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WO2004020664A2 - Loci microsatellites polymorphes dans des genes pour l'etablissement d'un diagnostic prealable - Google Patents

Loci microsatellites polymorphes dans des genes pour l'etablissement d'un diagnostic prealable Download PDF

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Publication number
WO2004020664A2
WO2004020664A2 PCT/EP2003/008822 EP0308822W WO2004020664A2 WO 2004020664 A2 WO2004020664 A2 WO 2004020664A2 EP 0308822 W EP0308822 W EP 0308822W WO 2004020664 A2 WO2004020664 A2 WO 2004020664A2
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loci
gene
locus
polymorphic
prp
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PCT/EP2003/008822
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WO2004020664A3 (fr
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Hermann Geldermann
Siegfried Preuss
Yihua Han
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Universität Hohenheim
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Priority to AU2003266268A priority Critical patent/AU2003266268A1/en
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Publication of WO2004020664A3 publication Critical patent/WO2004020664A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to a typing method for genes which have at least one microsatellite locus, a method for determining markers in genes which contain microsatellite loci with regard to the predisposition of diseases and a ner method for pradiagnostics of diseases which are associated with genes which have one or more microsatellite loci.
  • Microsatellites are common candidates for such investigations. However, it was assumed that they occur on average approximately every 10 to 50 kbp in DNA and only rarely within genes (cf., for example, Schlötterer 2000).
  • TSE Transmissible Spongiform Encephalopathies
  • BSE bovine spongiform encephalopathy
  • CJD Creutzfeldt-Jakob disease
  • Pathogens of such encephalopathies e.g. scrapie (sheep, disease known for over 250 years), bovine spongiform encephalopathy (BSE; cattle), Creutzfeldt-Jakob disease (CJD; human) or related diseases contain essentially no nucleic acids (neither DNA nor RNA ), but rather have an infectious character due to their protein component.
  • This type of pathogen was given the name "Prion” (proteinaceous infectious particles, Prusiner 1982).
  • TSE diseases can be transmitted horizontally from animal to animal. Injection of an extract from the brain of a diseased animal into a healthy animal leads to infection with subsequent manifestation of the typical clinical symptoms.
  • scrapie the fact that nucleic acids can have no significant significance for the infection was demonstrated by experiments with scrapie fir extracts which remained infectious, even though they had previously been treated with UV light and with short-wave ionizing radiation. This regularly damages or destroys nucleic acids (Prusiner 2000). Based on these and similar experiments, other hypotheses regarding the nature of the pathogen of TSE have been pushed into the background, e.g.
  • PrP gene The gene coding for the P ⁇ on protein (PrP gene) was found in the genome of all examined mammals. Its product, the cellular Pdon protein (PrP c ), is expressed especially in nerve cells, but also in cells of the placenta. So-called. "Knock-out" mice, in which the PrP gene was genetically eliminated or switched off, showed no phenotypic finding after injection of infectious material (Büeler et al. 1992, 1993). The function of the PrP (e.g. Cu transport, Signal transmission) has not yet been finally clarified.
  • TSE is probably transmitted by an isoform of the normal cell-specific PrP c , which is referred to as PrP Sc (Prusiner 1998).
  • PrP Sc PrP Sc
  • the two forms differ in their conformation, i.e. in the way they are spatially folded.
  • Fourier transform infrared spectroscopy with highly purified PrP c indicated a high proportion of ⁇ -helix, whereas PrP Sc , i.e. the infectious form, a large proportion of ß-sheet Structure (Pan et al. 1993, Caughey et al. 1991, Gasset et al. 1993).
  • the PrP Sc can multiply in the host animal by catalyzing the conformational reversal from PrP c to PrP Sc and triggering a cascade with domino effect.
  • PrP Sc capable of agglomeration, which cannot be broken down by proteinase K (resistance to proteases).
  • amyloid-like deposits form.
  • the pathogenicity of the pathogen is due to the disruption and impairment of the nerve tissue.
  • the sheep's PrP gene was developed in 1998 by Lee et al. sequenced.
  • the 31 412 bp sequence is stored in GenBank under the accession number U67922.
  • the PrP gene of the sheep (FIG. 2) contains 3 exons with lengths of 52, 98 and 4 028 bp and 2 introns (2 421 and 14 031 bp).
  • the database entry also comprises a region of 5,665 bp 5'-wards of exon 1 and a 3'-sided region of 5,117 bp.
  • the 5 C -UTR of the RNA encoded by the ovine PrP gene is encoded by exons 1, 2 and 3 and consists of a total of 160 bp (FIG. 3).
  • the 771 bp DNA section that is translated is located in exon 3 and encodes 256 amino acids.
  • Exon 3 also encodes the sequence of the 3'-UTR.
  • the 3'-UTR of the ovine PrP mRNA with 3246 bp is much longer than the z. B of humans and mice (Lee et al.
  • BovB Line, Long Interspersed Element
  • Bov-tA2 / -tA3 SINE, Short Interspersed Element
  • Mariner Element Lee et al. 1998
  • the protein PrP in sheep consists of 256 amino acids, including the 24 amino acid signal peptide.
  • the 5 repetitions of a motif mostly comprising 8 amino acids (octapeptide) from position 54 (Fig. 4) are striking.
  • At least 11 polymorphic positions in the ovine PrP gene are known in the prior art (Table 1). In all of the animals examined, only one deviation from the wild type was found, i.e. h a mutated position. Since the amino acid glycine at position 171 can be substituted by both arginine and histidine, there are a total of 12 different allelic variants of the ovine PrP gene. TSE can only be transferred if there is homology between the PrP of the donor and the PrP of the host, i.e. the amino acid sequences have a minimum correspondence (Wilkens 1997). In this context, one speaks in the case of transfers between species of “species barrier” and in the case of transfers within one species of “polymorphism barrier”.
  • the genetic predisposition therefore plays an important role in the transmission of the disease and its pathogenesis (see also Manson et al. 2000). Pronounced effects of DNA variants within the PrP gene were found in humans in Gerstmann-St Hurssler-Scheinker syndrome (Hsiao et al. 1989) and hereditary Creutzfeld-Jakob disease (Palmer et al. 1991).
  • haplotypes VRQ and ARQ are associated with an increased susceptibility to scrapie.
  • animals with the PrP ARR / ARR genotype with the exception of one case in Japan, showed no signs of scrapie disease.
  • the rarer haplotypes AHQ and ARH are classified as a middle range in terms of scrapie susceptibility.
  • the genotype is of paramount importance for the risk of infection with TSE. This results in the need to determine the genotype in animals and humans at an early stage in order to assess the risk of infection.
  • the division of the genotypes into risk classes forms the basis for breeding programs.
  • animals with the resistant genotype ARR / AR are to be bred with gradual elimination of PrP alleles associated with a very high scrapie susceptibility (Drögemüller and DisÜ 2001).
  • genotyping in animals and humans is carried out by sequencing, a method which is relatively expensive and time-consuming.
  • the object of the present invention is now to provide methods which permit simpler, faster and, in particular, more cost-effective genotyping in humans and animals. Furthermore, it is an object of the present invention to provide methods which make it possible to deal with diseases, e.g. TSE, to find related gene markers for susceptibility to such diseases and to use them for quick and inexpensive prediagnostics.
  • diseases e.g. TSE
  • a method of typing a gene having one or more polymorphic microsatellite loci in an individual comprising the steps of:
  • step (b) determining the length of the amplificate (s) from step (a).
  • the genotyping method according to the invention is based on the fact known per se that in the genome of eukaryotes, sections occur which are comparatively short and consist of sequence motifs repeated several times.
  • the number of repetitions (also known as "Repeats") of the motif can differ from individual to individual in these sections labeled "microsatellites".
  • the number of repetitions therefore also changes the length of the respective microsatellite locus.
  • the number of sequence motif repetitions and thus the length of the microsatellite locus between individuals or races is different, the microsatellite locus is referred to as "polymorphic".
  • the typing method to be typed ie the gene to be analyzed for belonging to a group with regard to a certain property has at least one microsatellite locus, advantageously a plurality of microsatellite loci, the length of which differs / differs between different individuals of a species differentiate, ie is / are polymorphic.
  • the length and thus indirectly the number of repetitions of the sequence motif of the respective polymorphic microsatellite locus is determined.
  • a section or region of the DNA containing the gene to be typified, which comprises the microsatellite locus to be analyzed is amplified for each microsatellite locus by means of PCR, i.e. amplified.
  • a sample of the individual in question contains a corresponding DNA which comprises at least a section of the gene to be typed with the polymorphic microsatellite locus or loci.
  • the amplification is carried out according to the invention by PCR (“polymerase chain reaction”), using selected primers (short DNA oligonucleotides as starter molecules) according to the DNA section or region to be reproduced.
  • PCR polymerase chain reaction
  • primers short DNA oligonucleotides as starter molecules
  • the technique of PCR is well known to a person skilled in the art and is described, for example, in Griffin and Griffin 2001, the disclosure content of which in this regard is fully included in the present invention.
  • the sample of the individual already set out above serves as a template, specifically the DNA contained therein.
  • Amplification by means of PCR has the advantages which are characteristic of this method, in particular a high speed and the fact that even the smallest amounts of sample DNA are required, ultimately a single DNA molecule containing the corresponding section of the gene is sufficient.
  • DNA-containing materials of the individual concerned can be used as sample sources. These include, for example, blood, serum, secretions, such as sweat, ejaculate, etc., synovial fluid, amniotic fluid, excrement, cerebrospinal fluid, etc.
  • secretions such as sweat, ejaculate, etc.
  • synovial fluid such as sweat, ejaculate, etc.
  • amniotic fluid such as sweat, ejaculate, etc.
  • individual cells or fragments thereof for example individual skin cells, hair root cells, which are preferably attached to torn or - fallen hair, etc. can be used.
  • the expression “gene which has one or more microsatellite loci” means that the section of the genome in question which contains the gene to be typed contains at least one microsatellite locus, this section in particular exon and intron regions of the gene and those associated with the gene , in particular inherited with it, 5 'and 3' regions, for example those regions which are involved in the regulation of the gene in question.
  • the size of the amplified DNA section or region is not particularly restricted according to the invention.
  • lengths of approximately 50 to approximately 500 bp, in particular approximately 50 to approximately 300 bp, are preferred.
  • step (b) of the genotyping method according to the invention the length of the ampH ficate or the amplificates obtained in step (a) is determined.
  • electrophoresis ie the separation of the amplified products with the aid of an electrical field, is advantageously carried out.
  • a gel in particular agarose or polyacrylamide gels, the production of which is familiar to a person skilled in the art, is preferably used as the separation medium; see. For example, Maniatis 2001.
  • Particularly preferred when the amplificates are from about 50 to about 300 bp in length, their separation in gels, which enable the fragments to be separated down to a few or even 1 bp.
  • Such gels are also used, for example, in the sequencing of DNA molecules.
  • the number of repetitions of the respective sequence motif is a rough indicator of the degree of polymorphism of the microsatellite locus.
  • microsatellite loci are generally all the more informative, that is, the more individual microsatellite loci are present, that is, the more individuals there are, the more the number of sequence motif repetitions in the microsatellite locus fluctuates between the individual individuals. Accordingly, it is preferred according to the invention if the polymorphic microsatellite locus has a sequence motif which is repeated at least three times, more preferably at least four times, particularly preferably at least five times.
  • Polymorphic microsatellite loci can be mentioned as examples, which are selected from the following consensus sequences: (ATA) 7 , (GA) 7 , (GT) 28 , (AGG) 4 , (AC) 5 , (TTTGT) 5 , ⁇ TA) 7 , (TC) 5 , (TAAA) 12 , (ATTTT) 12 , (TTTC) 18 , (CA) 18 , (CAGTT) 4 , (AGC) 5 , (GA) 5 , ⁇ TTTGT) 4 , CTCAGT) 4 , ( TTCAG) 4 , (ACTGA) 3 , (ACTGA) 4 , (CA) 12 , (ATC) 4 , (I) 25 , CTTG) 5 , (T) 29 , (CAA) 3 , (TTCAG) 3 , (AGTG ) 4 , (CAC) 5 , (AAAACA) 4 , (ACC) 4 , (ATCAG) 5 , (AC) 3 , (
  • the typing method according to the invention can be carried out for all genes which have at least one polymorphic microsatellite locus.
  • its sequence and its position and position in the gene to be typed can already be known.
  • the polymorphic microsatellite locus / loci can be determined in a preferred embodiment of the method according to the invention.
  • the identification of the polymorphic microsatellite locus / loci is preferably carried out as follows:
  • a first step (A) the gene to be typed is screened for microsatellite loci, ie screened.
  • Genomic sequence information relating to a large number of genes is known in the prior art and is stored in particular in the databases of GenBank and the EMBL under corresponding access numbers. For example, if only one cDNA sequence is available or only EST sequences, it is of course possible for a person skilled in the art to obtain the required genomic sequence information using the conventional molecular biological methods; see also Maniatis et al. 2001.
  • the genomic sequence is preferably screened for microsatellite loci using appropriately designed computer programs.
  • the programs can be found in the program package EMBOSS (from HGMP-RC, London), Sputnik (Abijian 1994) and Software RepeatMasker (Smit and Green, http :: //ftp.genome. Washington.edu/cgi- bin / repeatmasker).
  • EMBOSS from HGMP-RC, London
  • Sputnik Abijian 1994
  • Software RepeatMasker Smit and Green, http :: //ftp.genome. Washington.edu/cgi- bin / repeatmasker.
  • step (B) analogous to step (a) above of the genotyping method according to the invention, the microsatellite loci corresponding to the gene obtained during screening of the gene (one DNA section or area per microsatellite locus obtained) are obtained in a plurality of Individuals of the organism containing the gene to be typified were amplified by PCR, ie reproduced. With regard to the amplification, the statements made above for step (a) of the above typing method also apply.
  • step (C) analogously to step (b) of the typing method according to the invention - the length of the ampH ficate or the amplified products which were obtained in each individual is determined.
  • step (b) of the genotyping method according to the invention apply accordingly to the determination of the length of the AmpH ficates.
  • sequencing of the respective AmpHfikate or a section thereof, which, however, must contain at least the microsatellite locus itself can also be carried out. Suitable sequencing methods are well known to a person skilled in the art and can be reacted in particular by means of automated sequencing devices (eg ALF from Pharmacia).
  • the lengths of the AmpHfikate between the individuals or the nucleotide sequences obtained are compared.
  • the microsatel locus / loci is determined whose ampHficate (s) has a different length between the individuals or whose nucleotide sequence (s) is / are different between the individuals.
  • These microsatellite loci which differ between the individuals (with respect to which different axles therefore exist), are thus polymorphic.
  • the most different genes can be typed with the aid of the typing method according to the invention. The only requirement is that the gene to be typed contains at least one polymorphic microsatellite locus.
  • the typing method according to the invention is preferably used for genes which are directly or indirectly associated with the expression of a disadvantageous or advantageous characteristic of an organism.
  • genes are to be mentioned here in particular which are associated with a disease or other characteristics which are of medical or economic importance (for example in (useful) animals).
  • PrP BSE disorders such as scrapie in sheep, BSE in cattle, Creutzfeld-Jakob disease in humans
  • CFTR cystic fibrosis
  • genes associated with metabolic disorders in humans and animals such as ⁇ -Manosidosis, Pompe disease, citrullinemia and bovine leukocytic adhesion deficiency in cattle (each a defective gene, the variants of which can be distinguished at the DNA level), susceptibility to stress (MaHgnes hyperthermia Syndrome) in pigs (RYRI gene, variant T), genes that encode milk proteins, hormones or transcription factors, etc.
  • microsatellite loci can be found in all eukaryotic genes. Due to economic and scientific considerations, preferred examples of organisms include To name humans, monkeys, horses, sheep, cattle, pigs, goats, mice, rats, rabbits, guinea pigs, dogs, cats and chickens.
  • a particularly preferred target gene of the method according to the invention is the PrP gene already described above, which is associated with TSE diseases which occur in humans and other mammalian species.
  • the genotyping method according to the invention is applied to the human PrP gene, the sequence motif (s) of the polymorphic microscopic locus / loci being selected from the following consensus sequences: (ATA) 7 , (GA) 7 , ( GT) 28 , (AGG) 4 , (AC) 5 ,
  • the consensus sequences (ATA) 7 , (GA) 7 , (GT) 28 , (TAAA) 12 , (ATTTT) 12 , (CA) 18 , (A) 16 and (A ⁇ ) are particularly preferred.
  • the amplified DNA region (s) in step (a) of the genotyping method according to the invention contains nucleotides 3621 to 3641, 25415 to 25428, 39450 to 39505, 50315 to 50326, 58346 to 58385, 72392 to 72416, 78300 to 78313, 92615 to 92624, 108429 to 108476, 116907 to 116966, 125528 to 125599, 147980 to 148015, 55617 to 55632, 55806 to 55819, 64328 to 64349 and / or 64474 to 64489 and 69764 to 69886 according to GenBank accession number AL133396.
  • DNA regions for amplification according to step (a) are preferred which contain the nucleotides 3621 to 3641, 25415 to 25428, 39450 to 39505, 108429 to 108476, 116907 to 116966, 147980 to 148015, 64328 to 64349 and / or 64474 to 64489 according to GenBank accession number ALI 33396.
  • Preferred sequence motifs of the polymorphic microsatten locus / loci of the bovine PrP gene have the following consensus sequences: (CAGTT) 4 , (TC) 5 , (AGC) 5 , (GA) 5 , (TTGT) 4 , CTCAGT) 4 , T ⁇ CAG) 4 , (ACTGA) 3 , (ACTGA) 4 , (CA) 12 , (ATC) 4 , (T) 25 , (ITG) S - (T) 29 , (CAA) 3 , (TTCAG) 3 , ( AGTG) 4 , (CAC) 5 , (AAAACA) 4 , (ACC) 4 , (ATCAG) 5 and (AC) 3 .
  • the consensus sequences (AGC) 5 , (TTTGT) 4 , (CA) 12 , (T) 25 , (T) 29 , (TTCAG) 3 , (AAAACA) 4 and (ATCAG) 5 are particularly preferred.
  • DNA regions are preferably amplified which contain the nucleotides 444 to 463, 4121 to 4130, 7615 to 7629, 19808 to 19817, 25288 to 25307, 30633 to 30652, 32320 to 32339, 37891 to 37910, 39943 to 39957, 44220 to 44239, 44507 to 44530, 46259 to 46278, 48409 to 48420, 50471 to 50495, 53219 to 53233, 54990 to 55018, 60452 to 60460, 62703 to 62717, 64685 to 64700, 66178 to 66192, 68762 to 68785 68926 to 68937, 72474 to 72498 and / or 74333 to 74340 according to GenBank accession number AJ298878.
  • the amplified DNA regions of the bovine PrP gene comprise nucleotides 7615 to 7629, 25288 to 25307, 44507 to 44530, 50471 to 50495, 54990 to 55018, 62703 to 62717, 68762 to 68785 and / or 72474 to 72498 according to GenBank accession number AJ298878.
  • the gene to be typed is the PrP gene of the sheep.
  • the polymorphic microsatel loci preferably have the following sequence motifs (consensus sequences): (CAGTT) 4 , (TC) 5 , (AGC) 5 , (GA) 5 , CTTTGT) 4 , (TCAGT) ,, (ACTGA) 3 , (ACTGA ) 4 , (CA) 12 , (CTG) * (ATC) 4 , (GTT) 5 , (TTG) 5 , (T) 1S , (CAA) 3 , (CAA) 4 , (TTCAG) 3 , (AGTG) 4 , (CAC) 5 , (ACC) 4 and (AC) 4 .
  • Microsatelten loci in the sheep's PrP gene with the consensus sequences (GA) 5 , (TTTGT) 4 , (CA) 12 , (GTT) 5 , (T) 15 and (CAA) 4 are particularly preferred.
  • the DNA regions amplified according to step (a) above comprise the nucleotides 301 to 320, 590 to 613, 1033 to 1047, 2477 to 2496, 4651 to 4662, 6627 to 6641, 9433 to 9447, 11277 to 11291, 16748 to 16756, 18100 to 18119, 19372 to 19386, 21383 to 21398, 22853 to 22867, 25422 to 25433, 25580 to 25591 and / or 30168 to 30175 according to GenBank accession number U67922, as well as seven other genomic DNA areas that are 5 'of the in U67922 sequence is located and for which no GenBank entry exists.
  • DNA regions of the sheep PrP gene which are particularly preferably amplified comprise nucleotides 590 to 613, 6627 to 6641, 11277 to 11291 and / or 25422 to 25433 according to GenBank accession number U67922, and seven further genomic DNA regions which are 5 'of the are located in the sequence disclosed in U67922 and for which there is no GenBank entry.
  • microsatuites are available for the first time, particularly in the PrP gene of humans, cattle and sheep, which differ between the individuals of the individual species, i.e. are polymorphic, so there are several different oils related to this microsatellite loci.
  • the genotyping method described above is used in a method which is suitable for determining microsatellite markers for the predisposition of an individual to a disease, the disease being associated with a gene which has one or more polymorphic microsatel loci having.
  • a first step (i) of this identification method the corresponding gene is typed in a plurality of individuals according to the typing method described above.
  • this typing method reference is made to the above statements in the first aspect of the invention.
  • Each of the plurality of individuals is known, for example, to have or not to have the disease, for example the diseases mentioned above. Additionally or alternatively, for example from examinations of the respective known in the prior art
  • ORF polymorphisms e.g. ORF polymorphisms
  • step (i) At least 100, particularly preferably 500 or more individuals are genotyped.
  • the genotype frequencies for each polymorphic microsatelite locus in the individuals who are known to be ill or not or have a certain degree of predisposition to the disease are determined.
  • the polymorphic microsatel loci has the greatest statistical significance for the characteristic "sick” or “not sick” or for the known specific degree of predisposition, on the basis of the genotype frequencies determined in the previous step (H) . If, for example, an expression of the polymorphic microsatellite locus is found in healthy individuals, ie individuals who do not suffer from the disease in question, and the frequency of this genotype, ie this allele, is increased or particularly high in non-diseased individuals, the polymorphic microsatellite locus in question becomes a marker be suitable for a lack of predisposition to the disease and thus be selected.
  • a variant, ie an AHel, of a microsatellite locus, which is found exclusively or frequently in diseased individuals, is selected as a microsatellite marker for an increased predisposition to the disease in question.
  • AHel a variant of a microsatellite locus, which is found exclusively or frequently in diseased individuals.
  • the specific degree of predisposition can also be related according to the invention on the basis of other genetic markers which are known to correct with a specific degree of predisposition.
  • the ORF polymorphism known in the ovine PrP gene may be mentioned here.
  • ORF polymorphism in the ovine PrP gene known in the prior art, it can be assumed on the basis of the genotyping of numerous sheep of various breeds from various breeds and in different countries that the ORF AUele (ORF: open reading fiame, open reading frame (ie variants in protein-coding gene regions) VRQ and ARQ are associated with a greatly increased scrapie receptivity (ORF: open reading fiame, open reading frame (ie variants in protein-coding gene regions) VRQ and ARQ are associated with a greatly increased scrapie receptivity (ORF: open reading fiame, open reading frame (ie variants in protein-coding gene regions) VRQ and ARQ are associated with a greatly increased scrapie receptivity (ORF: open reading fiame, open reading frame (ie variants in protein-coding gene regions) VRQ and ARQ are associated with a greatly increased scrapie receptivity (ORF: open reading fiame, open reading frame (ie variants in protein-coding gene regions)
  • ARR / ARR genotype a particularly high scrapie resistance (with the exception of one observation in Japan, sheep with the ARR / ARR genotype were never diagnosed with scrapie disease).
  • the other ORF oils described are arranged in terms of scrapie susceptibility in between (see Dawson et al. 1998, Hunter et al. 2000).
  • the above-described polymorphic microsat position in the ovine PrP gene is in the region of this gene (provided that the ovine PrP gene has no insertions against the bovine PrP gene in this region, the most 5 'located microsatellite locus is only 29 kbp away from exon 1; the most distant microsatel focus lies even in exon 3) and its inheritance is therefore very closely linked to that of the ORF variants. Coupling breaks will therefore occur so rarely that they cannot be observed within races.
  • Another aspect of the present invention relates to a method for pre-diagnosing diseases.
  • the diseases are associated with a gene that has at least one polymorphic microsatten locus.
  • the prediagnostic method of the present invention initially again comprises a typing step (1) which is carried out with the aid of the genotyping method defined above.
  • the gene of an individual to be examined is related to one or more Microsatel locus / loci, which is a marker for the degree of predisposition to the respective disease.
  • the genotype obtained is then compared with the degree of predisposition to this disease that is assigned to this genotype.
  • the microsatelite markers are preferably determined with the aid of the identification method described above.
  • the statements made above from the viewpoint of the present invention apply.
  • the prediagnostic method according to the invention is used in particular for TSE prediagnostics, the human PrP gene, the bovine PrP gene and the ovine PrP gene being particularly preferred.
  • polymorphic microsattenoid loci are suitably used, which have one of the sequence motifs given above with reference to the typing method according to the invention.
  • Preferred amplified DNA regions in TSE diagnostics in humans are also given above from the point of view of the typing method according to the invention.
  • polymorphic microsatten loci are preferred which have the sequence motifs given above with regard to the typing method according to the invention.
  • DNA regions which are preferably amplified here comprise the positions defined above with regard to the typing method of the present invention.
  • scrapie prediagnostics sheep's PrP gene
  • polymorphic microsatel loci with sequence motifs are advantageously used, the consensus sequences of which have also been raised above in the typing process.
  • preferred positions of such microsatel loci in the sheep PrP gene which are suitably amplified in the genotyping carried out initially in the pradiagnostic method according to the invention, reference is also made to the above explanations regarding the typing method of the present invention.
  • 1 shows the tertiary structure of the prion protein (from: Pringle: BSE, Scrapie, CJD and the Prion Protein, http://www.uni-mainz.de/ ⁇ cfrosch/bc4s/prions.html).
  • the original form (PrP c ) is shown in FIG. 1 a
  • the pathogenic form of the protein in scrapie is shown in FIG. 1 b.
  • Fig. 3 shows the structure of the transcript of the ovine PrP gene (from Geldermann et al. 2002).
  • Figure 4 is a representation of the ovine PrP primary sequence (Geldermann et al., 2002).
  • the PrP protein of sheep therefore consists of 256 amino acids, including an N-terminal signal peptide of 24 amino acids.
  • 1J denotes a 5-fold repetition (5 ⁇ R) of a motif from 8th or 9 or 5 amino acids (PQGGGGWGQ, (PHGGGWGQ) 3 or PHGGG) which is conserved in the PrP protein.
  • the respective amino acid and its position of the PrP-AUele ⁇ ) are given (see Tab. 1).
  • the darkly marked amino acid variants were used in association analyzes with scrapie receptivity.
  • Fig. 5 the MicrosatelHtenloci in the ovine and bovine Prp gene are shown schematically. Microsatellite motifs with> 3 repeats (repetitions) and> 90% homology were identified with the help of the program etandem of the EMBOSS program package. Additional microsatuites were obtained using the information in Lee et al. (1998) (marked with *), the Sputnik program (Abajian 1994) (marked with *) or the RepeatMasker program (marked with 3 )). If a microsatellite focus was found in only one species with these measures, the homologous control in the DNA of the other species was also searched. Microsatelten loci that were identified using this method are marked with 4 ).
  • the reference scale in the middle refers to the sequences stored in GenBank (cattle: accession number AJ298878; sheep: accession number U67922).
  • GenBank accession number AJ298878; sheep: accession number U67922.
  • the 5 " ends of the ovine and bovine exons 3 are aligned with one another.
  • the dotted line in the sheep gene shows an area in relation to which no sequence information is available in GenBank, whereby only estimated positions of the microsatel loci are given. In both species homologous microsatel loci are given by the same numbers characterized.
  • the loci SOI to S06 and S09 in sheep were examined with primers designed for the PrP sequence in cattle.
  • the primers for the loci in cattle R07 and R08 gave no PCR products in sheep.
  • FIG. 6 shows an example of a comparison of all DNA sequences of selected polymorphic microsateloid loci.
  • the respective sequence from GenBank is given as reference (R) in the first line.
  • the different residues are marked in the oil sequences.
  • FIG. 6A shows the comparison of all DNA sequences for the Sll locus in the oval PrP gene.
  • 6B shows a corresponding comparison for a further polymorphic microsatten locus locus in the sheep's PrP gene (S15).
  • FIG. 6C shows a comparison of all DNA sequences of the R21 locus in the bovine PrP gene.
  • FIG. 7 shows a diagram in which the number of microsatellite motifs per kbp in the PrP gene of cattle and sheep (black bars) with the corresponding values in other genes (heUe bars) for different gene areas, namely exon, intron and 5 ' - Area, as well as the entire gene area are shown. What is striking is the significantly increased density of repeat DNA in exons compared to other genes in the ovine and bovine PrP gene (p ⁇ 0.05). The detailed data with respect to Fig. 7 are given in Table 6 described below.
  • Fig. 9 shows examples of electropherograms of the MicrosateUitenlocus S15.
  • Lane 1 shows a homozygous, lane 2 and 3 heterozygous genotypes.
  • Lane 10 shows examples of electropherograms from the MicrosateUitenlocus Sll. Lanes 1, 2, 3 and 6 show different animals with a heterozygous genotype, lanes 4 and 5 represent homozygous genotypes.
  • FIG. 11 shows examples of electropherograms of the S24 locus.
  • Lane 1 and 2 show homozygous, lane 3 a heterozygous genotype.
  • a PCR product appears in lane 4 with the length 152 in combination with the allele 147, in lane 5 it can be observed together with the alleles 144 and 147.
  • Fig. 12 is an AHgnment of the 3 sequenced S24 oils with a section of the corresponding database entry in GenBank.
  • the microsate unit of the database sequence consists of 4 repetitions of the CAA motif.
  • the 147 bp long nobility of sheep S102 corresponds to the database entry except for a point mutation from A to C. Due to this mutation, the allele 147 contains one more repetition of the motif CAA with the same length as the database sequence.
  • the microsateUit of AUels 144 from Schaf 33 consists only of 3 repetitions of the motif, which makes this fragment 3 bp shorter than the GenBank AUel.
  • an SNP from C to T occurs at position 57 (based on the GenBank sequence).
  • the AUel with fragment length 152 (sheep 311) is identical to AUel 147 in terms of microsatellite. However, between positions 29 and 30 there is a 5 bp insert. In addition, an A occurs at position 103 instead of a T.
  • FIG. 13 shows a schematic representation of the positions of the microsatellite loci in the human PrP gene.
  • Microsate motifs with> 4 repetitions (repeats) and> 90% homology were identified using the program etandem of the EMBOSS software package. 12 of these identified MicrosatelHtenloci (M01 to M12) over the entire Sequence distributed were selected for more detailed analysis.
  • 4 monorepeats (MM1 to MM04) and the octapeptide region MOct were genotyped in external samples. The polymorphic loci are highlighted by white letters on a black background. The detailed data of the microsatellite loci are given in Tables 7A and 7B.
  • Fig. 14 shows examples of all DNA sequences from human microsatel loci.
  • FaU of locus M02 different outputs arise due to a variable number of repeats (A).
  • At locus Mll there are different oils due to single nucleotide exchanges ("single nucleotide polymorphism", SNP), insertions and deletions within the repeat region (B).
  • SNP single nucleotide polymorphism
  • the sequences of the M12 locus differ by an AU-specific SNP within the repeat region and 2 SNPs in the flanking region of each AU (C).
  • Tables 1 to 26 in the appendix are referenced to the corresponding control units in the present description. In these tables, the principles on which the present invention is based and the results obtained in accordance with the following exemplary embodiments are given.
  • Tab. 1 summarizes the amino acid polymorphisms found in the PrP gene of sheep (according to Hunter 2000, supplemented).
  • Tab. 2 shows the risk classification (risk) due to the amino acid polyoiorphisms in positions 136, 154 and 171 of the sheep PrP, as it was carried out by the "Scrapie Information Group” (Dawson et al. 1998).
  • Table 3A shows the data from 24 microsatten loci with 3 or more motif repeats found in the bovine PrP gene.
  • Tab. 3B shows the corresponding data of the 23 microsatlten loci found in the PrP gene of the sheep. 9 primer pairs specific for the bovine DNA were also tested with sheep DNA as a template in order to analyze the previously unpublished region of the sheep sequence (about 44 kbp). Using this procedure, 7 microsatel loci could be identified in the sheep PrP gene. In the DNA area, in which both sheep and cattle Sequence information is available in GenBank, 13 of 16 of the loci examined in the sheep homologous microsate motifs were found in the bovine DNA sequence. These motifs consisted of 4-5 repetitions, in most cases with different sequences. Mononucleotide repeats found in introns 1 and 2 of the bovine gene and in intron 2 of the sheep gene showed 15 and more repeats.
  • Tab. 4 shows a summary of the results of an analysis of all PrP microsate DNA variants in the bovine (Tab. 4A) and ovine (Tab. 4B) PrP gene. Variants were obtained in about 30% of the analyzed loci. As summarized in Tab. 4, 4 of the 8 variable micros crizn loci showed 2 AUele in cattle, and 2 loci were highly variant (6 or 10 AUele). The data for the sheep gene in Table 4B show a locus with 2 AUelen, 2 loci with 3 AUelen and 3 Loci with 4 and more AUelen. Some of the variants were only found in one breed (9 out of 31 AUelen in cattle and 2 out of 22 AUelen in sheep), while the other variants occur in more than one breed.
  • the fragment length analysis with the ALF device showed 1-3 nucleotides shorter values (in 15 faults 1 Nucleotide, in 16 cases 2 nucleotides and in 4 cases 3 nucleotides less) than the results obtained by sequencing. These deviations are probably due to secondary structure ties due to the repeats in the microsatellite DNA fragments, which influence the mobility of the respective species. For the typing of genes, this deviation from the actual length does not matter because the fragment length measured by electrophoresis is constant for any given genotype and therefore a characteristic value is obtained for each AU using the same electrophoresis system.
  • Tab. 5 controls the microsateloid loci determined as polymorphic in the bovine (Tab. 5A) and ovine (Tab. 5B) PrP gene.
  • a frequency of the most common AU was found to be ⁇ 0.95.
  • a total of> 0.10 heterozygosity was observed in 11 of the 14 polymorphic loci found in both species.
  • Table 6 shows comparative data for the microsatten loci of bovine and ovine genes. Thereafter, microsateloid loci can be localized not only in the PrP gene of sheep and cattle but also in other genes of this species. On average, 1.12 microsatten loci per 1 kbp DNA were obtained in the 8 genes analyzed. The results according to Table 6 are shown graphically in FIG. 7 described above.
  • Tab. 7A shows the 16 microsatelten loci and the octapeptide repeat found in the human PrP gene, 9 of which showed more than 1 AUel in 18 healthy volunteers examined. The frequency of the prevailing AU in these was ⁇ 0.95. The observed degree of heterozygosity would be> 0.10 for external loci. Furthermore, the PCR conditions with regard to the attachment temperature and MgC ⁇ concentration as well as the primers used are given. The number and lengths of the individual oils found in the microsatel loci in the human PrP gene are combined in Table 7B.
  • Table 8 shows the number of sheep examined in Example 3 below and their ORF genotypes.
  • Table 9 shows the PCR approaches for ampHfication of the microsatel loci Sll, S24 and S15 in the PrP gene of the sheep,
  • Table 10 shows the PCR program carried out and
  • Table 11 shows the sequences of the primers used.
  • Table 12 shows the running conditions for the electrophoresis that was carried out to separate the microsate units and ampicules.
  • Tables 13 to 15 summarize information on the sequencing of cloned PCR products which resulted from the amplification of the microsatel locus S24 of the sheep's PrP gene.
  • Tab. 16 shows the results of the fragment length analysis of the microsattenoid locus S24 of the sheep PrP gene obtained by electrophoresis or sequencing (cf. also FIGS. 9 to 11 described above).
  • the frequencies and frequencies of the polymorphic microsatten loci in the PrP gene area for the 8 sheep breeds examined are shown in Table 17.
  • locus III the total fragment length 152 was determined as the most common oil with the lowest oil sequence 0.54 in the Suffolk breed and highest frequency 0.93 in the Merinoland and Schwarzkopf breeds. Another high oil frequency was found in the Suffolk breed for the total fragment length 158 at 0.45.
  • the other frequencies for this locus for the individual breeds ranged from 0.007 (4 observations of fragment length 154 for the Merino region) to 0.149 (26 observations of fragment length 158 for milk sheep).
  • fragment lengths 150 and 152 were detected in 8 races.
  • the fragment length 154 was only seen in the Merinoland breed.
  • fragment length was negated as information.
  • the analyzed fragment lengths 144 and 147 were determined in external breeds, whereby fragment 144 was most frequently represented in the Dorper, Isle de France, Merinoland, Schwarzkopf and Texel breeds with frequencies between 0.61 and 0.885 and fragment 147 in the Gotland, milk sheep breeds and Suffolk was the most common AUel with frequencies between 0.53 and 0.75.
  • the microsatellite locus S15 showed 3 aUele fragment lengths with the most common fragment 179 in outside races and frequencies between 0.57 (Gotland breed) and 0.98 (Dorper breed).
  • the fragment length 182 was represented in external breeds with low frequencies between 0.008 (1 observation for breed Suffolk) and 0.192 (33 observations for breed milk sheep). Fragment 173, which was absent in 4 races and was found with frequencies between 0.016 (2 observations of the Suffolk breed) and 0.286 (4 observations of the Gotland breed), was even rarer.
  • Frequencies and frequencies for the amino acid variants of the PrP genotypes are in Tab ' . 18 rose. Frequencies between 0.885 and 1.0 could be determined for the amino acids alanine (A) for the individual breeds for all variants of codon 136 (ORF1).
  • the amino acid valine (V) given in the literature with a high risk of scrapie susceptibility in animals with this expression, could only be observed with low frequencies up to 0.115 (Isle de France breed), with no animal with this variant in the Gotland and dairy sheep breeds occurred.
  • the all variant histidine (H) was present in very small numbers on codon 154 (ORF2 label) (highest frequency 0.258 in milk sheep). This amino acid expression was absent in all breeds, apart from Müchschaf (46 observations), Merinoland (40 observations) and Texel (1 observation). The amino acid arginine (R) was found at this position with frequencies above 0.74.
  • Codon 171 (ORF3 code) was able to determine 3 general values for the 8 breeds.
  • Amino acid expression could only be determined with one observation in the Merino Land breed.
  • the varieties glutamine (Q) and arginine (R) were represented in the breeds with very different frequencies.
  • the lowest observed degree of heterozygosity for the Dorper breed was found to be 0.13 for the MikrosatelHtenlocus Sll and the highest for the Suffolk breed to be 0.50.
  • the MicrosateUitenlocus S24 the lowest observed degree of heterozygosity was 0.231 for the Isle de France breed and the highest for the Suffolk breed was 0.594.
  • the MikrosatelHtenlocus S15 the values were between 0.044 for the Dorper breed and 0.714 for the Gotland breed.
  • the lowest mean observed degree of heterozygosity at 0.157 was found in the Schwarzkopf breed and the highest in the Gotland breed at 0.571. The observation values showed small, insignificant deviations from the expected values for all breeds.
  • the locus Sl 1 with 5 AUelen is the highest polymorphic locus in the investigation.
  • the genotype frequencies of the microsatel loci for equilibrium Hardy-Weinberg equilibrium
  • no deviations could be determined.
  • locus S24 with AUelen 144 and 147 the heterozygote animals were overrepresented in all breeds.
  • the genotype 144/147 was too common across the breeds with 23 observations (267 observed, 244 expected). However, this did not lead to a significant deviation of the genotype frequencies from an equilibrium situation in any race.
  • the locus S15 showed no deviation from the Hardy-Weinberg-Equüibrium (HWE) in one of the breeds in 3 AUelen with the rare AUelen 173 and 182.
  • HWE Hardy-Weinberg-Equüibrium
  • the ORF3 locus (codon 171 with amino acid variants H, R and Q (glutamine) showed a significant deviation (P ⁇ 0.05) from the HWE in the Merinoland breed with a significant underrepresentation of the type Q / R (14 observations too few) In the Suffolk breed this type occurred too frequently with 6 observations (not significantly different from the HWE).
  • the rare form H was almost exclusively present in the Texel breed and showed no abnormality in the frequencies.
  • Tab. 26 documents the individual data of the AUs of the microsatelten loci in the PrP gene which were examined in relation to an association between the polymorphic microsatel loci and various dementia groups in humans.
  • the examined microsatten loci within ORF genotypes are upgraded, within which the microsatel loci were sorted according to genotype in order S24, Sll, S15.
  • ORF type ARR / ARR associated with the lowest risk of disease.
  • all animals showed the same genotype combination of the 3 microsatten loci S24, Sll, S15 with 144/144, 152/152 and 179/179.
  • This homozygous occurrence at the three loci occurred in the entire data material with 188 observations with an anusly decreasing tendency up to risk level 4 with 47 observations (20% of 233 observations at this level).
  • This genotype combination was not represented in risk level 5.
  • Risk level 2 could be determined with 16 observations in the Merinoland and Müchschaf breeds; it was observed once in the Texel breed.
  • ORF type For this ORF type, a high proportion of the genotype combination of the microsateurs could be determined with 144/144, 152/152, 179/179 with 62 observations (37.6%). Within this type, the AUelel50, 154 and Locus S15 the AUel 182 each appeared for the first time in very low frequency.
  • the ORF type ARR / ARH, risk level 3 with 10 observations shows the rare expression H (histidine) on codon 171. With the exception of one exception in the Merino breed, ORF types with this amino acid were only determined in the Texel breed (see also ORF types ARH / ARH and ARQ / ARH in risk level 4 and ARH / VRQ in risk level 5).
  • the AUele 150 to locus Sll and 182 to locus S15 occurred in the subsequent risk levels with V (valine) at position 136 with even higher anteu (28% anteü for AUel 150 and 26% anteü for AUel 182).
  • the Locus S24 showed no abnormality in the frequencies of the microsattenoid genotypes in the highest risk level.
  • Genomic DNA sequences of the genes coding for the PrP protein are available in GenBank (accession numbers: U67922 for sheep and AJ298878 in cattle) and were generated on microsate motifs using the software etandem of the EMBOSS program package (HGMP-RC, London ) searched, the motif size window was 2 to 25 nucleotides and only those microsate motifs with at least 3 repeats (repeats) and 90% homology were evaluated.
  • microsatel loci were accessible through the Sputnik program (Abajian 1994), the RepeatMasker program (Smit and Green) at http://fdp.genome.washington.edu/cgi- bin / RepeatMasker) and Lee et al. (1998) found information found. If a microsate motif was identified in only one species, the DNA sequence of the other species was compared by means of a comparison and the homologous region was checked for the same microsate motif. Sequence comparisons were carried out using the GeneDoc program, which is accessible at the URL http: //www.psc.ecu/biomed/genedoc.
  • Blood samples were taken from 11 sheep breeds (3 animals per breed: Awassi, Changthangi, Dorper, Hu, Merinoland, Müchschaf, Gotland, Schwarzkopf, Shkodrane, Texel, White Karaman) and 8 cattle breeds (4 animals per breed: Busa, Holstein -Black-colored, Jersey, South Anatolian red cattle, Simmental, Nanjing cattle, Nguni, YerH Kara) collected.
  • DNA was isolated from EDTA-stabilized blood by chlorophore-phenol extraction or using IsoHêtskits (NucleonBAcc2, Pharmacia, Freiburg, or Nucleo Spin Blood Quick Pure, Macherey-Nagel, Düren). The quality of the DNA was checked by electrophoresis in 1% agarose gels and by photometric test at 260/280 nm. The DNA material obtained was stored in sterile TE buffer (10 fflM Tris-HCl, 1 mM EDTA, pH 7.5) at -80 ° C.
  • primer sequences were constructed using the Primer 3 software (Rozen and SHetsky 1998) and synthesized by Roth (Karlsruhe), with one fluorescent primer being controlled per locus.
  • PCR A standard protocol was used (initial denaturation for 5 min at 94 ° C followed by 30 cycles each with 1 min at 55 ° C, 1 min at 72 ° C and 1 min at 94 ° C, and a final extension for 15 min at 72 ° C), the reaction was carried out in a volume of 20 ⁇ i with 0.15 mM primer, 200 mM of each dNTP, 100 ng genomic DNA, 1.5 mM MgC ⁇ and 0.5 units Taq Polymerase (obtained from Roth, Düsseldorf). The efficiency and specificity of the PCR was optimized by changing the MgCl 2 concentration (from 1.5 to 4.5 mM) together with the annealing temperature (48-65 ° C) in a gradient thermal cycler.
  • the primers and further PCR conditions that were used for the fragment analysis according to embodiment 1 are given in Table 3.
  • Fragment length analysis Using an automatic laser fluorescence DNA sequencer (ALF Pharmacia) with 5% HydroHnk gels, the PCR products (0.5 ⁇ l each) were analyzed together with internal (99-198 nucleotides) and external (80-353 Standard fragments (prepared by PCR with ⁇ -DNA as a template) were analyzed. Before loading the gel, the fragments were denatured (2 min at 90 ° C and then ice cooling). The electrophoresis was carried out in 0.6% TBE buffer at 1500 volts, 45 mA and 50 ° C. The data analysis was carried out using the AUeleLinks software (Pharmacia, Freiburg).
  • microsate loci could be identified in the bovine and 23 in the ovine PrP gene.
  • the following conclusions can be drawn from the results which are shown in FIGS. 5 to 7 and Tables 3 to 6: Number of microsatellite loci in genomic DNA. An average of 1 microsate locus per 0.9 kbp DNA was obtained within the coding gene regions and the flanking regions. This frequency was also observed for other coding genes and is much higher than documented in the prior art (cf. e.g. Schlötterer 2000). This surprising difference can result from the fact that, in the prior art, information about microsat position in DNA is usually obtained by screening libraries of genomic DNA elements with microscopic motifs, and the number of repeat motifs in the genome was therefore underestimated.
  • Regulatory DNA sequences can even have a pattern of microsatters with an even higher density, since for example response elements often incorporate repeated nucleotides and variants in gene regulation become functionally important (Wales et al.
  • microsatellite motifs The high intragenic variability caused by microsatellite loci can stimulate the evolution of eukaryotic genes. It was determined that microsatuites in the PrP gene of humans and mice are very different (Li et al. 1998). Surprisingly, however, similar positions of the microsate loci were found in the closely related species cattle and sheep in the present invention, which were stable despite all variability within the species. This variability leads to genes which are polymorphic with respect to more than one locus ("hetero-oils”), whereby new oils can be generated by intragenic recombination. In these cases variants of individual loci can be found have proven to be successful during evolution, which results in new functional, possibly superior oils.
  • the variability of the ORF polymorphism is limited even in sheep, since the amino acid substitutions of the relevant codons (136, 154 and 171) only occur in 5 different haplotypes.
  • the microsate units shown in the present invention provide much more haplotypes and are therefore outstandingly suitable for typing individual individuals with respect to the PrP gene.
  • microsatellite markers in conjunction with ORF variants: In cattle, only a few polymorphic control units are available in the PrP gene for its typing (Hunter 2000). Since no informative markers in the PrP gene are known in the prior art, no detailed investigation of the relationship between BSE disposition and gene variability has been able to be carried out to date. The new microsate loci will therefore be used for further genetic testing of BSE, in particular they can be used for pradiagnostics related to this disease. In the sheep, 3 of the polymorphic MikrosatelHtenloci used together with the ORF variants to examine more than 600 animals of different breeds, which is described in Example 3 below.
  • 1 microsatelten loci within genes were analyzed using GenBank sequences of the bovine and ovine PrP gene. This procedure is used to screen a large number of polymorphic DNA markers per gene. This procedure was carried out using the PrP gene, but can be easily transferred to other eukariotic genes. When considering microsateUites with at least 3 repetitions, 24 loci in the bovine and 23 in the ovine PrP gene were identified. About 30% of the loci were polymorphic within the species. Of 32 cattle from 8 genetically different breeds, 4 polymorphic microsate units in 2.1 microsate units with 3, 1 microsate units with 4, 1 microsate units with 6 AUels and 1 microsate unit with 10 AUels occurred.
  • the data from 33 sheep from 11 genetically different breeds showed a locus with 2 AUelen, 2 Loci with 3 AUelen and 3 Loci with 4 and more AUelen. It has been determined that microsatten loci with at least 5 repeats have a higher degree of polymorphism than those with 4 or fewer repeats. In cattle, 9 of the 31 AUele were found only in one breed, in sheep it was 2 out of 22 AUele. Frequencies of the prevailing AUel of ⁇ 0.95 were observed in 6 microsatelten loci in cattle and in 6 microsatelten loci in sheep.
  • the fragment length analysis with an automatic DNA sequencer showed a length that was 1 to 3 nucleotides shorter than with the sequencing.
  • the deviations in the measured molecular size can be explained with secondary structures, which are built by microsatical motifs that influence the electrophoretic mobility of the fragment. All DNA fragments differed not only in the microsatel loci but also in their flanking areas. On average, a microsatellite locus occurred in 8 genes of cattle and sheep approximately 0.9 kbp, whereas the density of microsatel loci is higher in PrP exons. Microsateloid loci within genes can be functionally important in expression and can therefore be related to PrP accumulation in TSE-infected cells. The intragenic variability caused by microsatten loci can stimulate the evolution of eurocariotic genes. The intragenic polymorphic microsatten loci according to the invention allow, in particular, use in prediagnostics.
  • Example 2 Example 2:
  • the methods according to the invention were also applied to the human PrP gene.
  • the genomic DNA sequence (148,497 bp) of the human PrP gene was considered with regard to microsatical motifs using the etandem software of the EMBOSS software package (obtained from UK HGMP Resource Center, see http: / /www.hgmp.mrc.ac.uk).
  • the motif window size was 1 to 25 nucleotides and only those microsate motifs with at least 4 repeats (repetitions) and 90% homology were recorded.
  • the following PCR protocol was carried out as standard: initial denaturation for 2 min at 94 ° C, followed by 32 cycles, each with 1 min at 55 ° C, 30 or 45 s at 72 ° C and 45 s at 94 ° C, as well as one last extension phase for 5 min at 72 ° C.
  • the PCR was carried out in a reaction mixture of 20 ⁇ l, containing 0.15 ⁇ M primer, 200 ⁇ M of each dNTP, 100 ng genomic DNA, 1.5 mM MgC ⁇ and 0.5 units T ⁇ polymerase (Eppendorf,
  • the efficiency and specificity of the PCR was determined by changing the MgC ⁇ concentration (1.5 to 3.0 mM) and the annealing temperature
  • DNA sequencing of individual microsatellite alleles For the sequencing of individual AUele, 0.067 pmol of the purified PCR product was treated with a mixture for converting DNA ends (Novagen, Madison, WN, USA) and in the pT7- digested with Ec ⁇ KV to generate blunt ends. Blue- vector ligated. The heat shock transformation of Nova Blue E. ⁇ ? / Z host cells was carried out according to the manufacturer's instructions. Template DNA generated by means of vector PCR, starting from white clones, was purified using the Cycle Pure Kit (Peqlab, Er Weg, Germany). Both strands of DNA were sequenced by Biolux, Stuttgart, Germany. The sequences were analyzed with the hooves of the Chromas program (http://www.technelysium.com.au and Genedoc (http://www.psc.edu/biomed/genedoc).
  • Microsate positions and the octapeptide repeat are shown in Figure 13 and Table 7A.
  • oil variants occurred in 9 of the 17 Positions that were analyzed on (see Tab. 7A).
  • Four of the variable microsatellite positions showed two eyes, and the other loci occurred in between 4 and 9 eyes.
  • MicrosateUites with a higher repeat number were more polymorphic than those with a lower repeat number.
  • the 7 analyzed microsatten loci with 7 or fewer repeats occurred only in one or two external forms, whereas the 5 positions with> 12 repeats occurred in> 4 external oils. 4 loci had frequencies ⁇ 0.4 for the most common AUel. A degree of heterozygosity of> 0.10 was observed in all positions.
  • polymorphic microsate loci can thus also be used in humans for typing the PrP gene in order to diagnose AUele, from which a TSE predisposition of the wearer results.
  • the carriers of certain oils can then, for example, adjust their lifestyle habits, in particular eating habits, to the predisposition.
  • Genotypes of these loci were analyzed for the ORF genotypes determined in preliminary examinations.
  • the data collected included 8 breeds of sheep with 623 animals from 47 herds for which blood samples had been collected. In 573 sheep, 3 microsatten loci were clearly typed.
  • Eabormaterial The following chemicals or reagents or the following materials were used for the experiments:
  • CeHulose nitrate feeder SM 113 (pore size 0.45 ⁇ m), Sartorius, Göttingen
  • Aqua bidest was used to produce the following solutions and buffers. (manufactured by ultrafiltration, ⁇ 16 M ⁇ -cm). Unless otherwise stated, storage was at +4 ° C.
  • Bindesuan stock solution 40 ml ethanol; 98% (v / v); 150 ⁇ l of binding water; RT
  • Bindesüan solution 800 ⁇ l of Bindesüan stock solution; 200 ul acetic acid; 10% (v / v); RT
  • Loading buffer 50 ml formamide; 5 g mixed ion exchanger; 6 mg / ml
  • 10 x TBE buffer 1 M Tris-HCl; 830 mM boric acid; 10 mM EDTA; pH 8.2;
  • RT stop buffer 250 mM EDTA; 10% (w / v) FicoU 400TM; 1% (w / v)
  • Ethidium bromide solution 500 ⁇ g ethidium bromide / ml H 2 0 LB medium: 10 g tryptone; 5 g yeast extract; 5 g NaCl; 14 g agar; ad 1 1
  • DNA sequencer A.L.F. (Automated Laser Fluorescence) DNA sequencer with ALFWin and AUelelinks software, Pharmacia, Freiburg
  • Electrophoresis chambers DNA-Sub-CeUTM (15 x 15 cm), BioRad, Kunststoff Pipettes: Eppendorf “Research”, Hamburg Güson, “Pipetman”, Vü-
  • Amplification of the microsateUites by PCR In our own investigations, PCR approaches (Tab. 9), a PCR program (Tab. 10) and primers (Tab. 11) were used, which had been established in preparatory work for the loci.
  • Agarose gel electrophoresis of PCR products To prepare the agarose gels, 2 g of agarose and 100 ml of IxTAE buffer were heated in the microwave until the agarose was dissolved, 20 ⁇ l of ethidium bromide were added and the mixture was poured into the gel chamber. The slotformer was used immediately afterwards. Approximately 30 minutes later it was removed and the gel was placed in the electrophoresis chamber which was filled with IxTAE buffer. 5 ⁇ l PCR products were mixed with 2 ⁇ l stop buffer and pipetted into the pockets. The separation process took approx. 40 min. The gel was then viewed under UV light. The result was read by comparison with the marker.
  • DNA sequencing machine The PCR products were separated in 5% hydrogel gel with hooves of the A.L.F. The
  • Automatic sequencer consists of an electrophoresis and a detection unit. This contained a laser and 40 photodiodes, which are arranged above the individual gel tracks. Since the forward primers of the PCR and thus also the AmpH fikates were fluorescence-labeled, they were excited when they passed by at the level of the laser. This led to an electrical signal in the photodiodes, which was digitized and stored in the connected computer. The fragment lengths of the individual oils were calculated on
  • the HydroHnk gels were manufactured according to the instructions of the AX.F. HersteUers Pharmacia, Freiburg. After cleaning the glass plates with the fluorescence-free washing-up liquid Alconox, H 2 O and 98% ethanol with subsequent air drying, the upper area was coated with binding water to fix the gel pockets. The plates were then assembled with spacers and light couplers to form a gel chamber. For the solution, 60 ml of filtered (membrane with 0.45 ⁇ m pores) and degassed (6 min) Long Ranger HydroHnk solution were mixed with 200 ⁇ l APS and 50 ⁇ l TEMED. The well mixed solution was poured between the glass plates free of air bubbles using a 60 ml syringe.
  • a comb which was pushed into the gel chamber, was used to shape the gel pockets. After about 90 minutes the gel was gone RT polymerized and could be installed in the sequencers. After the gel reached 50 ° C, the comb was carefully removed. After the gel had been installed, 0.6 ⁇ TBE buffer was added to the electrode container and the gel was loaded with sample material. The gels were used three times and the gel pockets were rinsed out with TBE buffer in between. The first time, more precise results were achieved than in the following two runs
  • 0.1 - 4 ⁇ l DNA was mixed with 1 ⁇ l of a mixture of length standards and stop buffer in a Terazaki plate, depending on the PCR yield (1 ⁇ l DNA was used as standard).
  • the samples were then denatured for 2 min at 80 ° C and then controlled on ice. After rinsing out the gel pockets with TBE buffer, the prepared samples were pipetted into them.
  • End conversion reaction Incubation of 2 ⁇ l condensed PCR product as template in 2 ⁇ l water and 5 ⁇ l end conversion mix. Let it rest at RT for 30 min. Inactivate the mixture for approx. 5 min at 75 ° C, cool for 2 min on ice, then centrifugation.
  • the PCR products were checked using agarose gel electrophoresis.
  • the clones whose PCR products had the expected length were ampHfected with the fluorescence-labeled primer pair for the locus S24 (Tab. 11) and the products on the A.L.F. dargesteUt.
  • a clone whose fragment length matched the 152 bp length measured during genotyping was selected for sequencing.
  • a 100 ul PCR mix was made with primers T7 and U19 to get a sufficient amount of template for the sequencing reaction.
  • the length and the concentration of the PCR products was checked again on an agarose gel. This was followed by the purification of the PCR products using the 'TCR Purification Kit'. The samples were then sequenced.
  • pi frequency of the i-th AUel, p AA. Frequency of the genotype with AUele i or j, i, j: indices of AUele, k: number of AUele of a locus, N: number of examined individuals.
  • Deviations in genotype frequencies from the Hardy-Weinberg equilibrium were checked using the pooled ⁇ 2 test. Due to the low number of observations for individual genotypes (N ⁇ 5) with the problem of overestimating the significance with low genotype expectations in the ⁇ 2 test, three genotype classes (i, ii, iii) are built in the pool method:
  • H aUe heterozygous genotypes with the most common AUel
  • iii aUe ho o and heterozygous genotypes without the most common AUel.
  • each fragment length could be clearly assigned to a locus-specific AUel.
  • the first exception was the fragment length 146 at Locus S24, which occurred in a total of 6 cases with the breeds Schwarzkopf, Gotland and Suffolk.
  • the uniqueness of the different fragment lengths 146 and 147 was reliably controlled on the one hand by repeating the PCR application on different gels, and by arranging animals with AUel 146 and 147 in successive traces of a gel. A fragment length difference around the precise value of 1 could not be traced back to track effects with simultaneous internal marker alignment. It can only be proven by sequencing whether fragment 146 is a very rare additional AUel. This could e.g. B. from a deletion of a base outside the actual microsate area from the allele with the fragment length 147.
  • the information content of the 3 microsate loci showed mean degrees of heterozygosity> 0.35 for 4 of the 8 breeds considered (Gotland, Suffolk, Müchschaf and Texel) (Tab. 19). For the Dorper, Merinoland, Ile de France and Schwarzkopf breeds, the values were between 0.16 and 0.23. The information content was consistently lower for the ORF loci and showed values between 0.0 for Gotland and 0.29 for the Texel breed for the middle degrees of heterozygosity. These relatively low levels of information corresponded to the low number of observations by animals with a high risk of disease. In other words, low-risk genotypes have prevailed in the populations without the high-risk ones completely disappearing. This may be due to the fact that unfavorable PrP genotypes also have beneficial functions in the development of an animal, e.g. B. fertility or resistance to other pathogens.
  • the locus Sll showed in the analysis of variance only in the breeds Müchschaf and Texel a significant influence on the trait constructed via ORF haplotypes with a pronounced increase in risk for animals with AUel 150 in the Texel breed. This allele was not sufficiently frequent in the breed Müchschaf for an examination, however, in this breed there was a higher risk for animals with AUel 156 compared to animals with AUel 158 at this locus.
  • ORF genotypes showed only low information levels in the sheep breeds with values between 0.0 and 0.29 for the middle degrees of heterozygosity, which resulted in a value of 0.164 for the breeds. Nevertheless, 13 of a total of 15 ORF genotypes reported in the literature were found for the total material, with approx. 65% of the observations for the ARQ / ARQ (wüdtyp) and ARR / ARQ genotypes.
  • the rare one Expression V (valine) on codon 136 with a high susceptibility to scrapie disease was only represented in this study with an age sequence of almost 3% across the breeds.
  • microsate genotype sorted within ORF typing showed a uniform expression in the risk level 1 with the genotype combination 144/144, 152/152 and 179/179 for the loci S24, Sll and S15.
  • genotype combinations occurred in the higher risk classes. In these classes, it was not possible to assign certain microsate genotypes to the ORF genotypes discreetly, but there were clear clusters of certain oils in the risk classifications.
  • microsate unit loci in the human PrP gene recognized as polymorphic according to embodiment 2 were examined with regard to their association with different dementia groups in humans.
  • the microsatellite positions M01, M02, M09, MIO, Ml 2, M03, MM03, MM04 with regard to the occurrence of oil in people with CJD and other people with dementia (both groups included 100 people each).
  • a group of 18 healthy people (“Han”) served as a comparison group.
  • the octapeptide region already known from the prior art was included in the investigation.
  • the variant considered "pathogenic" in codon 129 (SNP 129) was used as a comparison. This variant has hitherto recognized the strongest associations with CJD.
  • the evaluation according to haplotypes compares the oil frequencies.
  • the further evaluation was carried out according to genotype frequencies. Microsatlet positions with many AUele were not included in the genotype evaluation.
  • Bossers et al. (1996): PrP genotype contributes to deteö ining survival times of sheep with natural scrapie. J. Gen. Nirol. 77, 2669-2673 Brown (1999) Modern Genetics. 2nd edition, Spektrum-Nerlag, Heidelberg
  • Prusiner (1998): Prions. Proc. Natl. Acad. Be. USA 23, 13363-13383
  • TBE buffer made from TRIS, boric acid and EDTA
  • TTTGT TTTGT 4 AAT CAG TGG GAT CAT AGA CTT TCA 55 2.0 TCC ATC TAG CTT CCA AAC TAT TGA C
  • TTCAG TCC TAT TCC TAG TCT GCT 55 1.5 TGG GAG TTG GTG ATA GAC AG
  • TTTGT TTTGT 4 AAT CAG TGG GAT CAT AGA CTT TCA 55 2.0 TCC ATC TAG CTT CCA AAC TAT TGA C
  • a AF532794 158 160 (AGC) 3 CGC (AGC) (R) kD 54
  • Locus AUel a accession no. b allele fragment sizes (bp) microsatellite sequence e differences in flanking regi number of observed alleles
  • a AF532818 139 141 (GA) 5 (R) 7 bp del., 1 bp ins., 5 SNP 35
  • Locus number of alleles Frequency of the previously observed degree indicates AU of heterozygosity
  • PrP 4242 9 2.12 15988 15 0.94 1000 1 1.00 78056 83 1.06
  • PrP Prion protein coding gene / AJ298878 (bovine), U67922 (sheep)
  • IGFBP3 Insulin-like growth factor binding protein-3 / AF305712
  • LZ Lysozyme gene / U25810 ⁇ -casein beta-casein gene / M55158 (bovine), X79703 (sheep) ACC- ⁇ acytyl-CoA carboxylase alpha gene / AJ292285
  • CFTR for regulator of transmembrane conductivity, involved in cystic fibrosis, coding gene (AF325412 - AF325422) 2) Length of the gene region searched
  • TTTGT TTTGT 5 GCACAGTCGAAAATCTGC 60 2.5 TGTAGTCCCAGCTACTCAGG
  • M microsate unit
  • MM mononucleotide microsateUit
  • MOcta Ocatapeptide Repeat
  • TTTTT TTTGT 3 (TTTTT)
  • Taq polymerase (5 units / ⁇ l) 0.5 4 0.5 4
  • Buffer 10 x (containing 15 mM MgCL) 2.5
  • V 13 0.110 0 0.000 3 0.115 1 0.002 0 0.000 1 0.011 2 0.016 16 0.113
  • ORF2 H 0 0.000 0 0.000 0 0.000 40 0.070 46 0.258 0 0.000 0 0.000 1 0.007
  • ORF3 H 0 0.000 0 0.000 0 0.000 1 0.002 0 0.000 0 0.000 0 0.000 30 0.211
  • B observed allele count
  • F frequency
  • ORF1, ORF2, ORF3 locus names for the genotypes at codons 136, 154 and 171
  • AS amino acid (A: alanine; H: histidine; Q: glutamine; R: arginine; V: valine)
  • ORF2 0.000 0.000 0.000 0.000 0.000 0.000 0.127 0.131 0.315 0.385 0.000 0.000 0.000 0.000 0.014 0.014
  • MH medium degree of heterozygosity
  • SE standard deviation
  • h (DC) degree of heterozygosity ("direct count estimate”)
  • h (unb.) degree of heterozygosity ("unbiased estimate")
  • Tab. 23 Relationships between microsatellite loci and the characteristic risk (see Tab. 2; evaluation separated by race according to model 1)
  • MQS minimum square estimate
  • S.E . Standard Error
  • ARQ / ARQ 4 144/144 150/150 182/182 1 4
  • ARQ / VRQ 5 144/144 150/152 173/182 4 10 144/147 150/152 173/182 1 6

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Abstract

L'invention concerne un procédé pour classer par types des gènes qui présentent au moins un locus microsatellite, un procédé pour déterminer des marquages dans des gènes pourvus de loci microsatellites, relativement à une prédisposition pour certaines maladies, ainsi qu'un procédé pour établir un diagnostic préalable concernant des maladies associées à des gènes qui possèdent un ou plusieurs locus microsatellites.
PCT/EP2003/008822 2002-08-09 2003-08-08 Loci microsatellites polymorphes dans des genes pour l'etablissement d'un diagnostic prealable WO2004020664A2 (fr)

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