EP1309722A1 - Use of microbial dna sequences for the identification of human diseases - Google Patents

Abstract

The use of DNA sequences comprising a fragment of a nucleic acid encoding a microbial virulence factor as means for the identification of diseases or a genetic predisposition thereof as well as its use for the development of disease animal models is disclosed.

Description

Use of microbial DNA sequences for the identification of human diseases

Cross Reference to Related Application

This application claims the priority of PCT patent application IBOO/01127, filed August 16, 2000, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to the use of a DNA sequence comprising a fragment of a nucleic acid encoding a microbial virulence factor as means for the identification of a disease or a genetic predisposition thereof as well as its use for the development of disease animal models .

Background Art The functional sequences of higher eukaryotes consist of genetic modules of at least two kinds. Modules of coding sequence are combined in many ways to produce proteins, whereas modules of non-coding sequences regulate the expression of genes. Some of the mutations cre- ated duplicates of entire genes, which have then evolved new functions, while others altered the expression of old genes by exposing them through gene shuffling to new regulatory sequences. By these means the human genome as a whole has evolved to its present day complexity. Since the 3' untranslated region (3'UTR) and especially the polyadenylation signal within the 3 ' UTR regulate the translation and expression of a gene, the entire gamut of molecular perturbations can be accomplished with the 3'UTR as a primary target. There is therefore a need for molecular tools allowing the detection of diseases or a predisposition thereof caused by mutations within the 3' UTR. Disclosure of the Invention

In the scope of the present invention it was now found that there are microbial DNA insertions in the 3 ' UTR of human genes which are associated with human diseases .

Hence it is an object of the present invention to provide the use of DNA sequences comprising a fragment of a nucleic acid sequence encoding a putative microbial virulence factor as means for the identification of diseases caused by bacterial mutations or a genetic predisposition thereof. The virulence factor stems preferably from a intracellular microorgansim and is located on a linear or circular chromosome or a plasmid, more preferably said virulence factor stems from a microorganism which is selected from the group consisting of Borrelia species, Chlamydia sp., Escherichia sp., Plasmo- dium sp. and Rickettsia. Even more preferably said nucleic acid encoding a virulence factor is selected from the group consisting of Seq. Id. No. 1 to Seq. Id. No. 17. Virulence factors stemmming from non-intracellular microorganisms which are part of a cluster shared by intracellular microonganisms are as well suitable for the use in the present invention. Another object of the present invention is a method for the identification of a disease or a genetic predisposition thereof, which comprises in a tissue or blood sample of a subject or in a fetal neuro-graft a mutation within a nucleic acid sequence selected from the group consisting of Seq. Id. No. 1 to Seq. Id. No. 17 and said sequence is inserted in a gene of said subject. Preferably said sequence is inserted in the 3'UTR of said gene and said mutation is found in the polyadenylation signal of said gene and said mutation preferably affects the expression of the protein encoded by said gene.

Another object of the present invention are transgenic non-human animals, which comprise in their ge- no e a partial or complete inactive endogenous gene which is selected from the group consisting of cannabinoid receptor 1 gene, MAP 2C gene, apolipoprotein E gene, prese- nelin 2 gene, integral membrane protein 2B gene, alpha synuclein gene, oligophrenin 1 gene and myotonin protein kinase gene. The gene is inactivated due to at least one mutation in its 3' untranslated region (3' UTR) and said mutation leads to an inhibition or suppression of protein expression. The term mutation as used herein encompasses any nucleotide change, insertion or deletion independent of their length that influences the activity, expression or regulation of a gene.

Although in the context of the present invention any mutation in the 3'UTR region leading to an inhi- bition or suppression of protein expression e.g. CBl protein expression, can be used, preferred are mutations located in the sequence following the polyadenylation signal, more preferably in the polyadenylation signal sequence of said gene. Any mutation in the polyadenylation signal sequence leading to an inactivation of said signal can be used. The mutation can e.g.be caused by a sequence of the same or a different microbial species e.g. by gene conversion or recombination.

The polyadenylation signal in eukaryotes has the following conserved sequence: AATAAA.

For the purpose of the present invention any non-human mammal can be used. Preferred are rodents e.g. mice or rats, which preferably harbor a homozygous or heterozygous CBl gene inactivation in their genome. Another object of the present invention is the use of the transgenic animals of the present invention for the identification of compounds that have an effect on the activity, expression or regulation of the gene encoded protein. The animals of the present inven- tion allow the identification of compounds which have a direct or indirect effect on CBl protein, MAP 2C protein, Apolipoprotein E, presenilin 2 protein, integral membrane

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Brief Description of the Drawings

The invention will be better understood and objects other than those set forth above will become ap- parent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings , wherein :

Figure 1 shows genetic exposure triggering gene conversion and further infectious recombination, Figure 2a shows genetic exposure leading to multiple translocations into the human genome and

Figure 2b shows genetic exposure leading to multiple translocations into the human genome .

Modes for Carrying Out the Invention

A gene construct for the production of a transgenic animal of the present invention, which comprises in its genome a partially or completely inactivated gene, can be prepared using standard genetic engi- neering technologies known in the art, such as described in Maniatis et al . , Molecular cloning: A Laboratory Manual, Cold Spring Laboratory, Cold Springs Harbor, N.Y. The starting material for said construct can be a portion of e.g. the genomic or cDNA CBl nucleotide sequence. In- troduction of the wanted mutation in e.g. the CBl sequence can be done by methods known to a person skilled in the art e.g. by site directed mutagenesis. The term mutation as used herein encompasses any nucleotide change, insertion or deletion independent of their length that influence the activity, expression or regulation of a gene .

A transgenic animal in accordance with the present invention can be made using generally known methods in the field. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986), Knock-out mouse models used to study neurobiological systems, Critical Reviews in rt μ- ≥J tr ø rt o μ- CO rt Hi > <! rt O Ω rt rt 0 3 rt tr 3 OJ to us Pi en < OJ en Hi tQ TJ g

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OJ ø 3 3 CO en ø KJ tr • ØJ μ- ø μ- H ø 0 ø TJ - ø φ CO o <J μ- tQ 0J P. TJ ø 3 rt OJ rt μ- Φ φ H- 0 CO DO 0 tQ eo ØJ o rt 0 μ- if μ- ø Φ ø TJ 0 3 rt φ tr μ- 0 ø - rt 5* •<; Cd • TJ φ rt ø tf ui 0 Ω ø 0J TJ en 0⁾ 0 tQ ii μ- o ø⁾ 0J μ- P. Φ TJ α OJ OJ t μ- p ø - 0J φ H ØJ OJ en Φ ~ Hi ft ii rt rt Φ Ω Hi ii 0 o μ- H ≥! ø μ- O 0 3 tQ φ CO H μ- TJ CO Φ μ- μ- en TJ μ- rt ø Ω TJ Φ > OJ rt OJ ø rr Ω ø OJ Φ Pi φ Hi tQ 0 0J o ii CO ø tQ O ^ OJ Ω ø φ OJ OJ Pi ø N 0 0 en 0 μ- CO μ- 0 Φ ii rt ø O 0 Φ ⁽Q Φ 0 cn ii 0 OJ Φ O

P. 3 ft μ- CO Pi TJ tr Hi Φ rt O CO Φ w ø 0 0 OJ rt 0 ø 0 rt Φ 3 ii i φ if en φ OJ tr Φ O tr μ- Ω μ- O en TJ Hi ø Φ M Φ Ω μ- O φ tr Φ TJ tQ O ø ø o TJ TJ ii φ μ- K. φ ø tr Pi Ω O TJ Φ Pi • Φ μ- Hi ø 3 tr o Φ ii

^■g 3 cn rt 0 H rX ϋ 3 μ- Φ en H. Ω ØJ tQ ø Φ μ- Φ rt cn * 0 tQ OJ φ co o cn φ μ- μ- φ φ Pi OJ ØJ TJ ø ø μ- Φ rt μ- en φ μ- ø o ø 0 0 0J ii tQ μ- 0 3 ø P ii μ- en ø - H. rt Ω rt Hi μ- ø rt 0 ø Cd Ω OJ 0 rt PJ ø ø H- en 0 μ- 1 Ω OJ en H- en en PJ Φ Φ 0 μ- O φ μ- N H O 0 0 ii en 3 1 0 φ ii 1 Φ ϋ 0 1 1 0 1 J PJ O ø 1 J ^ rt 1 Φ

1 en >< ø 1 i ø en 1

quences encoding microbial virulence factors and harboring at least one mutation affecting expression, activity or regulation of proteins encoded by said genes are preferred. As already mentioned above the present invention also concerns methods for the identification of a disease caused by bacterial mutations or a genetic predisposition thereof. Said methods comprise detecting the presence in a tissue-or blood sample of a subject a muta- tion within a nucleic acid sequence selected from the group consisting of Seq. Id. No .1 to Seq. Id. No. 17 and said sequence is part of a gene of said subject. The method of the present invention is e.g. suitable for the identification of one of the above mentioned diseases. Suitable methods for the identification of said diseases or a genetic predisposition thereof in humans are for example PCR techniques, DNA or gene chips, hybridisation techniques and Ligases chain reaction (LCR) . In a preferred embodiment said method comprises the following steps:

1. Blood or Tissue sample

2. DNA extraction

3. Amplification of at least one Sequence se- lected from the group consisting of Seq.

Id. No. 1 to 17 using flanking oligonucleotide primers

4. Analysis of amplification products by se quencing The sequencing step allows the identification of mutations present in the insertion sequence of interest. The design of the oligonucleotide primers is known to those skilled in the art and can be done using standard software. Examples

Example 1

Microbial sequences in 3'UTR polyadenylation regions of schizophrenia and dementia genes harbour microbial virulence factors and plasmids

DNA from Borrelia burgdorferi , Chlamydia and other intracellular microbes has inserted into ancestral 3 ' polyadenylation sites of the following candidate neu- rological genes: The central cannabinoid receptor gene (CBl) , Alzheimer disease and other dementia genes, Parkinson disease and myotonic dystrophy (see Seq. Id. No. 1 to 17 and Table 1A to IE) . Most insertions originate from microbial virulence factors, transposable elements and plasmids .

CBl and 5HT1E contain related insertions from B. burgdorferi . The human CBl gene is located at 6ql4, which has been reported as a candidate region for schizophrenia involving a translocation break-point co- segregating with schizophrenia, immediately adjacent to the 5-hydroxytryptamine (5HT1E) gene. The spirochaetal insertion into 5HT1E originated from a B . burgdorferi virulence factor, the flagellar basal-body rod protein (fbrp) , responsible for chemotaxis, locomotion and a sy- ringe mechanism for injection into cells. Through infectious recombination, B . burgdorferi introduced another nucleotide sequence containing pll5 and a polyadenylation site into our ancestral genome. As a result of this recombination, pll5 overlaps with fbrp. The fbrp section on the CBl gene originates from an ancient 5HT1E receptor already containing the spirochaetal insertion and not from a direct transposition of B . burgdorferi onto 6ql4, because the first three nucleotides of the overlapping sequence (see Table 1A) are on 5HT1E, and not on fbrp of B . burgdorferi . Gene conversion is the most likely explanation since CBl and human 5HT1E, on which fbrp is absent, are both located in tandem on 6ql4. OJ OJ t to I-¹ I-¹

Lπ o Lπ o LΠ o LΠ

netic knock-out of CBl. The transition of the parasite from arthropod vector to human host is accompanied by significant changes in gene expression, which has practical relevance for prevention, as well. The recently approved vaccine against Lyme disease consists of an immunogenic Osp A, that is expressed by the spirochaetal parasite while in the tick gut, turned off on entry into humans and then expressed again at a late chronic stage. After attachment of an infected tick and initiation of a blood meal, anti-OspA antibodies enter the tick gut and mediate killing of B . burgdorferi .

Example 2 Lateral gene transfer of B.burgdorferi into

5HT1E, gene conversion and homologous recombination with CBl

On the human CBl gene, located within a candidate region for schizophrenia at 6ql4 immediately adja- cent to the 5HT1E ( 5-hydroxytryptamine) gene, a nucleotide sequence of (pll5) originating from B. burgdorferi could be identified (Seq. Id. No. 1) . Another ancestral spirochaetal inclusion on 5HT1E originates from a B. burgdorferi virulence factor, the flagellar basal-body rod protein (fbrp) . It can still be found within the serotonin receptor IE gene (5HTIE) on the mouse (Mus muscu- lus) and rat (Rattus norvegicus) . During phylogeny, multiple recombinations between the spirochaetal fbrp and its pre-inserted fbrp templates have exposed the identi- cal sequences on the complementary strand on the double helix, including adjacent non-microbial nucleotides, to further recombination all over the human genome and a clustering of microbial virulence factors from Chlamydia uridarum iron binding protein, Plasmodiu falciparum rhoptry and Staphylococcus aureus penicilllin binding protein, a specific gene conversion occured (see Fig. 1 and Fig. 2). Through infectious recombination, B. burgdorferi subsequently introduced pll5 into our ancestral genome resulting in a genetic overlap with fbrp. Containing the poly-A signal (AATAAA) , pll5 thus introduced the code for the translation and actual genetic ex- pression of CBl.

Example 3

No point mutations within the polyadenylation signal (AATAAA)

With the introduction and natural selection of the inserted polyadenylation site, lateral gene transfer have effectively influenced the genetic expression of CBl . Presenting save havens for microbial DNA there are no point mutations on this important signal, for a change in the signal would disrupt the genetic expression of CBl . Suppose the mutually advantageous sequences on the polyadenylation signals recombine again with slightly dissimilar spirochaetal strands, the subsequent mismatch repair mutations will be deleterious for both hosts and parasites . Being located on an important 3 ' regulatory code, whose mutational rate runs slower than the molecular clock of silent point mutations in protein coding regions, microbial sequences are thus better protected from mutations and hence stabilised.

Example 4

DNA of B.burgdorferi underlies the schizo- phrenic genotype

That lateral gene transfer has influenced the evolution of higher eukaryotes, such as mammals is counter-current to established views. B . burgdorferi appears to be excluded from the benefits of the extensive lateral gene transfer between micro-organisms, however, as an intracellular parasite with an incomplete genome B. burgdorferi has a direct access to host genes, which it exploits for replication, and not to be recognised as foreign, the spirochete depends on its own sequences within the human genome. Through molecular mimicry of fbpr antigens within the host's CD45 leukocyte defence system and a structure homologous to nucleoprotein, B. burgdorferi might, for example, dispose of a protective shield at the DNA and protein level, respectively.

Dissimilarity and mismatch mutations between B . burgdorferi and its pre-inserted human templates may nevertheless occur, resulting from genetically induced variability by virulence factor operons, osp A and B, or, alternatively, from a genomic decay in plasmids (Casjens et al., Mol. Microbiol. 2000, 35, 490-516) borrelia reinfection causing putative mutations in AATAAA. Intrigu- ingly, by a single frame-shift mutation within the original translocase, the same process of genomic decay has apparently incapacitated B . burgdorferi to translocate further genetic templates into its hosts (Casjens et al . , Mol. Microbiol. 2000, 35, 490-516). Whereas point- mutations within the poly-A signal would impair the genetic expression, a change in the adenine content of polyadenylation tail (Alberts et al . , Molecular Biology of the Cell, 1994, Garland Publishing), would alternatively enhance or reduce the ribosomal translation of CBl. Several mutations at an early blastular stage can lead to different (chimeral) expressions of CBl (and perhaps other genes) and account for the reported continuum of major psychoses between schizophrenia and bipolar manic-depression . Dissimilarity and infectious recombination between protein coding sequences of the human chromosome associated protein hCAP and borrelia P115 (63% identities with P115) could, on the other hand, account for the altered leukocyte chromatin ultra-structure reported in schizophrenic patients.

Genetic epidemiology which has provided consistent evidence over many years that schizophrenia has a t t μ^»

Lπ σ LΠ σ Ul σ LΠ rt HI ii Ω TJ μ- tr tQ Hi 0 O TJ TJ rt n Cd cn _^— -_* Hi rt tQ Ω Ω μ- Ω

0 1 0 Φ H O ø μ- Φ tr 0 Cd if 0 tr td O Ω 0J 0 Φ Φ μ- Φ O ø O ι-3 ii Ω 0 en en ø 0 g » 0 en Φ ii ø rt ii Pi 0 rt ø 3 cn a rt Φ 0 Pi φ Φ ØJ Φ TJ tr • H μ- H rt Φ tr rt TJ Φ * f S 3 μ- Pi ii rt rt r Φ Φ rt cn ⁽Q Φ 0 - — - cn OJ H ii μ-

Φ μ- 0J ø^* ø rt μ- μ- pa en ft a - μ- TJ Φ H en Ω Pi Ω tr 0 Φ rt rt ø

0 μ- tQ rt Φ o Ω - ø $ o O μ- 0 μ- Ω tr O O 0 3 Φ OJ ø⁾ tr Pi ø tr Pi ø tr Φ 3 3 ø ⁽i Φ ø^j OJ μ- 0J 0 rt 0 φ Pi rt

Pi μ- ØJ ø⁾ Φ en Φ en Φ O ø O OJ 0 N Ω < i 1 ø μ- l_J. ø OJ rt P. Hi X ø^J Φ 0 H rt 0 Ω 0 tr 0 0 φ Φ 3 rt Hi 0

0J Hi μ- Ω tr tr TJ μ- _rO 0 ø⁾ Hi tr ii 0 PJ en TJ 0 ii 3 μ- ø^j tr

Ω rt rt ø

O 0⁾ O ϋ φ ii Ω ø Cd tQ rt 0⁾ μ- ii rt ø- rt en μ- Ω ii H en

Φ tr Φ ø Ω TJ rt ⁱTJ φ tr φ • O μ- Cd Φ tQ tQ H μ- H μ- Ω ^ TJ

0 Φ ii en Φ J 3 μ- cn ø 0 0 • rt μ- PJ 0J Φ rt O H o tr ft 0 rt φ tQ CO ø rt tr en ø 0J 0 o rt h-¹ 0 O ø 0 XX en rt Φ

TJ rt 3 ft Φ Φ Φ 0 μ- O H¹ 0 ^ tr H ØJ i μ- tr μ- rt Φ rt

0 ii tr μ- 3 ø ii O S •<; H ii 0 rt Hi _^~. tr ø^j Φ Hi μ- OJ ii 3 ^

0 Φ φ rt 0 φ TJ Φ ø tQ Φ μ- ii μ- Φ φ iϋ Φ • OJ h 0J

0 1 if O ø en ø⁾ TJ Φ

Cd Φ μ- PJ Ω ø tQ ø en ii 0⁾ Ω O μ-¹ ø Φ

1 μ- TJ 0 rt ØJ • O ø ø O 0 en PJ Ω μ- rt Hi to tr 3 0 Pi OJ ø

Φ ø 0 ØJ 1 ØJ rt Hi Ω rt ti 9 φ 0 μ-> Hi ^ rt O μ- 0⁾ 0 rt

^ cn μ- ø 3 li Φ tr ØJ O ii Hi iϊr H ϋ 0 H ø ø ø O cπ ø Ω O Φ rt

TJ Φ ø Ω μ- ^ 0 o PJ 0 φ μ- rt Hi en 0 rt en ø Ω rt M PJ H ø tr

O ii rt φ Ω O H ø) Cd Φ P. H ø μ- Φ μ- 3 tr P. φ if ^ Φ O Φ en rt CO ϋ en 0 tQ ø μ> CO ø μ- ØJ ø ii o Φ ø Φ ØJ O rt rt Ω rt ø

Φ Φ 3 0 rt PJ PJ • tQ μ- ø 0⁾ O 0 rt td ø rt tr en α P. 0 H tr ii ØJ O rt Φ TJ μ- •» 0 en ii ø tr Ω μ- Φ OJ TJ rt OJ μ- OJ 0 H NJ tr P. > 0 OJ O Ω TJ < Φ O O Φ P. ø μ-

Ω Hi OJ ø^j 0 Ω Hi tr φ μ» ø ø ø ØJ μ- μ- φ cπ 0 0 en φ Pi Ω ii

0 tr rt P. Φ φ rt cn rt ø Φ t tQ K Hi rt rt cn O φ O

0 ϋ μ- a cn H en H tr OJ ØJ o . 0 O μ- β μ- rt O H - 0 cn Ω ft TJ 0 a ø O rt μ- if μ- Φ 0⁾ 0 P. rt Ω Ω μ¹ H if ø⁾ XX rt tr ϋ 0 0 0 H o XX ø P. Pi cn ø tr ø td en φ O M rt ii ØJ

O O Ω ø⁾ Hi 0 TJ Pi μ- _rQ o cπ OJ en rt Cd O Φ ø⁾ Φ

H ø Hi rt a H tr cn O 3 rt H rt W Φ - — - 0 cn H¹ ϋ H rt ii Φ if n rt O rt μ- μ- l H3 rt Hi rt vQ ~

0 Φ cπ TJ if a Hi tr OJ en LΠ O 0^{^} 3 if en O . Hi ØJ if μ> 0 Φ cπ φ tα 3 H- rt tc 0J OJ ØJ φ 3 ø ii M tr rt ii (i ^ ii H¹ K

•9 >-3 CO μ- ι-3 OJ rt Pi OJ ØJ O 0 0 tr Φ tr rt μ- ] Hi ø (-^» 0⁾ O ¹ ø φ tr rt H tr 3 OJ rt rt Φ Φ Ω μ- H3 if X tr φ M Q tr Φ 0 M Pi μ- rt 0 O Ω en Φ ØJ Ω tr Φ td ii ø Φ OJ cn tr 0 ii •< n tr ØJ Φ ØJ li

Hi øJ H i 1 Pi ϋ Φ J TJ ø 3 ø ø tr μ- TJ

Ω ø en ^ ii μ- O O ø PJ cn μ- Φ

Φ H μ- φ Φ rt Φ 0 0! φ ii Φ <! P. OJ rt 0 Ω μ- OJ ϋ ø X OJ en 0 0 0 rt TJ cn Ω CO rt Φ ø Φ μ- PJ ØJ en p. H tr Φ Ω TJ ø

Φ Ω 0 tr Φ rt H μ- μ- TJ μ- H *« rt O Φ Φ OJ φ OJ Ω H 0 PJ

0 Ω Ω φ øJ TJ μ- OJ 0 øJ OJ ø rt φ Φ ø 0 rt cn 0 0 en

Hi O rt 0 Hi rt ii O rt ø μ- Φ tr Ω Ω — rt Φ tr 3 Pi Φ μ-

3 O ii ø μ- Φ Φ 0 μ- rt ii Φ rt o O Hi S μ- O r μ- PJ rt rt TJ H ii X Pi 1 en O en Φ μ- 0 ø tr ø i ii rt φ μ- ø cn tr 0⁾ rt φ rt ^ μ-¹ 0 μ- 3 3 ø - ft ft ϋ en Φ Φ tr Pi rt

Φ ϋ tr P. if < ii TJ ø tQ tQ 0 rt ⁴ li OJ rt TJ en ⁽Q ØJ if TJ μ- ^ φ μ- Φ øJ ø H rr 0 0 0J μ- tr 3 μ- M rt rt OJ φ i en en 3 tr H. ø Φ Ω 0 0⁾ ØJ OJ 0 0

0 ø φ O ø O O 0 μ- PJ Φ

OJ O μ- φ 1 X Pi rt ft 1 en μ- Hi 1 ø Ω O I

3 ø ø 1 1 3 φ μ- 1 J ØJ ø 0J φ 1 ø O

1 tQ 1 1

0 TJ TJ Ω ØJ 0 0⁾ tr

Hi 0 ii 0 p^j Ω O ii ro 3 Ω

H rt ø Φ 0 Φ μ-

0 Φ ØJ ft ii ii N

Hi PJ rt μ- cn rt O

Φ 0⁾ ø μ- μ- ø

Ω μ- H Hi X J Ω rt rt ø φ O ø ØJ OJ μ- to Ω μ- ii

0 if rt Pi μ- ø ø μ- ø rt rt en 3 tr o rt tQ ii ii

ØJ c ø μ- ØJ OJ α ø ii en Ω rt ø ø μ- ^ rX tr en en en tr Ω_ $ φ 3 Hi φ Φ 0 μ- μ- μ- φ

OJ μ- K. rt ø rt en Ω en ø Hi if rt H cn rt φ tQ Φ o O OJ μ- μ- en en H. ø ø 0 O

• μ. rt Φ en ø ø

—. tQ 3

TJ to μ- TJ ⁽Q μ- 0 0 tr rt ø H en en Hi Hi μ- Φ Hi μ- cn

Φ φ O 0⁾ μ- rt tQ

ØJ ii Ω ii ø o if φ i φ rt PJ ø Φ li φ μ- tr 3 μ- 0 H 0 PJ

TJ 0 0 0 OJ Hi μ- tr O H eo μ- μ- TJ tr Pi <! to φ ø ø⁾ ii 0⁾ ØJ ØJ Φ

• • μ- < 3 φ CO ø Φ Φ tr φ Ω

O Ω ØJ c • φ if H- μ- 3 i H¹

0 TJ ø - tr <Q >-3 M

H H P. μ- Pi if CO

Ω Φ Φ OJ Ω o μ- tr en φ ø tr ii en μ- 2

Pi Pi Hi - 0

H ø⁾ Ω ro 0

H ø tr cn 0⁾ ii μ- H

PJ Φ TJ 0 μ. ø PJ tr¹ Φ 0 μ- TJ ø ii rt Hi Hi

<! ii OJ tr ii 0⁾ Φ μ- ØJ ii P. 0 O Ω OJ ø Ω φ μ- cn 3 rt Pi tQ rt i Ω ^

1 μ- tr rt rt

Ω φ tr 0

Φ 1 Φ

stone, 2000, 2504-2518) . By adding genetically vulnerable cases to the population human germ-line transfection by B . burgdorferi would thus explain the continued presence of schizophrenia at high prevalence, despite the fact that the disease confers reduced procreational fitness and fertility.

The association between level of cannabis consumption and development of schizophrenia during a 15- year follow-up was studied in a cohort of 45,570 Swedish conscripts . The relative risk for schizophrenia among high consumers of cannabis (use on more than fifty occasions) was 6.0 (95% confidence interval 4.0-8.9) compared with non-users. Persistence of the association after allowance for other psychiatric illness and social back- ground indicated that cannabis is an independent risk factor for schizophrenia (Andreasson et al . , Lancet 1987 Dec 26; 2 (8574:1483-6) .

Example 5

Significant CBl-Dl protein homology versus CB1-D2 nucleotide homology

There are significant homologies, which cannot be reduced to lateral gene transfer. CBl shows sig- nificant homology with the dopamine Dl - a type I G- protein coupled receptor - at the amino-acid level (Score = 71.0 bits (171), Expect = 2e-ll, Identities = 75/320 (23%), Positives = 137/320 (42%), Gaps = 50/320 (15%) (Online Mendelian Inheritance in Man, OMIM 2000, Center for Medical Genetics, John Hopkins University

(Baltimore, MD) and National Center for Biotechnology Information, (Bethesda, National Library of Medicine, 2000) . Other homologous proteins comprise G-protein coupled receptors that receive information from outside the cell, such as olfaction or vision. However, owing to one specific sequence of 19 homologous base pairs at the nucleotide level, it is the dopamine 2 receptor (D2) - a type II G-protein coupled receptor - which shows significant DNA homology to CBl (Score = 37.2 bits (19), Identities = 19/19 (100%) . The circumscribed homology between CBl and the D2 receptor, which is an important phar acol- ogical target for the treatment of schizophrenia, encodes the seventh transmembrane loop which is known for its inhibitory-mode (i-mode) of metabotropic action. Apart from a silent point mutation from GTG to GTC in the rat D2 receptor gene (Rattus norvegicus) , which has occurred after the phylogenetic rat mouse divergence 35 million years ago, this homologous nucleotide sequence can be found an all sequenced primate (Macaca mulatta, Cercopithecus aethiops, H. sapiens; OMIM, 2000) and rodent (Mus muscu- lus) D2 receptor genes. An early prenatal event interfering with neu- ronal migration from inner to outer cortical laminae in mid pregnancy most likely underlies the consistent pattern of cellular disarray observed in schizophrenic brains. If the expression of higher levels of CBl within outer cortical compared to inter-cortical layers (Glass et al., 1997, Neuroscience, 77, 299-318) resulted from a CBl mediated migration of neurons, the inter-cortical disarray could be explained by a knock-out of CBl, whose metabotropic i-mode has recently been reported to be cru- cial for cellular migration (Song and Zhong, Journal of Pharmacology and Experimental Therapeutics, 2000, 294, 204-209) . The distribution of CBl, furthermore, exactly mirrors the macro-anatomic regions mainly affected in schizophrenia (Schultz and Andreasen, 1999, Lancet, 353, 1425-30) .

Through a dysinhibition of the i-mode, spatial memories (hippocampal long-term potentiation) of CBl knock-out mice are enhanced (Bohme et al . , 2000, Neuroscience, 95, 5-7) and goal directed, temporal memories decreased. This mnemonic effect, however, not only parallels the pattern of spatio-temporal distortions in schizophrenia. Despite the fact that patients with Alz- heimer's dementia and tertiary neurosyphilis do hallucinate, they apparently do not remember, or reconnect their hallucinations with a fixed delusion. The difference between dementia praecox (schizophrenia) and syphilitic de- mentia is that without memory hallucinations are lost, and that the thoughts of schizophrenics are flooded with fixed hallucinations, expanding into overt delusions. Furthermore, CBl knock-out mice do show reduced exploratory, goal-directed behaviours (Steiner et al . Proc. Natl. Acad. Sci. USA, 2000, 96, 5786-5790); symptoms that appear to be among the most robust indices in schizophrenia. Without time-bridging working memory, no creative speech and no logical thinking would be possible. A disruption of CBl, which reaches its highest levels in the left-hemispheric area of Wernicke, would thus account for the impairment of goal directed behaviour and speech - the highest form of sequential behaviour in man - being more stereotyped in schizophrenic patients than in healthy persons.

Example 6

The phylogenetic traces of microbial insertions into the human genome were investigated and the gene- tic mechanism were analysed, by which bacterial virulence factors and mobile elements from intracellular parasites could disrupt candidate genes for schizophrenia and dementia. This was done by co-incident DNA homology BLAST searches between neurotropic microorganisms, the central cannabinoid receptor CBl, and other known dementia genes, whose complete sequences with 3'UTR and polyadenylation signals are entered on Gene-Bank databases. Several such genes have now been characterised including those of ApoE4 Alzheimer Disease type II (AD2) and presenilin 2 (AD 4) . Positional cloning and sequencing has also been carried out in other dementias including Familial British Dementia (with mutations in the gene for integral membra- ne protein 2A) , Hereditary Multi-infarct Dementia (Notch- 3 gene) , primary X-linked mental retardation (oligo- phrenin 1 gene) , Frontotemporal Lobe Dementia, Autosomal Dominant Parkinson Lewy-Body Dementia and Familial Par- kinson disease type I (with mutations in the alpha synuclein gene) . The genomic facilities used are accessible at Online Mendelian Inheritance of Man (OMIM) and very much recommended to anybody interested in applied medical genetics . While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Genes Organism Sequence Seq. Id. No.

& Events

Table 1A SCHIZOPHRENIA

CBl 1st & 2nd borrelia integrations into ancestral 5HT-CB1 and introduction of poly-A signal

Borrelia (fbrp) (357) CATTTCTTCAACTAAATTAACATT ( 33 ) 5 ' 18 5HT1E/ AttCATTTCTTCAACTAAATTAACATT 19

CBl ( anc .1) AttCATTTCTTCAACTAAATTAACATT

6ql4 Borrelia (P115)(4133) GATTCAAATAAAAATTCTAAATTACCAT (4160) 3 ' 1

Chlamydia integration into ancestral rodent CBl Chlamydia (8221) TTACCTGGACTCAAATAAAAGT(8242) 3 ' 20

CBl (anc.2) (ibp) GAATCAAATAAAAATTCTAGATTACCATgaagaacata 22

CBl (rat) (5430) TTACCTGGAATCAAATAAAAGTTCTAGATTATCACg (5465)3' 21

Chlamydia uridarum integration into ancestral primate CBl

Chlamydia (8219) AGTTACCTGGACTCAAATAAA ( 8240 ) 3 ' 2

CBl (anc.2) (ibp) GAATCAAATAAAAATTCTAGATTACCATgaagaacata 22

CBl (human) /6ql4 (5484) AGTTACCTGGAATCAAATAAAAATTCTAGATTACCATgaagaacata(5530) 47

Chlamydia (1063) AGTTTAATAAAGATT (1049)5' 4

Apo E (anc.) GTTTAATAAAAATT 50

Apo E (4611) AGTTTAATAAAGATTca(4627)3' 27

borrelia infection - predicted mutations by virulence factors

Borrelia (acrB) (8106) TTTAAAAAAGATTCA (8120)3' 28

Borrelia (comp.loc) (11560) TTTAAAAAAGATTCA (11546)5' 28

PRESENILIN 2 (PS2) - Alzheimer's disease type 4 (AD4)

Borrelia burgdorferi (sgp) stability governing protein integrated within presenilin 2 and possible predicted mu- tation by Borrelia reinfection

Borrelia (sgp) (10599) ATACTAATATCAATAA (10614)3' 5

PS 2 (1651) ATACTAATATCAATAAa(1667)3 ' 29 Borrelia (sgp) reinfection (10599) ATACTAATATCAATAA (10615) 3 ' 30

AMYLOID PRECURSOR PROTEIN (APP) Alzheimer's disease type 1 (protease nexin-II)

No Borrelia but has Plasmodium falciparum inclusion and Salmonella typhimurxum transposable plasmid

APP (3547) TTTTCATGTAAATAAATACATTCT(3570)3' 31 Salmonella plasmid traJ (425) TGTAAATAAATACATTCT(442) 3 ' 48

Plasmodium chromosome 3 (79642) TTTTCATGTAAATAAATA (79625)5' 6

(hypothetical protein) (141360) TTTCAGGTAAATAAATA (141344)5' 32

■

FAMILIAL BRITISH DEMENTIA (xntegral membrane protexn 2B gene) Borrelxa burgdorferi Ixnear plasmxds lp25, lp36 and Plasmodium falσiparum major merozoite surface & receptor binding protein

FBD (1753) GATTTTTTCTTTAAATAAAAATAAGT(1778)3' 33

Borrelia lp25 plasmid (18048) TTTAAATAAAAATAAG (18032) 5 ' 7

Borrelia Ip 36 plasmid (9312) TTTAAATAAAAATAAG (9327)3' 51

Plasmodium (mmsp) (1083) TTTTTTTTTTAAATAAAAATA (1103)3' 34

Plasmodium (pfempl) (4682) GATTTTTTCTTTAGATAAAAATAAG (4658)5' 8

Oligophrenin 1 (OPHNl)

Borrelia burgdorferi inclusion from tryptophanyl-t-RNA synthetase

OPHN 1 (6648) CAAATAAAGTAGTAAAAGA (6666) 3^V 56

Borrelia (trsa) (101) CAAATAAAGTAGTAAAAGA (83) 5 17

HEREDITARY MULTI-INFARCT DEMENTIA (NOTCH 3 gene)

No Borrelia or plasmodxum, but has vibrio cholera virulenenσe factor and Clostrxdxum insertion

DHMI (8048) CCTAATAAAGGAATAGTTAAC (8068)3' 35

Vibrio (ntno) (4641) AATAAAGGAATAGTTAA (4657)3' 52 Clostridium (ctfA) (2544) CCTAATAAAGGAATAG (2559)3' 53

FRONTOTEMPORAL LOBE DEMENTIA (gene for microtubule associated protein tau)

No Borrelia or plasmodium, but has Staphylococcus aureus antxbxotxc resxstance plasmid

MAP Tau (2276)GCTAGTAATAAAATAT(2291)3' 36

Staph. Plasmid pS194 (2276) GCTAGTAATAAAATAT (2291) 3 ' 36

PARKINSON DISEASE

AUTOSOMAL DOMINANT LEWY BODY (PDLBD) (alpha synuclein gene) Borrelia burgdorferi inclusion from linear plasmid lp36

PDLBD

2nd poly A signal (1521) ACAATAAATAATATTC (1536) 3 ' 37

Borrelia lp36 (13825) ACAATAAATAATATTC (13810) 5 ' 9

PARKINSON DISEASE, FAMILIAL TYPE I (PDl) (alpha synuclein gene)

Borrelia burgdorferi linear plasmid lpl7, Plasmodium falciparum plasmid and Escherichia coli pilus protein PD - 1

3rd poly A signal (952) TAATAATAAAAATCATGCT (971) 3 ' 49

Borrelia lpl7 plasmid (9683) TAATAATAAAAATCAT (9668)5' 10 Plasmodium plasmid (10913) AATAATAAAAATCATG (10928)3' 12

Escherichia (pilus protein) (860) AATAATAAAAATCATGCTT(878) 3 ' 11

Table 1C

MYOPATHY

MYOTONIC DYSTROPHY, 3' UTR & TRIPLET REPEAT

Borrelia burgdorferi and Chlamydia muridarum integration into human myotonxn protein kinase (Mt-PK)

Borrelia (fbrp) (194) TCCGGAATAAAAGGCCCT(177) 5 ' 13

(Mt-PK, anc.) TCCGGAATAAAAGGCCCT 38

(Mt-PK) (2462) TCGCGAATAAAAGGCCCT(2479)3 ' 39

Chlamydia (3551) GCGAATAAAAGGCCCT (3536) 5 ' 14

Table 1 D

GLOBIN GENES - CONTROLS FOR MICROBIAL INSERTIONS β-globin (chromosome 11) β-globin chain versus β-thalassaemia

Poly-A cleavage site (1736 - 1737)

Normal β-globin (1711) AATAAAAAACATTTATtttcattgca atgATGTATTTAAATta (1753) .. 40

Rickettsia (232700) AATAAAAAACATTTAT (232685) 5 ' 15 Borrelia (oppAIV) (1143) ATGTATTTAAAT (1132) 5' 41

normal β- globin ;i754) tTTCTGAATATTTTACTAAAAA ( 1775 ) 3 ' 42 Borrelia (ospC) (364) TTCTGAAGATTTTACTAAAA (384)3' 43

β-Thalassaemia ( 1748 ) AATAAGAAACATTTATTTtcattgca ( 1773 ) 3 ' 44 Plasmodium falciparum (470) ATAAGAAACATTTATTT (486)3' 45

α-globin (chromosome 16) α-globin chain (NM_000558) in lower case letters versus Plasmodium bergei (L21708) within 3' α-globin in upper case letters 46

α-1 and α-2 1 actcttctgg tccccacaga ctcagagaga acccaccatg GTGCTGTCTC CTGCCGACAA 61 GACCAACGTC AAGGCCGCCT GGGGTAAGGT CGGCGCGCAC GCTGGCGAGT ATGGTGCGGA 121 GGCCCTGGAG AGGATGTTCC TGTCCTTCCC CACCACCAAG ACCTACTTCC CGCACTTCGA 181 CCTGAGCCAC GGCTCTGCCC AGGTTAAGGG CCACGGCAAG AAGGTGGCCG ACGCCCTGAC 241 CAACGCCGTG GCGCACGTGG ACGACATGCC CAACGCGCTG TCCGCCCTGA GCGACCTGCA 301 CGCGCACAAG CTTCGGGTGG ACCCGGTCAA CTTCAAGCTC CTAAGCCACT GCCTGCTGGT 361 GACCCTGGCC GCCCACCTCC CCGCCGAGTT CACCCCTGCG GTGCACGCCT CCCTGGACAA 421 GTTCCTGGCT TCTGTGAGCA CCGTGCTGAC CTCCAAATAC CGTTAAGCtg gagcctcggt 481 ggccatgctt cttgcccctt gggcctcccc ccagcccctc ctccccttcc tgcacccgta 541 cccccgTGGT CTTTGAATAA AGTCTGAGTg ggcggc 3 '

Table IE

VIRULENCE FACTORS AND OTHER MICROBIAL INSERTIONS

Borrelia burgdorferi (pll5) : related to human chromosome associated protein responsible for DNA and intracellular movement .

Borrelia b. (fbrp): flagellar basal rod protein for extracellular movement , chemotaxis , syringe mechanism for cell injection.

Borrelia b. (ospA) : outer surface protein A, antigen of genetically induced variation.

Borrelia b. (plasmid lp28-l) : pseudogene on linear plasmid size 28, group 1, being the result of genomic decay.

Borrelia b. (lip P) : lipoprotein P (homologous sequences on plasmids, i.e. Ip38), anti-genetic surface protein.

Borrelia b. (acrB) .-acriflavine resistance protein.

Borrelia b. (comp. loc) : competence locus with multiple homologous copies throughout genome. Borrelia b. (sgp) : stability governing protein for stabilisation of membrane.

Borrelia b. (oppAIV) : oligopeptide permease; immediately adjacent to 3 'poly-A cleavage site of (globin.

Borrelia b. (ospC) : outer surface protein C; 18 base pair distant from 3' poly-A cleavage site of (globin. Target of Borrelia vaccine.

Borrelia b. (pus) : pseudo uridylate synthase Borrelia b. (slipp) : surface lipoprotein p27

Borrelia b. (trsA) : tryptophanyl t-RNA synthetase

Borrelia garini (ospA) : outer surface protein A. Chlamydia muridarum (ibp) within CBl: iron binding protein to overcome host barriers of low iron levels .

Chlamydia m. within Mt-Pk: phosphocarrier protein. Chlamydia pneumonae: hypothetical protein.

Clostridium beijerinckii (ctfA): small subunit of coenzyme A transferase.

Escherichia coli pilus protein: responsible for cellular adherence and infection.

Plasmodium bergei within ( globin series: phosphoprotein mRNA.

Plasmodium falciparum within CBl: rhoptry associated protein (264) ATCAAATAAAAGTTCTA ( 280 ) 3 ' for erythrocyte penetration. Plasmodium f . within amyloid precursor protein: several sequences form chromosome 3 including hypothetical pro- teins .

Plasmodium f. (mmsp) :major merozoite surface protein, expressed during sexual stage.

Plasmodium f. (pfempl) : Plasmodium falciparum-encoded protein on the surface of infected erythrocytes mediates receptor binding.

Plasmodium f . in thalassaemia (globin: RNA polymerase III. Rickettsia prowazekii in (globin: proline-betaine transporter for the reduction of osmotic stress in host environment .

Vibrio cholerae (ntno) : Na+-translocating NADH-ubiquinone oxidoreductase enzyme complex involved in flagella rotation.

Microbial virulence factors within the 3' genetic hotspot of human disease. Base pairs originating form the late- ral gene transfer of microbial nucleotides are indicated in upper case, and non-microbial nucleotides in lower case letters. There are normally no point mutations on the polyadenylation signal, whose non-redundant code protects the microbial inclusions from mutations. Homologous recombination between B. burgdorferi pll5 and the bor- relia template fbrp originating from ancestral 5HT1E has introduced the polyadenylation signal AATAAA into CBl. Note that recombinational mismatch-repair has inserted an additional adenine into CBl. A point mutation from A to G has through a reduction of the adenine content led to a shortening of the 3' polyadenylation tail of the rat. Within human CBl and rat CBl, almost identical, but independent insertions of C. muridarium nucleotides must have occurred twice. This reoccurrence emphasises the attraction CBl and its polyadenylation signal exerts on microbi- al DNA to recombine . In the case of dissimilarity (i.e. AATATA) mismatch repair mutations might, in analogy to the genetic knock-out of globin in thalassaemia, result in a knock out of CBl and other neurological candidate genes .

3rd borrelia infection - predicted mutations by vxrulence factor or plasmid of human CBl poly-A signal

Borrelia burgdorferi (ospA) (41) ATAATAATTCTAAATTA (25 ) 5 ' 24 Borrelia garinii (ospA) (229) ATAATAATTCTAAATTA (213) 5' 23

Borrelia (plasmids ;^"i .e. Ip28-1) (7553) AATATAAATTCTATAT (7568)3' 25

Microtubule associated protein 2C (MAP2C)

Borrelia burgdorferi inclusion from pseudouridylate synthetase MAP2C (3634) AAACTCAGAAAATAAAATGT 3^λ (3653) 54

Borrelia (pus) (2645) AAACTCAGAAAATAAAATGT 5^l (2626) 16

Borrelia (slipp) reinfection (40665) CAGAAAATAAAAT 5 (40653) 55

Table IB

DEMENTIA

APOLIPOPROTEIN E (Apo Ξ) Alzheimer's diease type 2 (AD2)

Borrelia burgdorferi surface protein integrated within ancestral ApoEe4

Borrelia (lipP) (11) GTTTAATAAAAATT(24) 3' 3

Apo E (anc . ) GTTTAATAAAAATT 26

Chlamydia pneumoniae integration into ancestral ApoEe4

Claims

Claims

1. Use of a DNA sequence comprising a frag- ment of a nucleic acid encoding a putative microbial virulence factor as means for the identi ication of a disease caused by mutations or a genetic predisposition thereof .

2. Use of claim 1 wherein said virulence fac- tor is located on a linear or cirular chromosome or a plasmid.

3. Use of claim 1 or 2 wherein said virulence factor stems from a intracellular microorganism.

4. Use of claim 1 or 2 wherein said virulence factor stems from a non-intracellular pathogen and is part of a cluster shared by intracellular microorganisms.

5. Use of claim 1 wherein said microorganism is selected from the group consisting of Borrelia species, Chlamydia species, Escherichia sp., Plasmodium spe- cies and Rickettsia species.

6. Use of anyone of claims 1 to 5 wherein said fragment is selected from the group consisting of Seq. Id. No. 1 to Seq. Id. No. 17.

7. Use of anyone of claims 1 to 4 wherein said sequence comprises a mutation, either caused by by the same or a different species, preferably within the polyadenylation signal sequence.

8. Use of anyone of claims 1 to 7 wherein said disease is a human disease.

9. Use of claim 8 wherein said human disease is selected from the group consisting of schizophrenia, Alzheimer disease, Parkinson disease, Myopathy and other forms of dementias .

10. Use of claim 8 wherein said human disease constitutes a predisposition or a genetic variation, the pathological manifestation of which is triggered by medicaments or drugs .

11. Use of claim 10 wherein said drug is cannabis .

12. Use of claim 11 wherein said pathological manifestation comprises any form of dementia, schizophre- nia, or related psychatric disorders.

13. A method for the identification of a disease or a genetic predisposition thereof, which comprises detecting the presence in a tissue-or blood sample of a subject a mutation within a nucleic acid sequence se- lected from the group consisting of Seq. Id. No .1 to Seq. Id. No. 17 and said sequence is part of a gene of said sub ect .

14. The method of claim 13 wherein said tissue sample is a foetal graft for neurotransplantation.

15. The method according to claim 13 or 14, wherein said sequence is inserted in the 3'UTR of said gene .

16. The method according to anyone of claims 13 to 15, wherein said mutation is found in the polyade- nylation signal of said gene.

17. The method according to anyone of claims 13 to 16, wherein said mutation affects the expression of the protein encoded by said gene .

18. The method according to anyone of claims 13 to 17, wherein said gene is selected from the group consisting of Cannabinoid receptor 1 gene, MAP 2C gene, apolipoprotein E gene, presenilin 2 gene, integral membrane protein 2B gene, , alpha synuclein gene, oligo- phrenin 1 gene and myotonin protein kinase gene.

19. A transgenic non human animal whose genome comprises a partially or completely inactivated endogenous gene as defined in claim 18, wherein said inactivation is due to at least one mutation in its 3 ' untranslated region, said mutation leading to inhibition or suppression of the subsequent gene translation.

20. The transgenic non-human animal of claim 19, wherein the mutation is located in the nucleic acid sequence following the polyadenylation signal, more preferably in the polyadenylation sequence of said gene.

21. The transgenic non-human animal of claim 20, wherein said mutation is a point mutation.

22. The transgenic non-human animal according to anyone of claims 19 to 21, wherein said animal is a mammal, in particular a rodent.

23. The transgenic non-human animal of claim 22, wherein said animal is a mouse or a rat.

24. The transgenic non-human animal according to anyone of claims 19 to 23, wherein said inactivation is a homozygous or a heterozygous inactivation.

25. Use of a transgenic non-human animal according to anyone of claims 19 to 24 for the identifica- tion of compounds that have an effect on the activity, expression or regulation of the translated protein.

26. A method of screening compounds that have an effect on the activity, expression or regulation of a protein encoded by a gene according to claim 18 compris- ing introducing a compound in an animal according to anyone of claims 19 to 24 and monitoring behavioural changes in said animal as compared to a control animal .

27. Use of a transgenic non-human animal whose genome comprises a non-functional endogenous CBl gene for the identification of compounds that have an effect on the activity, expression or regulation of CBl protein.

28. A DNA and/or RNA chip comprising at least one of the nucleic acid sequences selected from the group consisting of Seq. Id. No. 1 to Seq. Id. No. 17.