WO1998059050A1 - Clonage d'une mutation genique permettant de deceler la maladie de parkinson - Google Patents

Clonage d'une mutation genique permettant de deceler la maladie de parkinson Download PDF

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WO1998059050A1
WO1998059050A1 PCT/US1998/013071 US9813071W WO9859050A1 WO 1998059050 A1 WO1998059050 A1 WO 1998059050A1 US 9813071 W US9813071 W US 9813071W WO 9859050 A1 WO9859050 A1 WO 9859050A1
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mutation
synuclein
nucleic acid
protein
disease
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PCT/US1998/013071
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WO1998059050A9 (fr
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Mihael H. Polymeropoulos
Christian Lavedan
Elisabeth Leroy
Robert L. Nussbaum
William G. Johnson
Roger C. Duvoisin
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The Government Of The United States Of America Represented By The Secretary, Department Of Health And Human Services
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Priority to AU81637/98A priority Critical patent/AU8163798A/en
Priority to US09/446,628 priority patent/US7001720B1/en
Publication of WO1998059050A1 publication Critical patent/WO1998059050A1/fr
Publication of WO1998059050A9 publication Critical patent/WO1998059050A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Parkinson's disease is a common neurodegenerative disorder with a lifetime incidence of approximately 2 percent.
  • a pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4.
  • This finding of a specific molecular alteration which is causative for PD will permit the detailed understanding of the pathophysiology of the disorder.
  • methods of screening nucleic acids for the presence of mutations in the synuclein gene to test for predisposition to Parkinson's Disease are now possible.
  • Parkinson's disease was first described by James Parkinson in 1817 (2).
  • the clinical manifestations of this l ⁇ . neurodegenerative disorder include resting tremor, muscular rigidity, bradykinesia and postural instability.
  • a relatively specific pathological feature accompanying the neuronal degeneration is the intracytoplasmic inclusion body, known as the Lewy body, which is found in many regions including the substantia nigra, locus ceruleus, nucleus basalis, hypothalamus, cerebral cortex, cranial nerve motor nuclei, and the central and peripheral divisions of the autonomic nervous system (1).
  • a heritable factor predisposes to the development of the clinical syndrome (2) .
  • PD including Lewy bodies, with the exception of a relatively earlier age of onset of illness at 46 ⁇ 13 years. In this family the penetrance of the gene has been estimated to be 85%, suggesting that a single gene defect is sufficient to determine the PD phenotype.
  • the Ala53Thr substitution is localized in a region of the protein whose secondary structure predicts an alpha helical formation, bounded by beta sheets. Substitution of the alanine with threonine is predicted to disrupt the alpha helix and extend the beta sheet structure. Beta pleated sheets are thought to be involved in the self aggregation of proteins which could lead to the formation of amyloid like structures (6).
  • NAC35 the 35 amino acid peptide derived from alpha-synuclein that was first isolated from plaques found in patients with Alzheimer's disease ( 4) .
  • NAC35 was shown to self aggregate and form amyloid fibril which shared the amyloid' characteristics of insolubility in aqueous solutions and green birefringence under polarized light, subsequent to Congo red staining (6).
  • NAC35 is located in the middle of the alpha synuclein molecule and extends from amino acid 61 to amino acid 95.
  • Residue 53 which is found to be mutated in PD, is outside the NAC35 peptide found in amyloid plaques.
  • NAC peptide involved in the plaques is not known since the protease used to isolate the peptide from AD tissue cuts at lysine 60 of the alpha synuclein protein. It is therefore possible that amino acid 53 may be part of the NAC peptide found in plaques.
  • Beta amyloid Abeta
  • residues 1-56 and 57-97 specifically bind amyloid and that a synthetic peptide consisting of residues 32-57 performed similarly.
  • Three members of the synuclein family have been characterized in the rat, with SYNl exhibiting 95% homology with the human alpha-synuclein protein (7) .
  • SYN 1 of the rat is expressed in many regions of the brain, with high levels found in the olfactory bulb and tract, the hippocampus, dentate gyrus, habenula, amygdala and piriform cortex, and with intermediate levels in the granular layer of the cerebellum, substantia nigra, caudate-putamen, and dorsal raphe (7).
  • This pattern of expression coincides with the distribution of the Lewy bodies found in brains of patients with Parkinson's disease.
  • the zebra-finch synelfin carries a threonine at amino acid 53, whereas both Bos taurus and Torpedo californica do not (10) .
  • phenotype could be explained by a combination of factors, including the following: the relative short life span of rodents may prohibit the observation of a late onset disorder, interaction with other cellular components not present in the rat may be required for the phenotype, absence of a critical environmental trigger in the rodents, or finally a heterozygous status Ala/Thr may be necessary for the production of a phenotype.
  • missense mutations can cause an adult onset neurodegenerative disorder. Of the 31 mutations described so far in the loci for presenilin 1 and 2, thirty were missense and one was a splice variant (11) . Missense mutations in the prion protein have also been implicated in the amyloid production seen in Gerstmann-Straussler-Scheinker and Creutzfeld-Jakob diseases, both forms of spongiform encephalopathy (12) . Studies in these neurodegenerative disorders have pointed to the importance of the physical chemical properties of mutant cellular proteins in initiating and propagating neuronal lesions leading to disease. Similar studies in the synuclein protein family may provide valuable insights into the etiology and pathogenesis of PD.
  • the mutation identified in the alpha synuclein gene is unlikely to account for the majority of sporadic and familial cases of PD. However, this mutation may account for a significant proportion of those families with a highly penetrant, early onset autosomal dominant PD phenotype.
  • the present invention includes an isolated nucleic acid comprising a mutated synuclein gene.
  • the isolated nucleic acid of the present invention contains at least one mutation in the alpha synuclein gene at base pair position 209 of Genbank # L08850, which, in particular, is a change from guanine to adenine.
  • PD Parkinson's Disease
  • the present invention also includes a method of using a synuclein gene sequence to identify specific PD mutations. Such mutations may occur in an unrelated population or in a family that demonstrates passage of PD within the family tree.
  • the present invention also includes oligonucleotides complementary to a portion of the synuclein gene, wherein said portion comprises or flanks a mutation associated with predisposition to Parkinson's Disease.
  • the oligonucleotides of the present invention will have a sequence that is complementary to a sequence from the alpha synuclein gene that includes or flanks base pair position 209.
  • this mutation is a change from guanine to adenine at this position.
  • Vectors comprising an isolated nucleic acid encoding a mutated synuclein gene will allow the production and isolation of the mutant protein in an appropriate host cell using techniques well known in the art.
  • peptides may be chemically synthesized using techniques also well known in the art. Isolation of such a protein or peptides thereof will allow the study of the molecular mechanisms which lead to development of Parkinson's disease.
  • the present invention also includes an isolated synuclein protein or peptide containing at least one mutation.
  • this mutation is at a position corresponding to the fifty-third amino acid in the native alpha synuclein protein, and in particular, this mutation is an alanine to threonine substitution.
  • Peptides corresponding to portions or the entirety of a synuclein gene may be useful as drugs for inhibiting the self-aggregation of mutant proteins that is thought to lead to Parkinson's disease.
  • the present invention includes a method of testing peptides and other compounds for the ability to interfere with this self-aggregation. Self- aggregation can be tested using a number of established methods, including Congo red staining, electron microscopy
  • Possession of isolated synuclein proteins or peptides will also allow the isolation of specific antibodies using techniques well known in the art. Such antibodies may distinguish a mutant synuclein protein from its wildtype counterpart, and therefor could also be used in diagnostic screens. Alternatively, such antibodies may also be used to inhibit the self-aggregation of proteins during the progression of Parkinson's disease. Accordingly, the present invention also includes antibodies specific for a mutated synuclein protein or peptide. It should be understood that useful derivatives of such antibodies, such as Fv fragments and Fab fragments, are also included. The above aspects of the present invention will allow methods of detecting subjects at increased risk for Parkinson's Disease.
  • Such a method comprises obtaining a sample comprising nucleic acids from the subjects, and detecting in the nucleic acids the presence of a mutation which is associated with Parkinson's disease.
  • the mutation detected by the method of the present invention is located on human chromosome four, preferably in the alpha synuclein gene.
  • the mutation causes an amino acid substitution at position 53 of the alpha synuclein gene, which is, in particular, an alanine to threonine substitution.
  • the detecting step of the method of the present invention may be accomplished several different ways as will be described in further depth below. All such methods are well known to those of ordinary skill in the art.
  • said detecting step may comprise combining a nucleotide probe which selectively hybridizes to a nucleic acid containing a mutation associated with a predisposition to Parkinson's disease, and detecting the presence of hybridization.
  • a probe may be an oligonucleotide that is complementary to a portion of the synuclein gene, wherein said portion comprises the mutation.
  • such an oligonucleotide is complementary to a mutated alpha synuclein gene having at least one mutation at base pair position 209. In particulc.r, this mutation is a change from guanine to adenine .
  • the detecting step of the method of the present invention may also comprise amplifying a nucleic acid product comprising said mutation, and detecting the presence of said mutation in the amplified product using any nucleic acid sequencing procedure known in the art.
  • the detecting step may comprise selectively amplifying a nucleic acid product comprising said mutation, and detecting the presence of amplification using any appropriate method known in the art.
  • Such methods include gel electrophoresis of amplified nucleic acids, and detection of radiolabeled amplified nucleic acids using autoradiographic film or any other detection method known in the art.
  • the amplifying step of the present invention may be performed using the polymerase chain reaction (PCR) , reverse transcriptase PCR (RTPCR) , or any other type of PCR reaction known in the art. Accordingly, such a step will comprise at least one annealing step whereby at least one oligonucleotide is annealed to said sample of nucleic acids.
  • said amplifying step uses two oligonucleotides. And in particular, the two oligonucleotides have the sequences given in SEQ ID NOs 2 and 3.
  • the detecting step of the method of the present invention comprises detecting the presence or absence of a restriction endonuclease site as detected by enzymatic digest of a nucleic acid sample.
  • a detecting means will be possible when a mutation associated with a predisposition to Parkinson's disease results in a sequence having a new restriction endonuclease cleavage site, or loss of a native restriction endonuclease site.
  • the mutation associated with Parkinson's disease results in the formation of a non-native Tsp45I restriction endonuclease site.
  • the detecting step of the present invention may be performed using a gene-specific primer and subsequent chain termination at the position of the mutation using DNA polymerase and labeled nucleotides or dideoxynucleotides .
  • the presence of nucleic acids in which a dideoxynucleotide corresponding to the mutation of interest is incorporated at the appropriate position may be detected by any means known in the art, including detection of radiolabeled dideoxynucleotides using, for example, autoradiographic film, or detection of fluorescently-labeled dideoxynucleotides .
  • the present invention also includes diagnostic kits which include the compounds of the present invention in a form that allows such compounds to be used quickly and easily for the designated purpose.
  • the inventors also contemplate that the isolated nucleic acid, oligonucleotides and antibodies of the present invention may eventually be used in methods directed at the correction or suppression of Parkinson's disease.
  • oligonucleotides or expression vectors designed from the synuclein nucleic acid sequences of the present invention may one day be used in antisense therapy directed at inhibiting expression of the mutated synuclein protein in patients with Parkinson's disease, or in individuals having a predisposition for Parkinson's disease.
  • antibodies specific for the mutated synuclein protein may be useful in therapies directed at inhibiting the self- aggregation of mutated proteins or peptides in patients having Parkinson's disease.
  • DNA sequence of the PCR product used for mutation detection (SEQ ID NO 1) .
  • Oligonucleotide primers are shown by arrows and the numerals 3 and 13 (SEQ ID NO 2 and 3) .
  • Intron sequence is shown in lower case and exon sequence in upper case.
  • Amino acid translation of the exon is shown below the DNA sequence.
  • the circled base represents the G209A change in the mutant allele.
  • the resulting amino acid Ala53Thr change is represented by the circled amino acid.
  • the newly created Tsp45 I site is indicated above the DNA sequence .
  • Mutation analysis of the G209A change is shown in a subpedigree of the Italian kindred. Filled symbols represent affected individuals. Numerical identifiers, denote the individuals immediately above. Tsp45 I digestion of PCR products is shown at the bottom of the figure, and fragment sizes are indicated on the right in base pairs.
  • FIG. 3 Mutation analysis of the G209A change in RT PCR products (7).
  • Lane 1 100 bp ladder, lanes 2 and 3 normal control, lanes 4 and 5 PD patient, lane 6 negative control without RT enzyme. Sizes are indicated on the right in base pairs.
  • Lanes 2 and 4 show uncut DNA and lanes 3 and 5 show DNA cut with Tsp 5 I .
  • FIG. 8 Sequence of BAC clone 139A20 for human beta synuclein. BAC clone was isolated using primers to known database sequences described in Figure 7. The sequence shown includes all coding exon sequences and some non-coding intronic sequences. (SEQ ID NO:ll)
  • This invention provides a method of diagnosing or predicting a predisposition to Parkinson's disease.
  • the method comprises detecting in a sample from a subject the presence of a mutation, for example, in nucleotide position 209 of the human alpha synuclein gene.
  • the presence of the mutation indicates the presence of or a predisposition to Parkinson's disease.
  • the term "gene” primarily relates to a coding sequence, but can also include some or all of the surrounding or flanking regulatory regions or introns .
  • gene specifically includes artificial or recombinant genes created from cDNA or genomic DNA, including recombinant genes based upon splice variants.
  • synuclein gene or protein may refer to the alpha synuclein gene or any homologue thereof.
  • a “homologue” is understood to mean any related gene or protein that is at least 25% homologous to the alpha synuclein gene or protein or performs a related function.
  • a synuclein gene or protein refers to alpha, beta or gamma synuclein, but most preferably refers to alpha synuclein.
  • an "isolated nucleic acid” is a ribonucleic acid, deoxyribonucleic acid, or nucleic acid analog comprising a polynucleotide sequence that has been isolated or separated from sequences that are immediately contiguous, i.e. on the 5' and 3' ends, in the naturally occurring genome of the organism from which it is derived.
  • the term therefor includes, for example, a recombinant nucleic acid which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule independent from any other sequences.
  • An isolated nucleic acid of the present invention may be "operatively linked” to an expression control sequence or regulatory region.
  • "operatively linked” means that the components are joined in such a way that the expression, transcription or translation of the sequence is under the influence or control of the regulatory region.
  • a "predisposition" to Parkinson's disease means an increased probability of developing Parkinson's disease during the subject's lifetime as compared to the average individual .
  • a LOD score is a measure of genetic linkage used herein, defined as the log 10 ratio of the probability that the data would have arisen if the loci are linked to the probability that the data could have arisen from unlinked loci.
  • the conventional threshold for declaring linkage is a LOD score of 3.0, that is, a 1000:1 ratio (which must be compared with the 50:1 probability that any random pair of loci will be unlinked) .
  • base pair position or “amino acid position” when referring to an isolated nucleic acid, probe, protein or peptide always indicates the relative position in the native gene or protein.
  • a “probe” refers to a nucleic acid which has sufficient nucleotides surrounding the codons at the mutation positions to distinguish the nucleic acid from nucleic acids encoding non-related genes.
  • the specific length of the nucleic acid is a matter of routine choice based on the desired function of the sequence. For example, if one is making probes to detect the mutation in base pair position 209, the length of the nucleic acid is preferably small, but must be long enough to prevent hybridization to undesired background sequences. However, if the desired hybridization is to a nucleic acid which has been amplified, background hybridization is less of a concern and a smaller probe can be used.
  • such a probe will be between 10 and 100 nucleotides, especially between 10 and 40 and preferably between 15 and 25 nucleotides in length. It is apparent to one of skill in the art that nucleotide substitutions, deletions, and additions may be incorporated into the polynucleotides of the invention. However, such nucleotide substitutions, deletions, and additions should not substantially disrupt the ability of the polynucleotide to hybridize under conditions that are sufficiently stringent to result in specific hybridization.
  • normal refers to a gene which encodes a normal protein.
  • normal means a protein which performs its usual or normal physiological role and which is not associated with, or causative of, a pathogenic condition or state. Therefor, the term “normal” is generally synonomous with the phrase "wild type”.
  • a multiplicity of normal allelic variants may exist, none of which is associated with the development of a pathogenic condition or disease state.
  • Such normal allelic variants include, but are not limited to, variants in which one or more nucleotide substitutions do not result in a change in the encoded amino acid sequence.
  • mutation generally refers to a mutation in a gene that is associated with a predisposition to Parkinson's disease.
  • “Mutant” can specifically refer to a mutation at nucleotide position 209 of the synuclein gene, and is in particularly a G to A transition.
  • other mutations in the synuclein gene or other genes which are associated with a predisposition to Parkinson's disease are also encompassed.
  • the term “mutation” is not limited to transition mutations, but can also mean a deletion, insertion or transversion as well.
  • mutant as it applies to synuclein genes, is not intended to embrace sequence variants which, due to the degeneracy of the genetic code, encode proteins identical to the normal sequences disclosed or otherwise enabled herein; nor is it intended to embrace sequence variants which, although they encode different proteins, encode proteins which are functionally equivalent to normal synuclein proteins.
  • mutant means a protein which does not
  • a mutated synuclein "protein” is understood to refer to the amino acid sequence resulting from any such mutation whether the resulting protein is shorter, longer or modified, i.e. due to an alteration in reading frame or generation of stop codon.
  • peptide is understood to refer to a portion of the mutated protein that is preferably at least five base pairs long, and more preferably at least 10 base pairs long. This portion may be derived from the amino or carboxyl terminus, or it may be an internal portion of the full length protein. As such, a peptide may be chemically synthesized using any method known in the art, or may be made using a recombinant DNA technology and an appropriate purification scheme or isolated from the native protein using enzymatic digestion.
  • the term "substantially pure” means a preparation which is at least 60% by weight the compound of interest. Preferably the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight the compound of interest . Purity can be measured by any appropriate method, i.e. column chromotography, gel electrophoresis or HPLC analysis.
  • Probe nucleic acid e.g., a nucleic acid which may include substitutions, deletions, and/or additions
  • a specific target nucleic acid e.g., a nucleic acid having the mutated sequence
  • the probe preferentially hybridizes to the specific target such that, for example, a band corresponding to the mutated DNA or restriction fragment thereof can be identified on a Southern blot, whereas a corresponding normal or wild-type DNA is not identified or can be discriminated from a variant DNA on the basis of signal intensity.
  • Hybridization probes capable of specific hybridization to detect a single-base mismatch may be designed according to methods known in the art (13-17) .
  • Stringent as it refers to hybridization conditions is a term of art understood by those of ordinary skill to refer to those conditions of temperature, chaotrophic acids, buffer and ionic strength which permit hybridization of a particular nucleic acid sequence to its complementary sequence and not to substantially different sequences.
  • the exact conditions which constitute “stringent” conditions depend on the nature of the nucleic acid sequence, the length of the sequence, and the frequency of occurrence of subsets of that sequence within other non- identical sequences.
  • Hybridization conditions may include temperatures of 20°C-65°C and ionic strengths from 5X to 0. IX SSC.
  • Highly stringent hybridization conditions may include temperatures as low as 40°C-42°C (when denaturants such as formamide are included) or up to 60°C-65°C in ionic strengths as low as 0. IX SSC.
  • the mutation can be detected by many methods.
  • the detecting step can comprise combining a nucleotide probe capable of selectively hybridizing to a nucleic acid containing the mutation with a nucleic acid in the sample and detecting the presence of hybridization.
  • the detecting step can comprise amplifying the nucleotides surrounding and including the mutation and detecting the presence of the mutation in the amplified product, or selectively amplifying the nucleotides of the mutation and detecting the presence of amplification.
  • the detecting step can comprise detecting the presence or absence of a restriction fragment created by an enzyme digest of the sample nucleic acid, or any other detection means known in the art.
  • sequence of various nucleotide probes can be determined from the known sequence of the relevant gene, especially the sequences surrounding the mutation.
  • oligonucleotide probes which may be prepared, for example, synthetically or by nick translation.
  • the probes may be suitably labeled using, for example, a radio label, enzyme label, fluorescent label, biotin-avidin label and the like for subsequent visualization by any appropriate assay, i.e. Southern blot hybridization.
  • the labeled probe is reacted with sample DNA that is bound, for example, to a nylon filter under conditions such that only fully complementary sequences hybridize.
  • the areas that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction.
  • the areas of the filter that exhibit such labeling may then be visualized, for example, by autoradiography .
  • Methods of manipulating hybridization conditions to achieve varying degrees of specificity are well known in the art.
  • tetra-alkyl ammonium salts may be used to bind selectively to A-T base pairs, thus displacing the dissociation equilibrium and raising the melting temperature.
  • 3M Me 4NC1 this is sufficient to shift the melting temperature to that of G-C pairs. This results in a marked sharpening of the melting profile.
  • the stringency of hybridization in such an experiment is usually 5°C below the Ti (the irreversible melting temperature of the hybrid formed between the probe and its target sequence) for the given chain length.
  • the recommended hybridization temperature is about 58°C.
  • the washing temperatures are unique to the sequence under investigation and need to be optimized for each variant.
  • the support to which DNA or RNA fragments of the sample to be analyzed are bound in denatured form is preferably a solid support and may have any convenient shape. Thus, it may, for instance, be in the form of a plate, e.g. a thin layer or a microtiter plate, a strip, a solid particle e.g. in the form of a bead such as a latex bead, a filter, a film or paper.
  • the solid support may be composed of a polymer, preferably nylon or nitrocellulose.
  • ligase chain reaction may involve the use of mismatch probes, i.e., probes which are fully complementary with the target except at the point of the mutation.
  • the target sequence is then allowed to hybridize both with oligonucleotides which are fully complementary and have oligonucleotides containing a mismatch, under conditions which will distinguish between the two.
  • mismatch probes i.e., probes which are fully complementary with the target except at the point of the mutation.
  • the target sequence is then allowed to hybridize both with oligonucleotides which are fully complementary and have oligonucleotides containing a mismatch, under conditions which will distinguish between the two.
  • PCR polymerase chain reaction
  • oligonucleotides can be prepared which are complementary to sequences which flank the DNA of interest . Each oligonucleotide is complementary to one of the two strands.
  • the DNA is denatured at high temperatures (e.g., 95°C) and then reannealed in the presence of a large molar excess of oligonucleotides.
  • oligonucleotides oriented with their 3 ' ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates .
  • the end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a DNA segment by more than one million- fold can be achieved.
  • the resulting DNA may then be directly sequenced in order to locate any genetic alteration.
  • direct visualization or allele-specific oligonucleotide hybridization (18) may be used to detect the Parkinson's disease point mutation.
  • PCR may be followed by restriction endonuclease digestion with subsequent analysis of the resultant products.
  • recognition site facilitates the detection of the Parkinson's disease mutation using restriction fragment length polymorphism (RFLP) analysis or by detection of the presence or absence of the restriction site in a PCR product that spans base pair position 209.
  • RFLP restriction fragment length polymorphism
  • DNA is obtained, for example from the blood cells of the subject suspected of having Parkinson's disease and from a normal subject, is digested with a restriction endonuclease, and subsequently separated on the basis of size by agarose gel electrophoresis.
  • the Southern technique can then be used to detect, by hybridization with labeled probes, the products of endonuclease digestion.
  • the patterns obtained from the Southern blot can then be compared.
  • an additional restriction endonuclease site such as a Tsp45I site, is detected by
  • primers for PCR are usually about 20 bp in length, and are most preferably 15-25 bp .
  • Denaturation of strands usually takes place at 94°C. and extension from the primers is usually at 72 °C.
  • the annealing temperature varies according to the sequence under investigation. Examples of reaction times are: 20 mins denaturing; 35 cycles of 2 min, 1 min, 1 min for annealing, extension and denaturation; and finally a 5 min extension step.
  • PASA PCR "amplification of specific alleles”
  • PASA is a rapid method of detecting single-base mutations or polymorphisms (22-28) .
  • PASA also known as allele specific amplification
  • PASA involves amplification with two oligonucleotide primers such that one is allele-specific .
  • the desired allele is efficiently amplified, while the other allele (s) is poorly amplified because it mismatches with a base at or near the 3' end of the allele-specific primer.
  • PASA or the related method of PAMSA may be used to specifically amplify the mutation sequences of the invention. Where such amplification is done on genetic material (or RNA) obtained from an individual, it can serve as a method of detecting the presence of the mutations.
  • LCR ligase chain reaction
  • SEQ ID NO: 29, 30 a method known as ligase chain reaction
  • LCR probes may be combined or multiplexed for simultaneously screening for multiple different mutations.
  • LCR can be particularly useful where multiple mutations are predictive of the same disease .
  • the Parkinson's disease mutation of the present invention may also be detected using chain termination with labeled dideoxynucleotides.
  • U.S. Patent No. 5,047,519 to Hobbs et al discloses fluorescently-labeled nucleotides as chain-terminating substrates for a fluorescence-based DNA sequencing method.
  • the subjects can participate in the screening of putative agents capable of treating Parkinson's disease.
  • This method comprises administering the test agent to the subject, which may be a human, which has a mutation in a gene associated with Parkinson's disease and monitoring the effect of the agent on the subject's condition. If the symptoms of Parkinson's disease improve, the agent can be used as a treatment for the disease.
  • transgenic model systems and/or cell lines containing the mutated nucleic acid(s) for use, for example, as model systems for screening for drugs and evaluating drug efficiency.
  • model systems provide a tool for defining the underlying biochemistry of, for instance, the mutated synuclein gene, thereby providing a rationale for drug design.
  • One approach to creating transgenic animals is to mutate the animal gene of interest by in vivo mutagenesis, transfer the mutant gene into embryonic stem cells by DNA transfection and inject the embryonic stem cells into blastocysts in order to retrieve offspring which carry the disease-causing mutation (31) .
  • viral vectors e.g., Adeno-associated virus
  • Adeno-associated virus can be used to deliver the mutated gene to a stem cell, or may be used to target specific cells of a fully developed animal (32,33).
  • the mutated gene product is a polypeptide, e.g. the 209 mutation, it can be used to prepare antisera and monoclonal antibodies using, for example, the method of
  • Mutant polypeptides can also be used to immunize an animal for the production of polyclonal antiserum (35) .
  • a recombinantly produced fragment of a variant polypeptide can be injected into a mouse along with an l ⁇ .' adjuvant so as to generate an immune response.
  • Murine immunoglobulins which specifically bind the recombinant fragment can be harvested from the immunized mouse as an antiserum, and may be further purified by affinity chromatography or other means.
  • spleen cells are harvested from the mouse and fused to myeloma cells to produce a bank of antibody-secreting hybridoma cells, which can then be screened for clones that secrete immunoglobulins which bind the recombinantly produced fragment with an increased affinity. More specifically, immunoglobulins that selectively bind to the variant polypeptides but poorly or not at all to wild-type polypeptides are selected, either by pre-absorption with wild-type proteins or by screening of hybridoma cell lines for specific idiotypes that bind the variant but not wild-type.
  • antibodies can be used to screen protein and tissue samples for the presence of mutated proteins.
  • a colored enzymatic reaction occurs when the specific antibody remains bound to its target protein, in situ, after thorough washing, as directed by established protocols.
  • the nucleic acid sequences of the present invention will be capable of expressing the desired mutant or normal polypeptides in an appropriate host cell.
  • the DNA sequences of the present invention will be operably linked to, i.e., positioned to ensure the functioning of, an expression control sequence.
  • such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts) , a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts.
  • the DNA sequence of the present invention may also be fused such that the reading frame is conserved to an appropriate signal sequence to facilitate export of the encoded protein across the cell membrane.
  • Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • suitable expression vectors are disclosed in Sambrook et al . (13) .
  • expression vectors will contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences.
  • E. coli is one prokaryotic host that is particularly useful for cloning and expression of the DNA sequences of the present invention because of the wide variety of available expression systems.
  • Vectors suitable for use in E. coli are known and are commercially available, i.e. pBR322 (13), pBLUESCRIPT (Stratagene), etc.
  • pBR322 13
  • pBLUESCRIPT pBLUESCRIPT
  • a variety of different types of expression systems may be used, including plasmids, cosmids, bacteriophage lambda, etc.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • Expression vectors for use in prokaryotic host cells will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) .
  • any of a variety of well-known promoters may be used, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • a promoter may optionally contain an operator sequence for regulatable gene expression, and will have a ribosome binding site sequence for the initiation of translation.
  • mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (36) .
  • Vectors for use in eukaryotic cells are known and commercially available, i.e. pcDNA3 (Invitrogen) .
  • Eukaryotic cells are actually preferred, and a number of suitable host cell lines capable of secreting intact human proteins have been developed in the art, including CHO cells, COS cells, HeLa cells, myeloma cell lines, Jurkat cells, etc.
  • Promoters for use in eukaryotic vectors may be cell -specific, or capable of being expressed in a wide variety of cells, i.e. viral promoters.
  • Expression vectors of the present invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. Kits
  • kit would comprise a carrier compartmentalized to receive in close confinement one or more containers wherein a first container may contain suitably labeled DNA probes .
  • Other containers may contain reagents useful in the localization of the labeled probes, such as enzyme substrates.
  • Still other containers may contain restriction enzymes (such as Tsp45I) ,
  • D4S421 and the PD locus were included because of prohibitive time run.
  • Multipoint analysis was performed on an IBM SP2 parallel computer and the SGI Challenge machine.
  • genomic DNA was amplified with oligonucleotides (3): 5' GCTAATCAGCAATTTAAGGCTAG 3' (SEQ ID NO:
  • Primers (IF) 5' ACGACAGTGTGGTGTAAAGG 3' (SEQ ID NO 9) and (13R) 5' AACATCTGTCAGCAGATCTC 3' (SEQ ID NO 10) corresponding to nucleotides 21-40 and 520-501 of genbank L08850 were used to amplify a product of 500 bp containing the mutation at nucleotide 209. PCR products were subjected to restriction digestion by Tsp45 I. The mutation at nt 209 creates a novel
  • Size standards used were the 100 bp ladder (Gibco BRL, Gaithersburg, MD) .
  • Genes previously mapped in the general region of linkage include the loci for alcohol dehydrogenase , formaldehyde dehydrogenase, synuclein, UDP-N- acetylglycosamine phosphotransferase and others.
  • chromosomal region 45
  • pathogenesis of diseases affecting the nigrostriatal pathway includes environmental influences, then a range of mutations affecting vulnerable sites in the electron transport chain or enzyme polymorphisms influencing neurotoxin metabolism may vary the penetrance of PD by altering an individual ' s resistance to exogenous or endogenous agents.
  • our finding of a highly penetrant genetic locus linked to PD suggested that abnormalities of a single gene may be sufficient to cause Parkinson's disease.
  • Alpha synuclein a presynaptic nerve terminal protein, was originally identified as the precursor protein for the NAC peptide, a non beta amyloid component of Alzheimer's disease (AD) amyloid plaques ( 4) .
  • the human alpha synuclein gene was previously mapped in the 4q21-q22 region (5) . We refined the mapping, and determined that the alpha synuclein gene is located within the non-excluded region harboring the PD gene in the Italian kindred. Thus alpha synuclein represented an excellent candidate locus for PD.
  • Protein sequence databases searched included the NR (non-redundant) and "month" databases of Genbank and Swiss Prot .
  • Several human clones were identified and characterized as alpha, beta and gamma clones as shown in Figure 7. Potential gamma clones were identified on the basis of homology to known rat and mouse sequences. Although gamma synuclein has been identified in species other than human, this is the first identification of the corresponding gamma synuclein from humans.
  • the beta gene contained one clone 139A20 has been sequenced as shown in Figure 8 (SEQ ID NO 11), which contains all coding exon sequences and some additional non-coding intronic sequence.
  • the gamma clone 174P13 has been sequenced and is available in GenBank: accession number AF044311. Sequence from the 5' end is given in Figure 9 (SEQ ID NO 12), and sequence from the 3' end is given in Figure 10 (SEQ ID NO 13) .
  • the human alpha synuclein gene has also been sequenced as shown in Figure 11, which provides the sequence of each separate exon region with some additional flanking intronic sequence for each exon. (SEQ ID NOs 14-19)
  • the three human homologues are highly conserved at the protein level.
  • the alpha and beta human homologues have about 60.4% similarity.
  • the gamma homologue is about 38.3% and 32.8% similar to the alpha and beta homologues, respectively, based on the portion of the coding sequence l ⁇ that we have obtained thus far.
  • mutations in either the beta or gamma synuclein gene may also result in Parkinson's disease.
  • ADDRESSEE SPENCER & FRANK
  • B STREET: 1100 New York Ave . Suite 300 East
  • ORGANISM Serinus canaria
  • C INDIVIDUAL ISOLATE: genbank L33860
  • GGGCATNTGC GTCCCGCGGG AGGGGCTGGG GTGAGAGTGC GGGGCCAGTG CACCGGTGCC CGTGTATCGC CCTCCCCAGG CCGCCAGGAT GGACGTGTTC ATGAAGGGCC TGTCCATGGC
  • GAAGACCAAG GAGGGCGTCC TCTACGTCGG TGGGCNGGGG GCNGGGTTTC TGGGGCTGCA
  • ACCCGCCCGC GTCCAACCCC GGGGCATGGA CAGGGCCAGG GTTGCGGTCG CGGCTGGGAG
  • MOLECULAR TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • IMMEDIATE SOURCE A) CLONE: human alpha synuclein gene/ exons 1 and 2 plus flanking intron sequences
  • POSITION IN GENOME A) CHROMOSOME/SEGMENT: 4
  • MOLECULAR TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • IMMEDIATE SOURCE (A) CLONE: human alpha synuclein gene/ exon 7 plus flanking intron sequences

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  • Medicinal Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La maladie de Parkinson est un trouble neurodégénératif commun ayant une incidence sur la durée de vie d'environ 2 pour cent. Récemment, il a été découvert qu'un gène de susceptibilité de la maladie de Parkinson se situe sur le bras long du chromosome humain 4. La présente invention concerne l'identification ultérieure d'une mutation dans le gène de synucléine alpha, qui code une protéine présynaptique, censée être impliquée dans la plasticité neuronale. La découverte d'une modification moléculaire spécifique responsable de la maladie de Parkinson permettra une compréhension détaillée de la physiopathologie du trouble qui rendra possibles des interventions thérapeutiques potentielles, ainsi que l'obtention de moyens permettant de diagnostiquer des individus présentant un risque accru de développer cette maladie.
PCT/US1998/013071 1997-06-25 1998-06-25 Clonage d'une mutation genique permettant de deceler la maladie de parkinson WO1998059050A1 (fr)

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AU81637/98A AU8163798A (en) 1997-06-25 1998-06-25 Cloning of a gene mutation for parkinson's disease
US09/446,628 US7001720B1 (en) 1997-06-25 1998-06-25 Cloning of a gene mutation for parkinson's disease

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US60/050,684 1997-06-25

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EP0908727A1 (fr) * 1997-09-19 1999-04-14 Neuropa Limited Dosage avec synuclein et synuclein
WO1999050300A1 (fr) * 1998-03-30 1999-10-07 The Trustees Of The University Of Pennsylvania Methode d'identification, de diagnostic et de traitement de troubles neurodegeneratifs a niveaux eleves de synucleine
EP1081225A1 (fr) * 1999-08-30 2001-03-07 Biofrontera Pharmaceuticals GmbH Modèle animal transgénique de maladies neurodégénératives
WO2001070944A2 (fr) * 2000-03-22 2001-09-27 Aventis Pharma Deutschland Gmbh Nematodes utilises comme organismes modeles servant a etudier des maladies neurodegeneratives, notamment la maladie de parkinson, et procede pour deceler des substances et des genes qui peuvent etre utilises pour le traitement de ces maladies, et identification d'un gene de nematode.
WO2002075317A2 (fr) * 2001-03-15 2002-09-26 Novartis Ag Gene associe a une maladie
EP1299411A1 (fr) * 2000-07-07 2003-04-09 Panacea Pharmaceuticals, LLC Procedes pour la prevention des degats relatifs aux tissus nerveux et pour le traitement des maladies liees a l'alpha-synucleine
WO2004041067A2 (fr) 2002-11-01 2004-05-21 Elan Pharmaceuticals, Inc. Prevention et traitement d'une maladie synucleopathique
WO2005041649A1 (fr) * 2003-10-30 2005-05-12 Taisho Pharmaceutical Co., Ltd. Mammifere non humain transgenique
WO2006020581A2 (fr) 2004-08-09 2006-02-23 Elan Pharmaceuticals, Inc. Prevention et traitement de maladies synucleinopathique et amyloidogenique
EP2450056A1 (fr) 2005-07-19 2012-05-09 Elan Pharma International Limited Prévention et traitement de la maladie synucléinopathique et amyloidogénique
EP2583978A2 (fr) 2007-02-23 2013-04-24 The Regents of the University of California Prévention et traitement de maladie synucléopathique et amyloidogénique
EP3067066A1 (fr) 2007-02-23 2016-09-14 Prothena Biosciences Limited Prevention et traitement de la maladie synucleinopathique et amyloidogenique

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EP0908727A1 (fr) * 1997-09-19 1999-04-14 Neuropa Limited Dosage avec synuclein et synuclein
WO1999050300A1 (fr) * 1998-03-30 1999-10-07 The Trustees Of The University Of Pennsylvania Methode d'identification, de diagnostic et de traitement de troubles neurodegeneratifs a niveaux eleves de synucleine
EP1081225A1 (fr) * 1999-08-30 2001-03-07 Biofrontera Pharmaceuticals GmbH Modèle animal transgénique de maladies neurodégénératives
WO2001016176A2 (fr) * 1999-08-30 2001-03-08 Biofrontera Pharmaceuticals Ag Modele d'animal transgenique destine a des maladies neurogeneratives
WO2001016176A3 (fr) * 1999-08-30 2001-09-27 Biofrontera Pharmaceuticals Ag Modele d'animal transgenique destine a des maladies neurogeneratives
WO2001070944A2 (fr) * 2000-03-22 2001-09-27 Aventis Pharma Deutschland Gmbh Nematodes utilises comme organismes modeles servant a etudier des maladies neurodegeneratives, notamment la maladie de parkinson, et procede pour deceler des substances et des genes qui peuvent etre utilises pour le traitement de ces maladies, et identification d'un gene de nematode.
WO2001070944A3 (fr) * 2000-03-22 2002-03-14 Elegene Ag Nematodes utilises comme organismes modeles servant a etudier des maladies neurodegeneratives, notamment la maladie de parkinson, et procede pour deceler des substances et des genes qui peuvent etre utilises pour le traitement de ces maladies, et identification d'un gene de nematode.
EP2319864A3 (fr) * 2000-03-22 2012-11-21 Sanofi-Aventis Deutschland GmbH Nématodes utilisés comme organismes modèles servant à étudier des maladies neurodégénératives, notamment la maladie de Parkinson, utilisation et procédé
US8193408B2 (en) 2000-03-22 2012-06-05 Sanofi-Aventis Deutschland Gmbh Nematodes as model organisms for the investigation of neurodegenerative diseases, in particular parkinsons disease, uses and methods for the discovery of substances and genes which can used in the treatment of the above disease states and identification of a nematode gene
US7399900B2 (en) 2000-03-22 2008-07-15 Sanofi-Aventis Deutschland Gmbh Nematodes as model organisms for the investigation of neurodegenerative diseases, in particular parkinsons disease, uses and methods for the discovery of substances and genes which can used in the treatment of the above disease states and identification of anematode gene
EP1299411A4 (fr) * 2000-07-07 2006-02-15 Panacea Pharm Llc Procedes pour la prevention des degats relatifs aux tissus nerveux et pour le traitement des maladies liees a l'alpha-synucleine
US7605133B2 (en) 2000-07-07 2009-10-20 Panacea Pharmaceuticals, Inc. Isolated peptides to treat alpha-synuclein diseases
EP1299411A1 (fr) * 2000-07-07 2003-04-09 Panacea Pharmaceuticals, LLC Procedes pour la prevention des degats relatifs aux tissus nerveux et pour le traitement des maladies liees a l'alpha-synucleine
WO2002075317A2 (fr) * 2001-03-15 2002-09-26 Novartis Ag Gene associe a une maladie
WO2002075317A3 (fr) * 2001-03-15 2003-11-06 Novartis Ag Gene associe a une maladie
WO2004041067A2 (fr) 2002-11-01 2004-05-21 Elan Pharmaceuticals, Inc. Prevention et traitement d'une maladie synucleopathique
EP2361629A1 (fr) 2002-11-01 2011-08-31 Elan Pharmaceuticals Inc. Prévention et traitement d'une maladie synucleopathique
JPWO2005041649A1 (ja) * 2003-10-30 2007-05-24 大正製薬株式会社 トランスジェニック非ヒト哺乳動物
JP4613824B2 (ja) * 2003-10-30 2011-01-19 大正製薬株式会社 トランスジェニック非ヒト哺乳動物
US7550649B2 (en) 2003-10-30 2009-06-23 Taisho Pharmaceutical Co., Ltd. Transgenic non-human mammal
WO2005041649A1 (fr) * 2003-10-30 2005-05-12 Taisho Pharmaceutical Co., Ltd. Mammifere non humain transgenique
WO2006020581A2 (fr) 2004-08-09 2006-02-23 Elan Pharmaceuticals, Inc. Prevention et traitement de maladies synucleinopathique et amyloidogenique
EP3369433A1 (fr) 2004-08-09 2018-09-05 Janssen Alzheimer Immunotherapy Prévention et traitement de la maladie synucléinopathique et amyloidogénique
EP2450056A1 (fr) 2005-07-19 2012-05-09 Elan Pharma International Limited Prévention et traitement de la maladie synucléinopathique et amyloidogénique
EP2583978A2 (fr) 2007-02-23 2013-04-24 The Regents of the University of California Prévention et traitement de maladie synucléopathique et amyloidogénique
EP3067066A1 (fr) 2007-02-23 2016-09-14 Prothena Biosciences Limited Prevention et traitement de la maladie synucleinopathique et amyloidogenique

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