WO1999031230A2 - Oligophrenin-1, its expression product, and the diagnostic and therapeutic applications thereof - Google Patents
Oligophrenin-1, its expression product, and the diagnostic and therapeutic applications thereof Download PDFInfo
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- WO1999031230A2 WO1999031230A2 PCT/EP1998/008557 EP9808557W WO9931230A2 WO 1999031230 A2 WO1999031230 A2 WO 1999031230A2 EP 9808557 W EP9808557 W EP 9808557W WO 9931230 A2 WO9931230 A2 WO 9931230A2
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- oligophrenin
- gene
- nucleic acid
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the identification of a new gene, called oligophrenin 1 , and its expression product, as well as to the diagnostic and therapeutic applications of these nucleotide and peptide sequences.
- X-linked mental retardation is acknowledged to be a major cause of severe learning difficulties. Surveys have shown an excess of males over females with severe mental retardation and later studies suggested that the excess was the result of an X-linked condition.
- X-linked mental retardation is a vastly heterogeneous group of disorders which can be roughly categorized as syndromic (MRXS) or non specific (MRX). Families with syndromic disorders usually have a quite distinct phenotypic presentation whereas families with non specific disorders present no distinctive somatic features.
- the authors of the present invention have thus isolated and characterized the oligophrenin 1 transcripts.
- Said transcripts contain an open- reading frame (ORF) which is encoded by exon 2 to exon 24.
- ORF open- reading frame
- This ORF is 2406 bases long and encodes a protein of 802 amino acids, called the oligophrenin 1 protein.
- a subject of the present invention is thus an isolated nucleic acid having a sequence selected from the group consisting of sequence SEQ ID n° 1 to SEQ ID n° 25, and a homologous nucleotide sequence thereof.
- SEQ ID n° 1 represents the 5' fragment of the genomic DNA of the human oligophrenin 1 gene.
- SEQ ID n° 2 to SEQ ID n°25 represent fragments of the genomic DNA of the human oligophrenin 1 gene including exons as shown in table 1.
- Table 1 identification of the sequences
- SEQ ID n° 26 represents the cDNA fragment corresponding to the common open-reading frame (ORF).
- a subject of the present invention is also an isolated nucleic acid having a sequence selected from the group consisting of exon 1 to exon 25 as identified in the sequence listing and in table 2, and a homologous nucleotide sequence thereof.
- Table 2 identification of exon sequences
- a homologous nucleotide sequence is understood as meaning a sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more bases.
- homologous sequences show at least 70 % of homology, preferably 80 % of homology, more preferably 90 % of homology with any of sequences SEQ ID n° 1 to SEQ ID n° 26.
- a polynucleotide of the invention, having a homologous sequence, hybridizes to the sequences to which it refers (any of sequences
- SEQ ID n° 1 to SEQ ID n° 26 preferably under stringent conditions.
- T m the temperature at which 50 % of annealed strands separate
- Tm is calculated as follows :
- T m 81.5 + 0.41 (% G + C) + 16.6 Log (positive ion concentration) - 0.63 (% formamide) - (600/polynucleotide size in base pairs) (Sambrook et al, 1989). For sequences comprising less than 30 nucleotides, T m is calculated as follows :
- T m 4(G + C) + 2 (A + T).
- hybridization temperature is around from 5°C to 30°C, preferably from 5°C to 10° C below the calculated T m
- hybridization buffer solutions that are used are preferably solutions with high ionic strength, such as an aqueous 6 X SSC solution for example.
- a nucleotide sequence homologous to the open-reading from SEQ ID n° 26 means a nucleotide sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more bases, or by the degeneracy of the genetic code so long as it codes for a polypeptide having the biological activity of oligophrenin 1 protein, as defined below.
- Said homologous sequences include mammalian genes coding for the oligophrenin 1 protein, preferably of primate, cattle, sheep, swine, or rodent, as well as allelic variants.
- the nucleic acid sequences of the invention are useful for the detection of an abnormality, such as a mutation, in the oligophrenin 1 gene or in the transcripts of the oligophrenin 1 gene.
- an abnormality such as a mutation
- a subject of the present invention is a method of in vitro diagnosis of a neurological disorder associated with an abnormality in the oligophrenin 1 gene or in the transcripts of the oligophrenin 1 gene, wherein one or more mutation(s), preferably inducing a modification of the expression of the oligophrenin 1 gene is detected in the oligophrenin 1 gene or in the transcripts of the oligophrenin 1 gene.
- a subject of the present invention is also a nucleic acid comprising a sequence identical to SEQ ID n° 26 or to homologous sequences thereof, except for a one base deletion of the nucleotide 1578 as shown in SEQ ID n° 26.
- the present invention relates to methods of in vitro diagnosis wherein the nucleic acid sequences of the invention or probes or primers derived thereof are used to detect aberrant synthesis or genetic abnormalities at the oligophrenin 1 gene level.
- the present invention is more particularly directed to a method of in vitro diagnosis comprising the steps of :
- the method of the invention can also be applied to the detection of an abnormality in the transcript of the oligophrenin 1 gene, by amplifying the mRNAs contained in a biological sample, for example by RT-PCR.
- Another subject of the present invention is a method of in vitro diagnosis, as previously defined comprising the steps of :
- This comparison of the amplified products obtained from the biological sample with the amplified products obtained with a normal biological sample can be carried out for example by specific probe hybridization, by sequencing or by restriction site analysis.
- a subject of the present invention is also a nucleic acid sequence which specifically hybridizes with a nucleic acid sequence of the invention as previously defined or with their complementary sequences.
- a sequence which specifically hybridizes varnish is understood as meaning a sequence which hybridizes with the sequences to which it refers under the conditions of high stringency (Sambrook et al, 1989). Such sequences are preferably oligonucleotides having at least 15, and more preferably at least 20 bases.
- sequences which are useful as primers or probes for the diagnosis methods according to the present invention may be preferably selected from the group consisting of nucleic acid fragments of SEQ ID N° 2 to SEQ ID N° 26 as shown in table 3, or the complementary sequences thereof.
- RNA chip Gel Electrophoresis
- SSCP method Single Strand Conformation Polymorphism
- RT-PCR method may be advantageously used for detecting abnormalities in the oligophrenin 1 transcript, as it allows to visualize the consequences of a splicing mutation such as exon skipping or aberrant splicing due to the activation of a cryptic site.
- This method is preferably followed by direct sequencing as well.
- the more recently developed technique using DNA chip can also be advantageously implemented for detecting an abnormality in the oligophrenin 1 gene (Bellis et al., 1997).
- the cloning of the oligophrenin 1 gene, as well as the identification of various mutations responsible for neurological disorders according to the invention, allow direct or semi-direct diagnosis.
- the specificity and reliability of such diagnosis methods are more particularly appreciable for prenatal diagnosis.
- the nucleic acid sequences of the present invention represent a highly interesting tool for genetic counseling.
- the oligophrenin 1 protein is a rho-GAP protein and that the constitutional loss of oligophrenin 1 activity in humans results in cognitive impairment. Defects in the oligophrenin 1 gene, or in the oligophrenin 1 gene product may cause inactivation of the oligophrenin 1 protein, which leads to constitutive activation of its target GTPases.
- the oligophrenin 1 gene would thus be involved in disorders due to an abnormal neurone migration.
- disorders include not only genetic disorders such as nonspecific X-linked mental retardation but also incurable cryptogenic epilepsies and neurodegenerative diseases, such as Alzheimer's disease and cognitive impairments related to aging.
- the present invention also provides transgenic non-human animals or cells thereof.
- Said transgenic animal can have a exogenous oligophrenin 1 protein of this invention due to the presence of a gene encoding and expressing that protein or part of that protein.
- Transgenic animals are generally well known, as are their methods of production.
- the present invention contemplates a non-human animal containing a oligophrenin 1 gene of the present invention integrated in the genome of the animal's somatic and germ cells, i.e., a transgenic animal, preferably transgenic mammals.
- transgenic mammals containing a transgene encoding a oligophrenin 1 protein of the present invention are typically prepared using the standard transgenic technology described in Hogan et al. (1987) and Palmiter et al. (1986). Production of transgenic mammals is also possible using the homologous recombination transgenic systems described by Capecchi (1989). Preparation of transgenic mammals has also been described in WO94/21670.
- One technique for transgenically altering a mammal is to microinject a rDNA into the male pronucleus of the fertilized mammalian egg to cause one or more copies of the rDNA to be retained in the cells of the developing mammal.
- Alternative methods for producing a non-human mammal containing a rDNA of the present invention include infection of fertilized eggs, embryo-derived stem cells, totipotent embryonal carcinoma (Ec) cells, or early cleavage embryos with viral expression vectors containing the rDNA (Palmiter et al., (1986)).
- a transgenic animal can also have a mutation in its own native oligophrenin 1 gene, thereby rendering the oligophrenin 1 protein nonfunctional (i.e., a "knockout" trangenic animal).
- Such an animal is useful as it presents the clinical conditions associated with the defects in the mutated oligophrenin 1 protein, and further can be a model for evaluation of candidate therapeutics that would treat subjects with defects in that protein.
- transgenic non-human animals or cells in culture that overexpress oligophrenin 1 protein or preferably express a native oligophrenin 1 protein that has been rendered non-functional (“knock-out" transgenic animal) may be useful in a method for screening chemical entities or drugs likely to act on the signaling pathway to which the rho-GAP MRX protein (oligophrenin) belongs.
- Transgenic non-human animals or cells thereof that overexpress oligophrenin 1 protein refer to animals or cells thereof that express an exogenous oligophrenin 1 protein of the invention in addition not the native protein.
- the screening method of the invention comprises the steps of : i) administering a drug to be tested to a transgenic non-human animal that overexpress oligophrenin 1 protein or preferably express a native oligophrenin 1 protein that has been rendered non-functional ; and ii) observing clinical expression of neuronal changes in vivo and/or in vitro culturing nervous cells from said animal and observing the stimulation or recovery of axon outgrowth or morphogenesis.
- the screening method of the invention comprises the steps of : i) contacting a drug to be tested with nervous cells or nervous tissue cultures that overexpress oligophrenin 1 protein or preferably express a native oligophrenin 1 protein that has been rendered non-functional ; and ii) measuring the axon outgrowth.
- said cells are either obtained from said transgenic animals or are established cell lines, such as neuroblastoma or primary cultures of neuronal cells, which have been transfected by a DNA construct, e.g by means of a viral vector, allowing the expression of exogenous oligophrenin 1 protein or rendering the native oligophrenin 1 non functional.
- Drugs selected by the methods of screening as above-defined, and pharmaceutical compositions containing such a drug in association with a pharmaceutically acceptable carrier, are also encompassed by the present invention.
- the ORF of the oligophrenin 1 gene as shown in SEQ ID n° 26 according to the invention encodes a protein of 802 amino acids with a relative molecular mass of 91 kD. Hydropathy analysis (Kyte and Doolittle, 1982) suggests that the oligophrenin 1 protein is hydrophilic. Based on consensus motifs in PROSITE database (Bairoch et al., 1997), many potential phosphorylation sites were predicted including r in a tyrosine kinase phosphorylation site at position 142. Comparison of the protein sequence with other sequences in the databases indicated that the oligophrenin 1 gene encodes a rho-GAP containing protein.
- Sequence alignment shown in figure 3b illustrates the remarkable similarity between the predicted oligophrenin 1 domain and other members of the rho-GAP subfamily. This similarity extends over 180 residue region localised in the central part of the predicted protein and concerns the three structurally conserved regions (SCRs) that are specific to the rho-GAP proteins.
- SCRs structurally conserved regions
- the oligophrenin 1 protein showed the greatest similarity to the chicken Graf protein (Hildebrand et al., 1996). This similarity extends on both sides of the rho-GAP domains, but oligophrenin 1 does not contain the SH3 domain reported for the Graf protein.
- the rho-GAP activity of the oligophrenin 1 protein is consistent with the functional analysis of the chicken Graf protein, which has both part of the N-terminal and rho-GAP domains identified in the oligophrenin 1 protein. Graf protein has been shown to preferentially stimulate the GTPase activity of the GTP-binding proteins RhoA and Cdc42 (Hildebrand et al., 1996).
- oligophrenin 1 protein does not match any known sequence, whereas the N-terminal domain of oligophrenin 1 protein is similar to a highly conserved protein, of unknown function, identified in C. elegans, mouse and human (Fig. 3c).
- This protein presents two isoforms identified as CELZK328 and CELT04C95 (Genbank), which correspond to two different ORF.
- a further subject of the present invention is thus an isolated oligophrenin 1 polypeptide substantially comprising the aminoacid sequence of SEQ ID n° 27 or a homologous aminoacid sequence thereof.
- substantially is understood as meaning that said isolated oligophrenin 1 polypeptide exhibits the same biological and/or immunological properties, as the native oligophrenin 1 protein.
- aminoacid sequence may be SEQ ID n° 26, or a homologous aminoacid sequence thereof.
- a homologous aminoacid sequence is understood as meaning a sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more aminoacids, without inducing modification of biological and/or immunological properties.
- Said derivative aminoacid sequence shows at least 60 % of homology, preferably 70 % of homology, preferably 80 % of homology with the oligophrenin 1 polypeptide of SEQ ID n° 26.
- biological properties of the polypeptides of the invention refer to the activity of the oligophrenin 1 protein, which enhances GTPase activity of small Ras-like GTPases and hence turns them off.
- the "immunological properties" of the polypeptides of the invention refer to the ability of the polypeptides of the invention to induce an immunological response mediated by antibodies which recognize the oligophrenin 1 polypeptide of the invention.
- polypeptides according to the invention can be obtained by any of the standard methods of purification of soluble proteins, by peptide synthesis or by genetic engineering.
- Said techniques comprise the insertion of a nucleic acid sequence coding for a peptide of the invention into an expression vector, such as a plasmid, and the transformation of host cells with the expression vector, by any of the methods available to the skilled person, like for instance electroporation.
- the present invention thus relates to vectors for cloning and/or expression comprising a nucleic acid sequence of the invention and to host cell transfected with these vectors.
- the expression vector according to the invention comprises a nucleic acid sequence encoding a polypeptide of the invention, said nucleic acid sequence being operably linked to elements allowing its expression.
- Said vector advantageously contains a promoter sequence, signals for initiation and termination of translation, as well as appropriate regions for regulation of translation. Its insertion into the host cell may be transient or stable. Said vector may also contain specific signals for secretion of the translated protein. These various control signals are selected according to the host cell which may be inserted into vectors which self-replicate in the selected host cell, or into vectors which integrate the genome of said host.
- Host cells may be prokaryotic or eukaryotic, including but not limiting to bacteria, yeasts, insect cells, mammalian cells, including cell lines which are commercially available.
- a subject of the present invention is also a method for producing a recombining oligophrenin 1 polypeptide, wherein said host cell is transfected with said expression vector and is cultured in conditions allowing the expression of a polypeptide according to the invention.
- the present invention also relates to monoclonal or polyclonal antibodies, or fragments thereof, or chimeric or immunoconjugate antibobies, which are capable of specifically recognizing a polypeptide according to the invention.
- Polyclonal antibodies can be obtained from serum of an animal immunized against the oligophrenin 1 , which can be produced by genetic engineering for example, as above described, according to standard methods well-known by one skilled in the art.
- Monoclonal antibodies can be obtained according to the standard method of hybridoma culture (Kohler and Milstein, 1975).
- the antibodies of the present invention can be chimeric antibodies, humanized antibodies, or antigen binding fragments Fab and F(ab')2. They can also be immunoconjugated or labelled antibodies.
- Said antibodies are particularly useful for detecting or purifying a oligophrenin 1 polypeptide according to the invention in a biological sample.
- Another subject of the present invention is a pharmaceutical composition
- a pharmaceutical composition comprising a purified oligophrenin 1 polypeptide of the invention and/or a homologous polypeptide thereof, in association with a pharmaceutically acceptable carrier.
- a further subject of the present invention is a pharmaceutical composition comprising a nucleic acid encoding said polypeptides and a pharmaceutically acceptable carrier. Said nucleic acid, preferably inserted in a vector, may be administered in a naked form or in association with transfection facilitating agents.
- a further subject of the invention is a pharmaceutical composition comprising an anti-sense sequence capable of specifically hybridizing with a nucleic acid sequence encoding said polypeptides, in association with a pharmaceutically acceptable carrier.
- a still further subject of the invention is a pharmaceutical composition comprising an antibody directed against said polypeptides, in association with a pharmaceutically acceptable carrier.
- the present invention is directed to a pharmaceutical composition
- a pharmaceutical composition comprising a purified oligophrenin 1 polypeptide of the invention and/or a homologous polypeptide thereof, in association with a pharmaceutically acceptable carrier.
- homologous polypeptide refers to a polypeptide with a homology of at least 40 %, preferably of at least 60 % in comparison to the oligophrenin 1 protein.
- homologous polypeptides include any known protein which exhibits a rho-GAP activity.
- homologous polypeptide is for example the protein CELZK328 or CELT04C95.
- compositions of the invention are useful for preventing and/or treating neurological disorders, wherein the oligophrenin 1 protein or a homologous protein thereof is implicated.
- the authors of the present invention have shown that defects in a Ras-like GTPase (rho-GAP) dependent signalling pathway are associated with cognitive impairment, resulting from misguided axon growth and/or defective cell migration. Consequently, the disorders which are more particularly aimed at are disorders of the central nervous system in connection with the axonal development, more particularly a disorder associated with cognitive impairment.
- Such disorders include nonspecific X-linked mental retardation, as well as cr ptogenic epilepsies or neurodegenerative diseases, such as Alzheimer's disease and cognitive impairments related to aging.
- Another subject of the invention is the use of a purified oligophrenin 1 polypeptide of the invention and/or a homologous polypeptide thereof, in association with a pharmaceutically acceptable carrier for the manufacture of a medecine for preventing and/or treating neurological disorders, wherein the oligophrenin 1 protein or a homologous protein thereof is implicated.
- compositions of the invention may be administered to a mammal, preferably to a human, in need of a such treatment, according to a dosage which may vary widely as a function of the age, weight and state of health of the patient, the nature and severity of the complaint and the route of administration.
- the appropriate unit forms of administration comprise oral forms such as tablets, gelatin capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, subcutaneous, intramuscular, intravenous, intranasal or intraoccular administration forms and rectal administration forms.
- a further subject of the present invention is a method of preventing and/or treating neurological disorders resulting from defects in the oligophrenin 1 gene or in the oligophrenin 1 protein or in a homologous gene or protein thereof, which comprises administering to a subject in need of a such treatment an amount of a pharmaceutical composition as above defined effective to prevent and/or alleviate said neurological disorders.
- FIG. 1 (a) Physical map of the Xq12 locus and genomic structure of the oligophrenin 1 gene spanning the X-chromosomal breakpoint. YAC, PAC and cosmid contigs are indicated with lines and STSs by vertical bars. The STS C16T3 was generated from the distal end of the 9 kb Hindlll fragment containing the X-chromosomal breakpoint (represented by an arrow).
- the oligophrenin 1 gene spans at least 300 kb and it consists of 25 exons of which 23 are coding (white boxes), (b) Southern blot analysis of Hindlll digested genomic DNAs from the patient exhibiting the (X;12) translocation and a normal female using the STS C16 T3 as probe. The junction fragment is indicated by JF.
- FIG. 1 Fetal and multiple adult tissue northern blots containing poly(A) + RNA (Clontech®) hybridized with C2 cDNA probe. A 7.5 kb transcript was observed after an overnight exposure at - 80°C. Hybridization of Northern blots with an actin probe was performed to assess differences between amounts of loaded poly(A) + RNA samples.
- FIG. 3 (a) Coding part of the cDNA and deduced amino acid sequences of the oligophrenin 1 gene. Nucleotides in bold correspond to exon-exon boundaries. The GAP domain is underlined, Primers used in RT-PCR to study the expression of the gene in the patient with the X;12 translocation are double underlined and the internal primer used for hybridization to ascertain PCR products is shown in dotted line. The deleted nucleotide at position 1578 is indicated in italic bold case, (b) Sequence alignment of the GAP domain present in oligophrenin 1 and different rhoGAP proteins reviewed in Aelst et al., 1997.
- the GAP domain contains three Structurally conserveed Regions (SCRs) (Boguski et al., 1993).
- CELZK328 corresponds to an ORF predicted from sequences of the C.elegans genome, (c). Sequence alignment showing the high conservation of oligophrenin 1 N-terminal domain.
- CELTO4C95 corresponds to another C. elegans ORF, different from CELZK328.
- EST483210 is part of the mouse homologue oligophrenin 1 cDNA and EST387042 corresponds to a human EST that was localized on chromosome 11 by the Whitehead Institute.
- Figure 4. (a). Study of oligophrenin 1 transcript in the patient with the (X;12) translocation and in a normal female used as control (XX). Southern blot of PCR products amplified from total RNAs isolated from EBV transformed lymphoblastoid cell lines, (b) Nucleotide sequences showing the one base pair deletion. Direct sequencing of PCR products corresponding to exon 19 of IV-3 proband.
- YAC clones of the Xq12 locus were obtained from the UK HGMP Resource Centre.
- PAC clones were obtained from the German resource center (RZPD).
- Primer sequences corresponding to STSs are available in Genome Data Base.
- STSC16T3 is a 189 bp fragment amplified with the primers: C16T3F (5' CACAGCAAGCAATAAGCACT 3') and C16T3R (5' TGTCTCCTGTGCTCTTTCCA 3'). Overlaps between clones and STS mapping were performed by a combination of STS/EST amplification and hybridization approaches.
- Cosmid 2C6, 4D2 and 35 shown in figure 1 a are from a cosmid library corresponding to the YAC 4690. Exon-intron boundaries were identified through sequence comparisons between cDNA and genomic DNA clones. To generate genomic sequences, DNAs of cosmid clones were used as templates and primed with exonic oligonucleotides.
- ICRF coordinates of cosmid clones shown in figure 1 are as follow: cos12: ICRFc104J1515Q8, cos15: ICRFc104K1628Q8, cos7: ICRFc104P0212Q8, cos3: ICRFc104B1719Q8, cos11 : ICRFc104F178Q8, cos5:
- Figure 1 a depicts the location of the translocation breakpoint on the normal X and summarizes YAC, PAC and cosmid contigs spanning the breakpoint.
- the probe, STSC16T3, which detects the junction fragment and localized the breakpoint to a 9 kb Hindlll fragment (fig 1b) was isolated from the cosmid clone 4D2 (Fig 1a). Aberrant bands confirming the latter results were also obtained by hybridization of the same probe to a Southern blot containing DNA from the patient digested with several other enzymes.
- the predicted polypeptide corresponding to this EST revealed a significant homology with the human EST 387042 localised on chromosome 11 and with the C. elegans ORF CELT04C95. Further investigations suggested that the C. elegans ORF is represented on the genomic sequences derived from the PAC clone by 8 different potential exons scattered over 130 kb (Fig. 1a). These predictions were confirmed by RT-PCR experiments using primers located within the potential exons and human fetal brain total RNA. Furthermore, hybridization of the RT-PCR products to a Northern blot containing polyA + RNA detected a 7.5 kb transcript most highly expressed in fetal brain (Fig. 2). Together, these results indicated the presence of a candidate gene located in the vicinity of the translocation breakpoint.
- the structure of the gene including the exon-intron boundaries was determined through sequence comparisons between cDNA and cosmid genomic DNA clones isolated either from a cosmid library generated from YAC clone 4690 or the ICRF flow sorted X-specific cosmid library (fig 1a).
- the physical mapping of the 25 exons allowed to demonstrate that the candidate MRX gene is transcribed from telomere to centromere and the translocation breakpoint maps within the second intron leading therefore to a displacement on the derivative chromosome 12 of the first two exons including the one containing the putative translation initiation codon.
- the normal X chromosome was found to be late replicating, indicating preferential X-inactivation of the normal X chromosome (Bienvenu et al., 1997), and the MRX candidate gene was found to undergo X-inactivation.
- Figure 4(a) represents a Southern blot of PCR products amplified from total RNAs isolated from EBV transformed lymphoblastoid cell lines. RT- PCR were performed with (+) or without (-) reverse transcriptase and cDNAs were amplified for 40 cycles with primers located in exon 1 and 2 (figure 3, double underlined nucleotide sequences). After gel electrophoresis, the Southern blot was hybridized with an internal oligonucleotide (dotted line on figure 3). The 650 bp fragment corresponds to an amplification from the contaminating genomic DNA (intron 1 is 140 bp long). The lane labelled DNA, corresponds to the PCR product obtained from a female total genomic DNA. The negative control indicated by Ct(-) corresponds to a PCR experiment without template. RT-PCR amplification of the ubiquitously expressed transcript produced by the distal part of the dystrophin gene was used as internal standard.
- the mutation to co-segregate with the mental retardation phenotype as shown on figure 4c was detected by denaturing gradient gel. electrophoresis of PCR products corresponding to exon 19 of the oligophrenin 1 gene. Exon 19 was amplified by PCR with primers 19F (5' GTT AAT CTT GCC CCT TTT CT 3') and 19R (5' Psoralen- TA GGA AGA CAG GTA GTG AGA AT) yielding a 221 bp product. 10 ⁇ l of each amplified product was mixed with 10 ⁇ l of normal control PCR product. Heteroduplexes were generated by denaturing for 10 min, and subsequent reannealing for 45 min at 56°C. The samples were electrophoresed through a 25-65% denaturant 6% polyacrylamide gel for 7.5h at 60°C and 160V. The characteristic shifted profile displayed by the mutated allele allow an easy study of the familial segregation.
- C2 cDNA clone was isolated from a fetal brain cDNA library.
- C2 DNA probe was used to hybridize poly(A) + RNA (Clontech ⁇ ) contained in fetal and multiple adult tissues. A 7.5 kb transcript was observed after an overnight exposure at - 80°C. Hybridization of Northern blots with an actin probe was performed to assess differences between amounts of loaded poly(A) + RNA samples.
- the oligophrenin 1 transcript was most abundant in RNA from fetal brain. A lower level of expression was also detected in several other tissues including adult brain.
- in situ hybridization was used to examine the expression of the mouse homologous gene in embryonic days (E) 10.5, E12.5, E14, E18 and in postnatal day 1 of mouse embryos and postnatal tissues. In addition to a low expression in all tissues with no significant differences, it was found that the gene is expressed at a higher level in all parts of the developing neuroepithelium of the neural tube. During later differentiation stages and in the mature brain a significant level of expression is visible in different structures of the brain with no striking distribution of the mRNA expression.
- oligophrenin 1 Several lines of evidence show that defects in oligophrenin 1 are responsible for X-linked non-specific mental retardation.
- the oligophrenin 1 gene maps to a potential mental retardation genetic locus in Xq12 identified by linkage analyses (Lubs et al., 1996, The European XLMR Consortium, 1997).
- literature reports (Davies, 1997) concerning two patients with complete androgen insensitivity syndrome (CAIS) and mental retardation showed the presence of deletions which include several markers both proximal and distal to the AR gene and extend to DXS905 and DXS908. The above mapping data showed that these markers are located within the second and fifth intron of the oligophrenin 1 gene (Fig.
- deletions in two CAIS patients without mental retardation do not extend to the oligophrenin 1 gene as deletions are limited to the androgen receptor gene itself (Davies, 1997).
- investigation of this gene in the female patient with mental retardation and a t(X;12) demonstrated an absence of expression of both alleles resulting from the disruption of one allele by the translocation breakpoint and a preferential inactivation of the second allele.
- Kel, A.E., et al. SITEVIDEO a computer system for functional site analysis and recognition. Investigation of the human splice sites. Comput Appl Biosci 9, 617-27 (1993).
- Vits L. et al., MASA syndrome is due to mutations in the neural cell adhesion gene LICAM. Nature Genet 7, 408-413 (1994).
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JP2000539130A JP2002508177A (en) | 1997-12-15 | 1998-12-14 | Novel gene called oligofrenin 1, its expression product and its diagnostic and therapeutic use |
CA002315268A CA2315268A1 (en) | 1997-12-15 | 1998-12-14 | Oligophrenin-1, its expression product, and the diagnostic and therapeutic applications thereof |
EP98966703A EP1037993A2 (en) | 1997-12-15 | 1998-12-14 | A new gene called oligophrenin 1, its expression product, and the diagnostic and therapeutic applications thereof |
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