225 ATT-ACT Ile-Thr LPL

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92. 239 TGCt-TGA Cys term LPL 93. 243 gCGC-TGC Arg-Cys LPL 94 . 243 CGC-CAC Arg-His LPL 95. 244 cTCC-ACC Ser-Thr LPL 96. 249 ATC-ACC Ile-Thr LPL 97. 250 cGAC-AAC Asp-Asn LPL 98. 251 TCT-TGT Ser-Cys LPL 99. 252 CTG-CGG Leu-Arg LPL 100 252 tCTG-GTG Leu-Val LPL 101 259 AGTa-AGA Ser-Arg LPL 102 259 aAGT-GGT Ser-Gly LPL 103 261 gGCC-ACC Ala-Thr LPL 104 264 TGCa-TGA Cys term LPL 105 286 CTG-CCG Leu-Pro LPL 106 291 AAT-AGT Asn-Ser LPL 107 301 ATG-ACG Met-Thr LPL

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108 302 TACc-TAA Tyr-Term LPL 109 303 CTG-CCG Leu-Pro LPL 110 334 gGCC-ACC Ala-Thr LPL 111 365 CCTA-GTA Leu-Val LPL 112 382 TGGa-TGA Trp term LPL 113 410 aGAG-AAG Glu-Lys LPL 114 410 GAG-GTG Glu-Val LPL 115 418 TGT-TAT Cys-Tyr LPL 116 421 gGAG-AAG Glu-Lys LPL 117 447 TCA-TGA Ser-Term LPL 118 51 CGG-CCG Arg-Pro MTHFR 119 52 CGA CAA Arg-GIn MTHFR 120 116 cGCC-ACC Ala-Thr MTHFR 121 157 CGG-CAG Arg-GIn MTHFR

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122 183 cCGA-TGA Arg term MTHFR 123 222 GCC-GTC Ala-Val MTHFR 124 227 ACG-ATG Thr-Met MTHFR 125 251 CCC-CTC Pro-Leu MTHFR 126 323 CTC-CCC Leu-Pro MTHFR 127 324 AAC AGC Asn-Ser MTHFR 128 325 cCGC-TGC Arg-Cys MTHFR 129 335 gCGC-TGC Arg-Cys MTHFR 130 339 gTGG-GGG Trp-Gly MTHFR 131 357 gCGC-TGC Arg-Cys MTHFR 132 358 cCGA-TGA Arg term MTHFR 133 374 TACa-TAG Tyr-Term MTHFR 134 377 cCGT-TGT Arg-Cys MTHFR 135 387 GGC-GAC Gly-Asp MTHFR

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136 429 GCA-GAA Ala-Glu MTHFR 137 567 gCGA-TGA Arg term MTHFR 138 572 CCC-CTC Pro-Leu MTHFR 139 584 gAAG-TAG Lys term MTHFR 140 586 cGAG-AAG Glu-Lys MTHFR 141 55 cATG-TTG Met-Leu PONl 142 192 CAA-CGA Gl&eegr;-Arg PONl

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B) Promoter mutations

No. sequence position gene 1. CTTGGCCCCCAGAATGGAGGAGGGTGTC
TG(G>T)ATTACTGGGCGAGGTGCCCTC
CCTTCCTG -216 rela
tive to
Trans
scription
begin APOE 2 . GTTTCACCATGTTGGCCAGGCTGGTCTC
AA(A>T)CTCCTGACCTTAAGTGATTCG
CCCACTGTG -491 relay
tive to
Trans
scription
begin APOE 3 . GCAGCCAATGATCTCAGAGGCTGTATAC
CC(C>A)CCCAGAGTTATTTTATGCATA
TCAAGGAAA -629 rela
tive to
Trans
scription
begin CETP 4 . A>C -204 rela
tive to
Trans
scription
begin CYP7Al 5. ATAGCCAATAGGTGATGAGGTTTATTTG
CA(T>C)ATTTCCAGTCACATAAGCAGC
CTTGGCGTG -39 rela
tive to
Trans
crypt
onsstart LPL 6. TTTGCTCAACGTTTAGAAGTGAATTTA
GG(T>G)CCCCTCCCCCCAACTTATGATT
TTATAGCCA -93 rela
tive to
Trans
crypt
onsstart LPL 7. GGACAGCGTGG(G)GGAGTCAGCC 4G/5G Pro
engine PAIl 8th. T>C -107 rela
tive to
Start codon PONl 9. G>A -162 rela
tive to
Start codon PONl 10. G>A -830 relay
tive to
Start codon PONl 11. C>G -907 relay
tive to
Start codon PONl

C) Splicing defects

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number Intron-
No. Type d.
splice
Job position
d.
mutation Base-out
exchange gene 1. 5 ds + 1 G-T ApoB 2. 24 ds + 2 T-C ApoB 3. 3 as -2 A-G ApoE 4. 10 ds +2 T-G CETP 5. 14 ds + 1 G-A CETP 6. 9 as -6 G-A ITGB3 7. 9 ds + 1 G-T ITGB3 8th. 1 ds + 1 G-C LPL 9. 2 as -1 G-A LPL 10. 2 ds + 1 G-A LPL 11. 3 as -6 C-T LPL 12. 6 as -3 C-A LPL 13. 6 as -3 C-T LPL 14. 1 as -1 G-T MTHFR 15. 4 ds + 1 G-A MTHFR

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D) Small deletions

No. Position/Codon mutation gene 1. 1423 AGTCTCAAAAgGTTTACTAAT APOB 2 . 1727 TGACCACACAaacaGTCTGAACAT APOB 3 . 1793 CCCTGAAGCTgCATGTGGCTG APOB 4 . 1828 AGCAGACACTg t TGCTAAGGTT APOB 5. 2182 GAAAAATTAAaAAGTCTTGAT APOB 6 . 2356 CTACAACAAGt t aagATAAAAGATT APOB 7 . 3039 GTTAACAGGGaAGATAGACTT APOB 8th. 3385 CATAACAGTAcTGTGAGCTTA APOB 9. 3876 GTTTGAAAAAcAAAGCAGATT APOB 10 3942 CTGCTTTCAGgGAATGGGAAG APOB 11 4033 TTGGGAAGAAgAGGCAGCTTC APOB 12 30 ACTGGCACTGgGTCGCTTTTG APOE 13 141 CCCACCTGCGcaagctgcgTAAGCGGCTC APOE 14 208 CGGGCCCAGGcctggggcgaGCGGCTGCGC APOE 15. 210 GAAGAAGCAGagTGTGTCACGG ITGB3 16. 324 GTGAGCTCATcccaggGACCACAGTT ITGB3 17. 650 CTGGCAAGGAtgcagtgaattGTACCTATAA ITGB3 18. 17 TGCCCTAAGGacccctgaagaCACAGCTGAG LPL 19. 68 GCCAAAACTTgtGGCCGCCCTG LPL 20. 69 AAACTTGTGGccgcCCTGTACAAG LPL 21. 119 GAGGAGTTTAactaCCCTCTGGAC LPL 22. 220 CATTGGAGAAgCTATCCGCGT LPL 23 . 250 CTTCATCGACtcTCTGTTGAAT LPL 24 . 290 TATGAGATCAaTAAAGTCAGA LPL 25. 395 CTGGTGGAGCagtcccGGCTTCGCCA LPL

E) Other mutations

No. Type of mutation gene 1. Deletion of 694 bp including exon 21 APOB 2 . Duplication of 21 bp at codon 121-12 7 APOE 3 . Insertion T at exon3 / intron 3, position +3 CETP 4 . G>A in Intron 1 CETP 5. Deletion 11.2 kb including exon 10 to exon 13
(partial) ITGB3 6. Deletion of 1 kb, inversion of 15 kb ITGB3 7. Insertion at codon 34: A LPL 8th . Codon 69: deletion AAACTTGTGGccgcCCTGTACAAG;
Insertion gg LPL 9. Deletion of 2.1 kb including exon 9 LPL 10. Insertion of 2 kb in Ex. 6 / Intron 6 LPL 11. GC>TT at codon 149; G149V MTHFR 12. Insertion at nucleotide 4977 TA PAIl

Example 2:

If only a limited number of relevant mutations should or can be examined - or a limitation with regard to the mutations that undoubtedly correlate with an increased risk of arteriosclerosis - is to be made, the following polymorphisms are examined using the method according to the invention in a preferred embodiment (Table 4):

Table 4:

No. Codon Sequence-
mutation amino acid gene 1. 3500 CGG-CAG Arg->Gln APOB 2 . 3500 aCGG-TGG Arg->Trp APOB 3 . 3531 aCGC-TGC Arg->Cys APOB 4. 112 qTGC-CGC Cys->Arg APOE 5. 158 qCGC-TGC Arg->Cys APOE 6. - A-204C in
promoter - CYP7A1

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7 . 291 AAT-AGT Asn->Ser LPL 8th. 447 TCA-TGA Ser->Stop LPL 9. 22 GCC-GTC AIa->Val MTHFR 10. nucleotide
C-629A in
promoter CETP 11. G->A in
first
Intron CETP 12 . 222 C->T AIa->Val MTHFR 13 . - 4G/5G in
promoter - PAIl 14 . 33 CTG-CCG Leu->Pro ITGB3 15. 192 CAA-CGA Gln->Arg PONl

Claims (10)

1. Nucleotide carrier for determining the genetic risk of arteriosclerosis, characterized in that the nucleotide carrier has oligonucleotide probes for examining the genes from Table 1 from a patient sample, which are each at least partially identical or complementary to sections of the genes mentioned in Table 1. 2. Nucleotide carrier for determining the genetic risk of arteriosclerosis, characterized in that the nucleotide carrier comprises probes of 10- to 25-mer oligonucleotides. 3. Nucleotide carrier according to claim 1 or 2, characterized by 15- to 18-mer oligonucleotide probes. 4. Nucleotide carrier according to one of claims 1 to 3, characterized by 15-mer oligonucleotide probes. 5. Nucleotide carrier according to one of claims 1 to 4, characterized in that the oligonucleotide probes are each at least partially identical or complementary to reference sequences from Table 2. 6. Nucleotide carrier according to one of claims 1 to 4, characterized in that the oligonucleotide probes are each at least partially identical or complementary to mutations of Table 3. 7. Nucleotide carrier according to one of claims 1 to 4, characterized in that the oligonucleotide probes are each at least partially identical or complementary to polymorphisms of Table 4. 8. Nucleotide carrier according to one of claims 1 to 7, characterized by oligonucleotide probes with sequence segments which are at least 90% identical or complementary to a selection of the mutations indicated in Table 3 or 4. 9. Nucleotide carrier according to one of claims 1 to 8, characterized in that the nucleotide carrier comprises oligonucleotide probes for examining one or more of the following genes: cholesterol 7-alpha-hydroxylase (CYP7A1), plasminogen activator inhibitor-1 (PAI1) and paraoxonase 1 (PON1). 10. Nucleotide carrier according to one of claims 1 to 9, characterized in that it additionally comprises further probes, preferably those which are at least partially identical or complementary to the wild-type sequences of the respective reference sequence.