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  • G01N2800/2857—Seizure disorders; Epilepsy

Definitions

• the present invention relates to nucleotide and amino acid sequences corresponding to a complete protocadherin 19 (PCDHJ 9) open reading frame (ORP), and mutant nucleotide sequences encoding nonfunctional or altered PCDH19 mRNA or non-functional or altered PCDH19 protein which can result in illnesses related to PCDH19 protein deficiency or altered function in human subjects, in particular EFMR (Epilepsy and Mental Retardation limited to Females).
• PCDHJ complete protocadherin 19
• ORP open reading frame
• Inherited diseases caused by mutations on the X chromosome are generally characterised by the affected status of carrier males and sparing of carrier females.
• EFMR Epilepsy and Mental Retardation limited to Females
• EFMR is a unique X-linked condition which, by contrast, spares carrier males and is expressed in females (Ryan SG et al, 1997).
• EFMR is a rare condition characterised by seizure onset in early childhood (6-36 months) and cognitive impairment. The phenotype is restricted to females with males apparently spared, demonstrating normal cognitive function and absence of seizures.
• PCDHl 9 protocadherin 19
• PCDH 19 is the first cadherin to be implicated in epilepsy and mental retardation.
• the expression analysis described herein shows a role for PCDH 19 in normal neuronal development.
• a mechanism of phenotype rescue that saves transmitting males (ie carrier males) from clinically expressing the disorder is suspected, through a related male-specific human gene, protocadherin 1 IY (PCDHIlY) (Blanco P et ah, 2000). This mechanism is consistent with the remarkable mode of inheritance observed in EFMR.
• PCDHIlY protocadherin 1 IY
• the studies described herein have identified nucleotide and amino acid sequences corresponding to a complete PCDHl 9 open reading frame (ORF) as well as mutant sequences encoding non-functional PCDH19 mRNA or non-functional PCDH19 protein.
• PCDH 19 protein deficiency or altered function such as epilepsy and mental retardation, in particular EFMR.
• male carriers of the PCDH 19 deficient genotype have been shown to be rescued from the disease phenotype by the male-specific protocadherin PCDHl IY.
• the identification of the complete PCDHl 9 ORF and the identification of mutations in the nucleotide sequence causing a disease state provide for methods for diagnosis of illnesses related to PCDH19 protein deficiency or altered PCDH 19 protein function, methods for the identification of a predisposition to such illnesses, methods of screening to identify carriers of such illnesses methods, and agents for the therapeutic or prophylactic treatment of PCDH 19 deficiency.
• the present invention provides a method of diagnosing an illness related to functional protocadherin 19 (PCDH 19) protein deficiency or altered PCDH 19 protein function, or assessing a predisposition to an illness related to functional PCDH 19 protein deficiency or altered PCDH19 protein function, or screening to identify carriers of illnesses related to functional PCDH19 protein deficiency or altered PCDH 19 protein function, wherein said method comprises the step of:
• the invention provides a kit for diagnosing an illness related to functional protocadherin 19 (PCDH 19) protein deficiency or altered PCDH 19 protein function, or assessing a predisposition to an illness related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, or screening to identify carriers of illnesses related to functional PCDH19 protein deficiency or altered PCDH 19 protein function, wherein said kit comprises one or more of the following: an antibody or fragment thereof which specifically binds to PCDH19 protein or polypeptide, or a fragment or variant thereof; and an oligonucleotide probe/primer molecule which specifically hybridises to a polynucleotide molecule encoding PCDH 19 protein or polypeptide, or a fragment or variant thereof under high stringency conditions.
• PCDH 19 functional protocadherin 19
• the present invention provides for the use of: a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to a complete protocadherin 19 (PCDHl 9) open reading frame (ORF) nucleotide sequence according to SEQ ID NO: 1, wherein said nucleotide sequence encodes a functional PCDH 19 protein or polypeptide, or functional fragment or functional variant thereof encoded by a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete PCDHl 9 ORF nucleotide sequence according to SEQ ID NO: 1; in the treatment of PCDH19 protein deficiency or altered PCDH19 protein function in a subject.
• PCDHl 9 complete protocadherin 19
• ORF open reading frame
• the present invention provides a method for the therapeutic or prophylactic treatment of protocadherin 19 (PCDH19) protein deficiency or altered PCDH19 protein function in a subject, wherein said method comprises the step of:
• a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete protocadherin 19 (PCDHl 9) open reading frame (ORP) nucleotide sequence according to SEQ ID NO: 1, wherein said nucleotide sequence encodes a functional PCDH 19 protein or polypeptide, or a functional fragment or functional variant thereof; a functional PCDH 19 protein or polypeptide, or functional fragment or functional variant thereof encoded by a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete PCDH19 ORF nucleotide sequence according to SEQ ID NO: 1; and/or an agent that compensates for the loss of PCDH 19 protein function; optionally in combination with a pharmaceutically-acceptable carrier.
• PCDHl complete protocadherin 19
• ORP open reading frame
• the present invention provides an agent capable of treating a deficiency in functional protocadherin 19 (PCDH 19) protein or altered PCDH 19 protein function in a subject.
• PCDH 19 functional protocadherin 19
• the present invention provides a method for identifying an agent capable of treating a deficiency in functional protocadherin 19 (PCDH 19) protein or altered PCDH 19 protein function, wherein said method comprises the steps of;
• the present invention provides a kit for use in the method of the sixth aspect, wherein said kit comprises instructions for the operation of the method together with one or more containers and/or vessels containing one or more cell(s) or animal(s) comprising a polynucleotide molecule comprising a mutant sequence of the protocadherin 19 (PCDHl 9) ORF nucleotide sequence shown as SEQ ID NO: 1.
• the present invention provides a kit for identifying an agent capable of treating a deficiency in functional protocadherin 19 (PCDH 19) protein or altered PCDH 19 protein function, wherein said kit comprises;
• control cell or animal comprising a polynucleotide molecule comprising a wild-type form of the complete PCDHl 9 ORP nucleotide sequence shown as SEQ ID NO: 1, said wild-type form encoding a functional PCDH 19 protein or polypeptide, or a functional fragment or functional variant thereof.
• the present invention provides an isolated protein or polypeptide comprising an amino acid sequence encoded by a nucleotide sequence showing at least 70% sequence identity to a complete protocadherin 19 [PCDHl 9) ORF nucleotide sequence according to SEQ ID NO: 1, or a functional fragment or variant thereof.
• the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to a complete protocadherin 19 (PCDHl 9) ORF nucleotide sequence according to SEQ ID NO: 1 or a complementary sequence thereto.
• PCDHl 9 complete protocadherin 19
• the present invention provides a cell transformed with the polynucleotide molecule of the tenth aspect.
• the present invention provides a non-human animal comprising the polynucleotide molecule of the tenth aspect.
• Figure 1 shows pedigrees of the seven EFMR families assessed in the studies described herein.
• a specific mutation in the PCDHl 9 gene, responsible for EFMR in each family, is indicated alongside the corresponding sequence chromatogram section showing the location of the mutation.
• Females presenting with EFMR are represented by filled circles and carrier males are represented by small circles within squares.
• FIG. 2 shows a schematic diagram of the PCDH19 protein with the signal peptide, extracellular cadherin (EC), transmembrane (TM) and cytoplasmic (CM) domains indicated. The relative locations of mutations found in the EFMR families are also shown.
• Figure 3 shows the partial alignment of the human PCDHl 9 with orthologues of PCDH19 from other species and other human protocadherins. The high conservation of residues affected by two missense mutations, V441E (top panel) and N557K (bottom panel) are indicated by rectangular boxes. The calcium ion-binding acidic residues are also indicated by a bracket against both alignments.
• Figure 4 shows Northern blot (Clontech Laboratories, Inc., Mountain View, CA, United States of America) analyses of PCDHl 9 and PCDHl 1X/Y in various human brains tissues.
• the position of the -9.8 kb PCDH19 transcript is indicated by an asterisk, while the position of the smaller -9.5 kb PCDHl 1X/Y mKNAs is shown by an arrowhead.
• the brackets indicate either non-specific binding of the PCDHl 9 probe or PCDH19 degradation products.
• Figure 5 shows a section of a nucleotide sequence chromatogram from an EFMR affected female, indicating the detection of a mutation, 253OT, in genomic DNA (gDNA) (top panel), the absence of the mutant sequence in fibroblast cDNA (middle panel) and the presence of both the mutant and wild-type cDNA after the treatment of fibroblasts with cyclohexamide (bottom panel). The position of the mutation is boxed.
• Figure 6 shows the expression of PCDH19 in murine central nervous system (CNS) at 15.5 days postcoital (a-f) and postnatal day 2 (g-1).
• CNS central nervous system
• Arrowheads in c indicate PCDH 19 expressing cells within the cortex; the asterisk in e highlights the dorsolateral wall of the lateral ventricle, (g, h) are adjacent sections stained with Haematoxylin and Eosin and processed for PCDHl 9 in situ, respectively, (i) is a posterior brain section (to h) highlighting PCDH19 expression, (j, k, 1) are higher magnification images of the boxed regions in (g, h, respectively).
• Cx/P cortical plate
• Hn Hippocampal neuroepithelium
• Iv lateral ventricle
• Th thalamus
• Hy hypothalamus
• icf intercerebral fissure
• Ob olfactory bulbs
• Ne nasal epithelium.
• Magnification bars (a, b, f-i) represent 200 ⁇ M
• bars in (c-e; and j-1) represent 50 ⁇ M.
• Figure 7 shows a diagrammatic representation of the expected mechanism underlying the inheritance of EFMR whereby PCDHl IY functionally rescues PCDH19 mutations in transmitting males.
• Figure 8 shows expression profiles of PCDH19 (top panel), PCDHl IY (middle panel) and PCDHl IX (bottom panel) in various regions of adult human brain tissues.
• the present invention generally relates to methods and kits for the diagnosis of illnesses related to PCDH 19 protein deficiency or altered PCDH 19 protein function, methods and kits for the identification of a predisposition to such illnesses, methods of screening subjects to identify carriers of such illnesses, and methods and kits for the therapeutic or prophylactic treatment of PCDH 19 deficiency or altered PCDH 19 protein function.
• the present invention also relates to nucleotide and amino acid sequences corresponding to a complete PCDHJ 9 ORF, mutant sequences encoding non-functional PCDH19 mRNA (eg which may be degraded by nonsense mediated mRNA degradation (NMD) processes) or altered PCDH19 mRNA, or non-functional PCDH19 protein or altered PCDH19 protein causative of illnesses related to PCDH19 protein deficiency or altered function in human subjects, and transformed cells and transgenic non-human animals comprising wild-type or mutant PCDHl 9 ORF nucleotide sequences.
• NMD nonsense mediated mRNA degradation
• the present invention provides a method of diagnosing an illness related to functional protocadherin 19 (PCDH19) protein deficiency or altered PCDH19 protein function, or assessing a predisposition to an illness related to functional PCDH19 protein deficiency or altered PCDH 19 protein function, or screening to identify carriers of illnesses related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, wherein said method comprises the step of:
• Illnesses resulting from PCDH19 deficiency or altered PCDH19 protein function include epilepsy and/or mental retardation, in particular EFMR.
• the method of the first aspect provides a method of diagnosing EFMR, assessing a predisposition to EFMR or screening carriers of EFMR, in particular, male carriers of EFMR.
• preferred methods include prenatal diagnosis or screeing of EFMR.
• the detection of a loss of PCDH 19 protein function or altered PCDH 19 protein function can be used, in the case of a subject for which EFMR has not previously been diagnosed, either on its own or in combination with other tests, to diagnose EFMR in the subject.
• the detection of a loss of PCDH 19 protein function or altered PCDH 19 protein function can be used in an assessment of a predisposition to EFMR or carrier status of EFMR.
• the detection of a loss of PCDH 19 protein function or altered PCDH 19 protein function can involve one or more of detecting a mutant sequence in a PCDHl 9 ORF of the subject which encodes non-functional or altered PCDH19 mRNA or non-functional or altered PCDH19 protein (eg by genotyping the subject) or causes reduced expression of PCDH19 protein (eg mutations of the PCDHl 9 gene expression control sequences). Conveniently, this may be achieved by amplifying the PCDHl 9 nucleotide sequences (or a target region thereof) within a suitable biological sample and, thereafter sequencing the amplication product.
• the detection of a loss of PCDH 19 protein function involves detecting a mutant sequence in the extracellular (EC) domain-encoding region of a PCDHl 9 ORF of the subject such as, for example, a mutant sequence causing an amino acid substitution within or adjacent to a calcium ion- binding site (eg within 20 amino acids of a calcium ion-binding site) such that calcium ion binding is impaired, or a mutant sequence comprising a premature termination codon (PTC).
• a mutant sequence in the extracellular (EC) domain-encoding region of a PCDHl 9 ORF of the subject such as, for example, a mutant sequence causing an amino acid substitution within or adjacent to a calcium ion- binding site (eg within 20 amino acids of a calcium ion-binding site) such that calcium ion binding is impaired, or a mutant sequence comprising a premature termination codon (PTC).
• a mutant sequence in the extracellular (EC) domain-encoding region of a PCDHl 9 ORF of the subject such
• the detection of a loss of PCDH 19 protein function or altered PCDH 19 protein function may also be indirectly achieved by conducting, for example, assays for functional PCDH 19 protein or polypeptide.
• Assays for detecting functional PCDH 19 protein or polypeptide preferably comprise the use of an antibody or fragment thereof that is capable of specifically binding with PCDH19 protein or polypeptide, or a functional fragment or functional variant thereof, to determine the relative amount of the protein or polypeptide that is present in a suitable biological sample taken from the subject. This can involve the use of any of the methods well known to persons skilled in the art (eg standard ELISA-based methods or in situ immunofluorescence using tissue section samples).
• the relative amount of functional PCDH19 protein or polypeptide can be determined by quantitatively detecting the protein or polypeptide with a specific antibody or fragment thereof (ie a primary antibody) which is either directly conjugated to a detectable label or is otherwise detected via a secondary antibody or fragment thereof directly conjugated to a detectable label.
• a detectable label include chromophores, fluorophores (eg fluorescein or FITC), radiolabels (eg 125 I), and enzymes such as horseradish peroxidase.
• Functional PCDH 19 protein or polypeptide may be characterised as being encoded by a nucleotide sequence showing at least 70% sequence identity, preferably at least 85% sequence identity, and, more preferably, at least 95% sequence identity to a complete PCDH19 open reading frame (ORF) nucleotide sequence according to:
• PCDH 19 protein or polypeptide is characterised by comprising an amino acid sequence according to:
• KRLKDIVL 1148 (SEQ ID NO: 2).
• assays for detecting loss of PCDH 19 protein function may comprise the use of an antibody or fragment thereof that is capable of distinguishing between, for example, a wild-type PCDH19 protein or polypeptide and a non-functional variant thereof. This may or may not result in the identification of the particular form of the PCDH 19 protein or polypeptide that is present in the biological sample taken from the subject.
• assays for detecting loss of PCDH19 protein function may comprise determining the relative amount of messenger RNA (mRNA) encoding functional PCDH 19 protein or polypeptide in a suitable biological sample taken from the subject.
• the relative amount of mRNA encoding the protein or polypeptide may be determined by any of the methods well known to persons skilled in the art including Northern blot (by comparison to reference samples) and PCR-based mRNA quantification methods (eg using RT-PCR with primers conjugated to a detectable label).
• the relative amount of mRNA encoding the protein or polypeptide will be determined by comparison against the amount, or range of amounts, present in "normal samples” (eg samples from equivalent biological samples taken from normal subject(s)).
• the detection of a loss of PCDH 19 protein function comprises detecting a mutant sequence encoding a non-functional variant of the PCDH19 amino acid sequence shown as SEQ ID NO: 2.
• Said mutant sequence may comprise any one or more of the nucleotide changes, relative to the nucleotide sequence shown as SEQ ID NO: 1, as follows: 1322 T>A, 253 OT, 2012 C>G, 2030_2031insT, 1671 OG, 357delC and 1091_1092insC.
• Methods for the detection of such nucleotide changes may comprise the step of detecting any hybridisation of a suitable oligonucleotide probe/primer molecule under high stringency conditions to the mutant sequence present in a suitable biological sample.
• High stringency conditions are well known to persons skilled in the art, and are typically characterised by high temperature (ie high annealing temperature) and low ionic strength (ie low salt concentration, especially OfMgCl 2 , KCl and NaCl). Thus, high stringency conditions may vary according to the circumstances of the hybridisation (ie for probe hybridisation, PCR amplification, etc).
• high stringency conditions is to be understood as referring to such conditions applicable to probe hybridisation (eg conditions which: (1) employ low ionic strength and high temperature for washing, for example, 15 mM NaCl/1.5 mM sodium citrate/0.1% NaDodSO 4 at 50 0 C; (2) employ, during hybridisation, a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5X SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS
• an oligonucleotide molecule useful in the detection of a mutant sequence according to the present invention may be suitable for use as, for example, a probe or primer sequence.
• the oligonucleotide molecule will consist of 10 to 50 nucleotides and, more preferably, about 15 to 30 nucleotides.
• the oligonucleotide molecule is derived from the nucleotide sequence shown as SEQ ID NO: 1 or a complementary sequence thereto, or the nucleotide sequence as shown as SEQ ID NO: 1 but incorporating one or more of the nucleotide changes mentioned above (ie 1322 T>A, 253 C>T, 2012 C>G, 2030_2031insT, 1671 OG, 357delC and 1091_1092insC) or a complementary sequence thereto.
• a suitable biological sample taken from the subject may be selected from, for example, tissue biopsies and fixed sections (eg formalin fixed or paraffin embedded) or fixed cell samples prepared therefrom, including epithelial samples, smear samples, blood samples, faecal samples, urine samples or buccal samples.
• the sample may be pre-treated by, for example, filtration, separation or extraction methods to partly or completely purify or isolate cells, proteins, polypeptides, polynucleotide molecules, oligonucleotide molecules or fragments thereof or fractions containing these components.
• the invention provides a kit for diagnosing an illness related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, or assessing a predisposition to an illness related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, or screening to identify carriers of illnesses related to functional PCDH19 protein deficiency or altered PCDH19 protein function, wherein said kit comprises one or more of the following: an antibody or fragment thereof which specifically binds to PCDH 19 protein or polypeptide, or a fragment or variant thereof; and an oligonucleotide probe/primer molecule which specifically hybridises to a polynucleotide molecule encoding PCDH 19 protein or polypeptide, or a fragment or variant thereof under high stringency conditions.
• kits may comprise, for example, instructions for the operation of the method and, optionally, for thereafter diagnosing an illness related to functional PCDH19 protein deficiency or altered PCDH19 protein function, or assessing a predisposition to an illness related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, or identifying carriers of illnesses related to functional PCDH 19 protein deficiency or altered PCDH 19 protein function, together with one or more containers or vessels containing said antibody or fragment thereof and/or said oligonucleotide probe/primer molecule.
• said antibody or fragment thereof will bind to a protein or polypeptide comprising an amino acid sequence showing at least 70% sequence identity to the PCDH 19 amino acid sequence according to SEQ ID NO: 2.
• said oligonucleotide probe/primer molecule will hybridise to a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete PCDHl 9 ORF nucleotide sequence according to SEQ ID NO: 1.
• the present invention provides for the use of: a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete protocadherin 19 (PCDHl 9) open reading frame (ORF) nucleotide sequence according to SEQ ID NO: 1, wherein said nucleotide sequence encodes a functional PCDH19 protein or polypeptide, or a functional fragment or functional variant thereof; or a functional PCDH 19 protein or polypeptide, or functional fragment or functional variant thereof encoded by a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete PCDHl 9 ORF nucleotide sequence according to SEQ ID NO: 1; in the treatment of PCDH19 protein deficiency or altered PCDH19 protein function in a subject.
• PCDHl 9 complete protocadherin 19
• the said nucleotide sequence shows at least 85% sequence identity, and, more preferably, at least 95% sequence identity to the complete PCDH19 ORF nucleotide sequence according to SEQ ID NO: 1.
• the said functional PCDH19 protein or polypeptide comprises an amino acid sequence according to SEQ ID NO: 2.
• the term "functional fragment” as used herein is to be understood as referring to a fragment which exhibits biological activity that is substantially equivalent to a protein or polypeptide comprising the complete PCDHl 9 amino acid sequence shown as SEQ ID NO: 2.
• variant as used herein in relation to an amino acid sequence, is to be understood as referring to a protein or polypeptide, or fragment thereof, comprising an amino acid sequence showing a high level of sequence identity to the corresponding complete (or part thereof as the case may be) of the amino acid sequence shown as SEQ ID NO: 2, but which includes one or more variations in the sequence which do not result in any significant alteration of the biological activity of its derivative protein or polypeptide (ie a protein or polypeptide comprising the complete PCDH 19 amino acid sequence shown as SEQ ID NO: 2) or which otherwise results in enhanced or reduced biological activity (eg variants may include one or more amino acid substitutions, additions or deletions, or may include the addition or deletion of a sequence of amino acids, which enhances or reduces biological activity).
• a variant with enhanced or reduced biological activity can therefore be regarded as a "functional variant", whereas a variant which has no or minimal biological activity can be regarded as a "non-functional variant".
• Variations that do not result in any significant alteration of the biological activity may include conservative amino acid substitutions.
• Exemplary conservative amino acid substitutions are provided in Table 1 below. Particular conservative amino acids envisaged are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, N ⁇ -alkylamino acids.
• the present invention provides a method for the therapeutic or prophylactic treatment of protocadherin 19 (PCDH19) protein deficiency or altered PCDH19 protein function in a subject, wherein said method comprises the step of:
• a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete protocadherin 19 (PCDHl 9) open reading frame (ORF) nucleotide sequence according to SEQ ID NO: 1, wherein said nucleotide sequence encodes a functional PCDH 19 protein or polypeptide, or a functional fragment or functional variant thereof; a functional PCDH 19 protein or polypeptide, or functional fragment or functional variant thereof encoded by a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete PCDH19 ORP nucleotide sequence according to SEQ ID NO: 1; and/or an agent that compensates for the loss of PCDH 19 protein function; optionally in combination with a pharmaceutically-acceptable carrier.
• PCDHl complete protocadherin 19
• ORF open reading frame
• the method comprises administering a functional PCDH 19 protein or polypeptide comprising an amino acid sequence according to SEQ ID NO: 2, or a functional fragment or functional variant thereof.
• the method comprises administering an agent that compensates for the loss of PCDH 19 function in the subject.
• an agent is a protein or polypeptide that compensates for PCDH 19 function, such as another protocadherin/cadherin protein, or a functional fragment or functional variant thereof.
• An example of a preferred compensatory agent is PCDHl IY.
• the method of the fourth aspect may include the administration of whole cells or recombinant delivery vehicles (eg viral vectors).
• Suitable polynucleotide molecule-delivery vectors include those suitable for the chromosomal integration of the polynucleotide molecule of the present invention (eg retroviral vectors), or simply for the non- integrated expression of the polynucleotide molecule (eg plasmid vectors).
• a polynucleotide molecule encoding a PCDH 19 protein or polypeptide, or functional fragment or functional variant thereof may involve any of the methods and/or agents for the delivery of "naked DNA” to cells well known to persons skilled in the art (eg liposomes, lipoplexes, polyplexes, gold microparticles, and conjugation to mannose and the like).
• the present invention provides an agent capable of treating a deficiency in functional protocadherin (PCDH 19) protein or altered PCDH 19 protein function in a subject.
• PCDH 19 functional protocadherin
• Preferred agents according to the fifth aspect include: a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity, preferably at least 85% sequence identity, and, more preferably, at least 95% sequence identity to the complete PCDH19 ORF nucleotide sequence according to SEQ ID NO: 1; or a functional PCDH19 protein or polypeptide, or functional fragment or functional variant thereof encoded by a polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity, preferably at least 85% sequence identity, and, more preferably, at least 95% sequence identity to the complete PCDHl 9 ORF nucleotide sequence according to SEQ ID NO: 1.
• the agent is a polynucleotide molecule comprising a nucleotide sequence encoding a PCDH 19 protein or polypeptide comprising the amino acid sequence shown as SEQ ID NO: 2; or a functional PCDH19 protein or polypeptide comprising the amino acid sequence shown as SEQ ID NO: 2.
• Further preferred agents include an isolated or recombinantly expressed PCDHl IY protein or polypeptide, or a functional fragment or functional variant thereof.
• Homologues, analogues, orthologues or mimetics of PCDH19 or PCDHl IY may also be suitable and, indeed, these may possess further desirable characteristics for use as therapeutic agents, for example in vivo stability, safety and toxicity, pharmaceutical acceptability and the like.
• the selection of preferred homologues, analogues, orthologues or mimetics of PCDH19 or PCDHl IY according to desirable characteristics may be readily determined by methods well known to persons skilled in the art.
• agents according to the fifth aspect are agents that are capable of providing treatment to EFMR or prophylactic treatment to subjects predisposed to EFMR.
• Agents of the fifth aspect may be administered with a pharmaceutically acceptable carrier, and/or formulated into any suitable pharmaceutical/veterinary composition or dosage form (eg compositions for oral, buccal, nasal, intramuscular and intravenous administration).
• a composition or dosage form will be administered to the subject in an amount which is effective to treat EFMR or provide prophylaxis to a subject predisposed to EFMR, and may therefore be provided at between about 0.01 and about 100 ⁇ g/kg body weight per day of the agent, and more preferably, at between 0.05 and 25 ⁇ g/kg body weight per day of the agent.
• a suitable composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective result.
• the present invention provides a method for identifying an agent capable of treating a deficiency in functional protocadherin 19 (PCDH 19) protein or altered PCDH 19 protein function, wherein said method comprises the steps of;
• the method of the sixth aspect may identify agents capable of providing a treatment of illness caused by a deficiency in functional PCDH19 protein or altered PCDH19 protein function, or which may be capable of providing a prophylactic treatment of functional PCDHl 9 protein deficiency or altered PCDH 19 protein function.
• control response referred to in step (iii) of the method may include a baseline response detected in said cell or animal without exposure to the test agent or, alternatively, the control response may be a response following exposure to the test agent in cells or animals comprising a normal or wild-type complete PCDH19 ORF nucleotide sequence.
• test agent may be selected from known and novel compounds, complexes and other substances which may, for example, be sourced from private or publicly accessible agent libraries (eg the Queensland Compound Library (Griffith University, Nathan, QLD, Australia) and the Molecular Libraries Small Molecule Repository (NIH Molecular Libraries, Bethesda, MD, United States of America).
• the test agent may therefore comprise a protein, polypeptide or peptide (eg a recombinantly expressed PCDH 19 or PCDHl IY protein or polypeptide, or a functional fragment or functional variant thereof), or a mimetic thereof (including so-called peptoids and retro-inverso peptides), but more preferably comprises a small organic molecule and especially one which complies or substantially complies with Lipinski's Rule of Five for "druglikeness" (Lipinski, CA et al., 2001).
• the test agent may also be selected on the basis of structural analysis of known or novel compounds or may otherwise be designed following the further structural analysis of PCDHl 9 or PCDHl IY binding sites, particularly calcium ion binding sites.
• the method of the sixth aspect may be adapted for high-throughput screening of large numbers of test agents.
• the step of comparing a response in said cell or animal with a control response may be conducted using one or more standard binding assay formats (eg ELISA-based or competition-based assays).
• the test agent will be labelled with a readily detectable label (eg a fluorochrome or radioisotope) to allow detection of binding to, for example, a calcium channel receptor.
• a readily detectable label eg a fluorochrome or radioisotope
• a change in activity may be observed in such assays by using standard methods including spectrophotometric, fluorimetric, calorimetric or chemi luminescent means preferably providing for the automation or partial automation of the detecting step (eg by a microplate reader or use of a flow cytometer).
• kits for use in the method of the sixth aspect wherein said kit comprises instructions for the operation of the method together with one or more containers and/or vessels containing one or more cell(s) or animal(s) comprising a polynucleotide molecule comprising a mutant sequence of the protocadherin 19 (PCDHl 9) ORF nucleotide sequence shown as SEQ ID NO: 1.
• the present invention provides a kit for identifying an agent capable of treating a deficiency in functional protocadherin 19 (PCDH 19) protein or altered PCDH 19 protein function, wherein said kit comprises;
• control cell or animal comprising a polynucleotide molecule comprising a wild-type form of the complete PCDH19 ORF nucleotide sequence shown as SEQ ID NO: 1, said wild-type form encoding a functional PCDH 19 protein or polypeptide, or a functional fragment or functional variant thereof.
• the kit of the seventh or eighth aspect may further comprise means for comparing a response in said mutant cell or animal with a control response (eg as caused by a test agent), means for detecting a response (eg adhesiveness of PCDH 19 or impaired calcium ion binding in the mutant cell or animal) and, for example, a test agent, and other components as are well known to persons skilled in the art including, for example, wash buffers, stabilisation buffers or other reagents.
• a control response eg as caused by a test agent
• means for detecting a response eg adhesiveness of PCDH 19 or impaired calcium ion binding in the mutant cell or animal
• a test agent eg adhesiveness of PCDH 19 or impaired calcium ion binding in the mutant cell or animal
• the present invention provides an isolated protein or polypeptide comprising an amino acid sequence encoded by a nucleotide sequence showing at least 70% sequence identity to a complete protocadherin 19 (PCDH19) ORF nucleotide sequence according to SEQ ID NO: 1, or a functional fragment or variant thereof.
• PCDH19 complete protocadherin 19
• the term "isolated”, when used in relation to a protein or polypeptide, or a functional fragment or variant thereof, or when used in relation to a polynucleotide molecule, is to be understood as referring to the protein, polypeptide, functional fragment, variant or polynucleotide molecule in a form that is essentially free of whole cells, components thereof and/or other exogenous cellular or biological materials such as exogenous proteins, polypeptides, peptides and nucleic acid molecules.
• an isolated protein, polypeptide, functional fragment, variant or polynucleotide molecule in accordance with the present invention, may be present in a preparation comprising no more than 10% (by weight) of exogenous cellular or biological materials, and may be prepared by any of the methods well known to persons skilled in the art.
• the protein or polypeptide comprises an amino acid sequence showing at least 75% sequence identity to the complete PCDHl 9 amino acid sequence shown as SEQ ID NO: 2 and at least 65% sequence identity to the amino acid sequence corresponding to the extracellular cadherin (EC) domain.
• the protein or polypeptide, or functional fragment or variant thereof comprises an amino acid sequence showing at least 85% sequence identity and, still more preferably, at least 95% sequence identity to the complete PCDH19 amino acid sequence shown as SEQ ID NO: 2.
• the protein or polypeptide comprises an amino acid sequence according to SEQ ID NO: 2.
• the present invention provides a variant, preferably a nonfunctional variant, of the amino acid sequence shown as SEQ ID NO: 2 including one or more amino acid mutations.
• Particularly preferred mutations included within such a variant include one or more amino acid mutations selected from the following changes to the amino acid sequence shown as SEQ ID NO: 2; V441E, Q85X, S671X, L667fsX717, N557K, I119fsX122 and P364fsX375.
• the isolated protein or polypeptide, functional fragment or variant thereof may be isolated from tissues derived from whole organisms (eg biopsied tissues), from cultured tissues (eg cultured fibroblasts), or from other recombinant expression systems. This may involve any of the methods for isolating proteins or polypeptides well known to persons skilled in the art, including ion exchange, chromatography electrophoresis, isoelectric focusing, adsorption chromatography, paper chromatography, reverse-phase chromatography, hydrophobic interaction chromatography, dialysis, ultrafiltration, gel electrophoresis, gel filtration, and ultracentrifugation.
• Suitable techniques for expressing a recombinant protein or polypeptide, functional fragment or variant thereof according to the present invention are well known to persons skilled in the art and include, for example, techniques for expressing recombinant His-tagged PCDH 19 from a suitable expression vector or cassette using a suitable host cell (eg CHO cells and BL21 cells). Thereafter, the His-tagged expression products can be readily isolated using affinity chromatography (eg using a Ni-NTA column (Qiagen Inc, Valencia, CA, United States of America)). Where a functional fragment is to be provided, isolated recombinant protein or polypeptide may be cleaved using a proteolytic enzyme (eg trypsin).
• a proteolytic enzyme eg trypsin
• Proteins, polypeptides, variants or, in particular, functional fragments according to the present invention may optionally be incorporated into synthetic proteins or polypeptides such as fusion proteins.
• Fusion proteins may include components that assist in the production or downstream processing (eg a protein, polypeptide, functional fragment or variant thereof, may be linked to a secretory signal peptide, affinity purification tag or the like).
• the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence showing at least 70% sequence identity to the complete protocadherin 19 ⁇ PCDH19) ORF nucleotide sequence according to SEQ ID NO: 1 or a complementary sequence thereto.
• the polynucleotide molecule comprises a nucleotide sequence showing at least 85% sequence identity and, more preferably, at least 95% sequence identity to the complete PCDH19 ORF nucleotide sequence shown as SEQ ID NO: 1. Most preferably, the polynucleotide molecule comprises a nucleotide sequence as shown as SEQ ID NO: 1.
• a polynucleotide molecule of the present invention may encode a protein or polypeptide comprising the amino acid sequence according to SEQ ID NO: 2, or a functional fragment or variant thereof.
• a polynucleotide molecule of the present invention may otherwise encode a non-functional PCDHl 9 mRNA (eg an mRNA including a premature termination codon which is degraded by NMD processes) or altered PCDH 19 mRNA.
• polynucleotide molecules according to the present invention are oligonucleotide probe/primer molecules which consist of 10 to 50 contiguous nucleotides and, more preferably, about 15 to 30 contiguous nucleotides of the complete PCDHl 9 ORF nucleotide sequence shown as SEQ ID NO: 1.
• polynucleotide molecule comprises a nucleotide sequence showing at least 70% sequence identity, preferably at least 85% sequence identity and, more preferably, at least 95% sequence identity to the nucleotide sequence shown as SEQ ID NO: 1, it will be appreciated that such a polynucleotide molecule may vary from that nucleotide sequence only in minor changes which, for example, do not result in a significant alteration in an encoded protein or polypeptide due to degeneracy in the DNA code or which may be required in order to enhance expression in a particular system (ie to comply with preferred codon usage).
• polynucleotide molecule may otherwise encode a variant of a protein or polypeptide comprising the amino acid sequence shown as SEQ ID NO: 2 which shows enhanced or reduced biological activity (eg a variant including one or more amino acid mutations in the extracellular cadherin (EC) domain and showing reduced adhesiveness through impaired calcium ion binding).
• SEQ ID NO: 2 which shows enhanced or reduced biological activity
• the present invention provides a polynucleotide molecule encoding a variant, preferably a non-functional variant, including one or more amino acid substitutions, additions or deletions in the extracellular cadherin (EC) domain and showing reduced adhesiveness through impaired calcium binding.
• mutations encoded by such polynucleotide molecules include one or more of the following changes to the amino acid sequence shown as SEQ ID NO: 2; V441E, Q85X, S671X, L667fsX717, N557K, Il 19fsX122 and P364fsX375.
• the mutations responsible for such amino acid sequence changes may be, respectively; 1322 T>A, 253 OT, 2012 OG, 2030_2031insT, 1671 OG, 357delC and 1091_1092insC.
• the polynucleotide molecule of the present invention may be used to express an encoded protein or polypeptide, or functional fragment or variant thereof, by recombinant methodologies involving cloning of the polynucleotide molecule into a suitable expression cassette or vector and thereafter introducing the expression cassette or vector into a suitable host cell.
• Suitable expression vectors may include functional sequences such as a multiple cloning site, a detection tag (eg glutathione-S-transferase (GST) or green fluorescent protein (GFP)), a tag for downstream purification (eg a histidine tag (His)), linker and fusion sequences.
• the present invention provides a cell transformed with the polynucleotide molecule of the tenth aspect.
• the polynucleotide molecule may comprise a mutant sequence of the PCDH19 ORF nucleotide sequence shown as SEQ ID NO: 1, and thereby encode non-functional or altered PCDHl 9 mRNA, non-functional PCDH 19 protein or a PCDH 19 protein with altered function, or which otherwise causes reduced expression of PCDH 19 protein, or a complementary sequence thereto.
• the polynucleotide molecule may also consist or encode an antisense RNA, ribozyme, DNAzyme or interfering RNA molecule (eg siRNA) targeted to PCDH19.
• the transformed cell may be selected from bacterial cells, insect cells and mammalian cells.
• the cell may be transformed using any of the methods well known to persons skilled in the art including direct uptake, transduction, f-mating or electroporation.
• the transformed polynucleotide molecule may be maintained in a non-integrated form (eg in a non-integrated plasmid expression vector), or alternatively, may be integrated into the genome of the transformed cell.
• the transformed cell can be employed in a variety of applications that will be readily apparent to persons skilled in the art, in particular, for the generation of an isolated recombinant protein or polypeptide, or functional fragment or variant thereof according to the present invention.
• the expression of the recombinant product can be determined by, for example, Western immunoblot assays for the direct detection of the protein or polypeptide, functional fragment or variant thereof, or for detection of an expression tag (eg a His tag).
• an expression tag eg a His tag.
• the present invention provides a non-human animal comprising the polynucleotide molecule of the tenth aspect.
• the polynucleotide molecule may comprise a mutant sequence of the PCDH 19 ORF nucleotide sequence shown as SEQ ID NO: 1, and thereby encode non-functional or altered PCDHl 9 mRNA, non-functional PCDH 19 protein or a PCDH 19 protein with altered function, or which otherwise causes reduced expression of PCDH 19 protein, or a complementary sequence thereto.
• the polynucleotide molecule may also consist or encode an antisense RNA, ribozyme, DNAzyme or interfering RNA molecule (eg siRNA) targeted to PCDH19.
• the polynucleotide molecule is preferably uniformly integrated throughout the animal's tissues. Where a chimeric animal is provided, the polynucleotide molecule is preferably present in cells of the animal's nervous tissues.
• the polynucleotide molecule may be introduced into the animal by any of the methods of transformation or transgenesis well known to persons skilled in the art.
• transformation methods include DNA transfection (via electroporation, liposome or protoplast fusion, or microinjection), infection with viral delivery vectors (ie vectors that facilitate genomic integration such as adenoviral and retroviral vectors), or via random mutagenesis followed by the selection of desired mutations by screening.
• the animal of the present invention will generally be preferably generated by micro-injection methodologies.
• micro-injection is preferably performed at the one-cell embryo stage by any of the methods well known to persons skilled in the art.
• Preferred transgenic animals include rodents, in particular mice and rats.
• the animals of the eleventh aspect can be employed in a variety of applications that will be readily apparent to persons skilled in the art, in particular, for use as in vitro or in vivo disease models for use in methods or kits for screening potential agents to compensate for PCDH 19 protein deficiency or altered PCDH 19 protein function.
• the present invention provides an antibody or fragment thereof which specifically binds to the protein or polypeptide, functional fragment or variant thereof, of the ninth aspect.
• Suitable antibodies include monoclonal and polyclonal antibodies.
• Suitable antibody fragments include fragments produced by enzymatic cleavage of antibodies such as Fab and F(ab') 2 fragments, and recombinant antibody fragments such as single chain Fv (scFv) fragments.
• the antibody or fragment thereof may be capable of distinguishing between, for example, a wild-type PCDH19 protein (ie comprising the complete PCDH19 amino acid sequence shown as SEQ ID NO: 2) and a variant thereof, particularly, a non-functional variant thereof.
• the present invention extends to an antibody or fragment thereof that specifically binds to a variant of the complete PCDH 19 amino acid sequence shown as SEQ ID NO: 2.
• Families 1-4 are described in Scheffer IE et al. (2007).
• Family 5 was screened on the basis of one sister having infantile seizures and Asperger syndrome and her sister having epilepsy and mild intellectual disability.
• the clinical details of members of Family 6 and Family 7 are described elsewhere (Juberg RC and Hellman CD, 1971; Fabisiak K and Erickson RP, 1990; and Ryan SG et al, 1997).
• forward primer - 5'CCGGATTCTTGGCCACTCTGACS' (SEQ ID NO: 3); and reverse primer - 5'CAATGGTGTAAGACACGGAAGS' (SEQ ID NO: 4).
• the 374 bp probe was labelled with radioactive ⁇ 32P-dCTP (Perkin Elmer, Waltham, MA, United States of America) using the Mega prime DNA labelling system (GE Healthcare, Piscataway, NJ, United States of America).
• cDNAs were amplified with Taq DNA polymerase (Roche, Basel, Switzerland) and specific single-stranded DNA primers for 35 cycles (denaturation, 94 0 C for 30 seconds; annealing, at specific Tm for each pair of primers for 30 seconds; extension, 72 0 C for 30 seconds).
• PCR products were separated by agarose gel electrophoresis and stained with 1% ethidium bromide for visualisation under UV.
• a 3 mm skin biopsy excised from the upper arm of each subject was cut finely and transferred to a tissue culture flask.
• the biopsy was cultured in RPMI medium with 20% foetal calf serum (FCS) (further supplemented with 4 mM L-Glutamine, 0.017 mg/ml benzylpenicillin) and grown at 37 0 C with 5% CO 2 .
• FCS foetal calf serum
• Cvcloheximide treatment of skin fibroblast cell lines Primary fibroblast cells were seeded 1x 10 4 /cm 2 in RPMI with 10% FCS and incubated with 50 ⁇ g/ml cycloheximide (Sigma-Aldrich Co, St Louis, MO, United States of America), or media alone, for 6 hrs. Fibroblasts were harvested using a sterile cell scraper (Techno Plastic Products AG, Trasadingen, Switzerland), then washed once in phosphate buffered saline (PBS) prior to total RNA extraction and reverse transcription to generate cDNA as described above.
• PBS phosphate buffered saline
• Gene expression profiles were generated using Rapid-Scan Gene Expression Human Brain cDNA panels (Origene Technologies, Inc, Rockville, MD, United States of America) containing first strand cDNA prepared from polyA+ RNA.
• the cDNA panels allow semi-quantitative analysis due to the cDNAs being serially diluted over a 4-log range.
• the profiles were obtained from panels containing approximately 1 ng of first strand cDNA.
• the PCR primer pair X-RT4F2 - 5' GTA ACA AGT GTA CCT GGT ATG GAC T 3' (SEQ ID NO: 5) and X-RT5R2 - 5' TCA ACC TTT ACT TTC ATC ACG 3' (SEQ ID NO: 6), were used to amplify the PCDHl IX sequence to yield a 683 bp product, and the primer pair; Y-RT4F - 5' TAC AAC AAA CTG TCA CAA GTG TTT 3' (SEQ ID NO: 7), and Y-RT5R2 - 5' TCA ACC TTT ACT TTC ATC ACA 3' (SEQ ID NO: 8), were used to amplify PCDHUY to yield a 681 bp product.
• WLR - 5' CTG CAG ATG GTC ACA TCG AC 3' (SEQ ID NO: 10), were used to amplify PCDH 19 to yield a 626 bp product.
• the PCR conditions used to amplify the PCDH19 product comprised an initial step at 94 ° C for 3 minutes, followed by 35 cycles at 94 0 C for 30 seconds, 60° for 30 sec and 72° C for 2 mins.
• the PCDHl IX and PCDHl 1 Y products were amplified using Hotstar Taq (Qiagen) according to the Hotstar recommended cycling conditions (94 0 C for 15 minutes followed by 10 cycles at [94° for 30 sec, 60° for 30 sec, 72° for 1 min] and then 30 cycles at [94° for 30 sec, 55° for 30 sec, 72° for 1 min] followed by 72 0 C for 10 min).
• accession numbers corresponding to these sequences are:
• GenBank library can be accessed at the following URLs;
• the EFMR gene was identified from re-sequenced 737 VEGA annotated X-chromosome genes in probands from three families. In all three families, each of the X chromosomes encoded protocadherin 19 (PCDH 19) mutations (X m ) which were found to co-segregate with the EFMR clinical phenotype.
• the PCDH19 gene was known to be located at Xq22 (Ensembl) within the original linkage region (Ryan SG et al, 1997).
• An example of a sequence chromatogram of a PCDHl 9 mutation as detected in an affected female from each family is shown alongside the pedigrees in Figure 1.
• N-terminal sequencing of family members identified a missense change, 1322T>A (residue V441E) in Family 1, a nonsense change, 253OT (residue Q85X) in Family 2 and a putative frameshift change, 2030 2031 insT (residues L667fsX717) in Family 4.
• the N-terminal PCDHl 9 region in affected females from an unreported Irish EFMR family (Family 5) was also sequenced resulting in the identification of a frameshift change, 375delC (residues Il 19fsX122).
• PCDHl 9 ORF nucleotide changes were identified in all seven of the assessed EFMR families. All seven nucleotide changes segregated with the clinical phenotype in each EFMR family and were not identified in 250 male probands from families with putative X-linked mental retardation (XLMR) or in 750 control X chromosomes. The positions of the PCDH 19 mutations in conjunction with alignments showing the location and conservation of the two missense mutations further demonstrate that PCDH 19 mutation is causative of EFMR.
• Rett syndrome is a female specific disease known to present with a similar phenotype to EFMR. 46 females with apparent Rett syndrome, who were negative for mutations in the two Rett associated genes, MECP2 and CDKL5, were investigated. No nucleotide changes were found in the Rett syndrome cohort, suggesting that PCDHl 9 mutations are unlikely to commonly contribute to Rett syndrome. Since autistic features were commonly seen in affected EFMR females, a cohort of 55 females with autism and seizures were screened for changes in PCDHl 9. The absence of mutations in this cohort suggests that PCDH19 mutations also do not commonly contribute to autism.
• PCDH 19 gene which consists of 6 exons was annotated (GenBank accession number EF676096).
• the full-length processed PCDH19 mRNA is 9.765 kb long, this was confirmed by Northern blot analysis which showed a transcript size of approximately 9.8 kb using a PCDHl 9 specific probe on combined male and female mRNA from various areas of the adult human brain ( Figure 4, PCDHl 9 mRNA is indicated by an asterisk).
• PCDHl 9 exon 2 was found to be alternatively spliced (results not shown).
• PCDHl 9 encodes an 1148 amino acid protein belonging to the protocadherin (PCDH) 52 subclass within the cadherin superfamily.
• the PCDH19 protein contains a signal peptide, six extracellular cadherin (EC) repeats, a transmembrane (TM) domain and a cytoplasmic region with conserved CMl and CM2 domains.
• FIG. 2 a schematic diagram is shown, illustrating the locations of the PCDH 19 amino acid sequence changes of each family with respect to the signal peptide, the extracellular cadherin domain (comprising ECl, EC2, EC3, EC4, EC5 and EC6), the transmembrane domain (TM) and the cytoplasmic (CMl and CM2) domains of the PCDHl 9 protein. All seven EFMR mutations identified are located in the large extracellular domain.
• PCDH 19 The biological role of PCDH 19 is not known; however, members of the PCDH family are predominantly expressed in the nervous system and are postulated to be involved in the establishment of neuronal connections and in signal transduction at the synaptic membrane (Wu Q and Maniatis T, 1999; Yagi T and Takeichi M, 2000).
• the equivalent asparagine residue of ECl of classical cadherins eg NlOO of N- cadherin; Patel SD et al, 2006
• protocadherins eg NlOl of CNR/Pcdh ⁇ ; Morishita H et al, 2006
• tissue mosaicism of PCDH 19 negative and PCDH 19 wild-type cells scrambles cell-cell communication manifesting clinically as EFMR.
• valine residue at position 441 (EC4 in Figure 2, or the equivalent of V96 of ECl of N-cadherin; Patel SD et al, 2006; or V97 of ECl of CNR/Pcdh ⁇ ; Morishita H et al,
• the PCDH19 mutations 253OT, Q85X (Family 2) and 2012OG, S671X (Family 3) introduce a premature termination codon (PTC) into the PCDH19 mRNA.
• PTC premature termination codon
• Such PTC-containing mRNAs are usually recognised by the NMD surveillance complexes and efficiently degraded (Maquat LE, 2004).
• the consequences of the PCDHl 9 mutations 253OT, Q85X (Family 2) and 2012OG, S671X (Family 3) were examined on the stability of their respective mRNAs by detecting PCDHl 9 mRNA in primary skin fibroblasts collected from biopsied patients by RT-PCR.
• Figure 5 shows a sequence chromatogram from an EFMR affected female from Family 2 showing the detection of the mutation 253OT, Q85X, in the genomic DNA (gDNA) (top panel), the absence of the mutant sequence in fibroblast cDNA (middle panel) and the presence of both mutant and wild-type cDNA after the treatment of fibroblasts with cyclohexamide (bottom panel), which inhibits the pioneer round of translation and leading to NMD. Similar results were found in tissues collected from EFMR affected members of Family 3 (2012OG mutation, S671X) (data not shown). The inhibition of NMD by cycloheximide treatment of skin fibroblast cells was found to preserve PTC mutation-containing mRNA.
• FIG. 6 shows the expression of PCDHl 9 in the developing murine CNS at 15.5 dpc ( Figures 6a to 6f) and P2 ( Figures 6g to 61).
• Figures 6a and 6b show adjacent sections stained with Haemotoxylin and Eosin and processed for PCDH19 mRNA in situ, respectively.
• PCDHl 9 mRNA was expressed in a widespread, symmetrical pattern in the embryonic forebrain and frequently localised to discrete cell clusters within the cortex (CxP, cortical plate), thalamus (Th) and hypothalamus (Hy).
• the lateral ventricle (Iv) and hippocampal neuroepithelium (Hn) are also indicated.
• Figures 6c, 6d and 6e show higher magnification images of the boxed regions in Figure 6b.
• the arrowheads in Figure 6c indicate PCDH19-expressing cells within the cortex.
• expression was restricted to the cortical plate and extended medially into the intercerebral fissure (icf) (Figure 6d).
• the asterisk in Figure 6e highlights the dorsolateral wall of the lateral ventricle, robust expression was also detected in the ganglionic eminence that abuts the dorsolateral wall of the lateral ventricles.
• Figures 6g and 6h show adjacent sections stained with Haemotoxylin and Eosin and processed for PCDHl 9 mPvNA by in situ hybridisation, respectively.
• Figure 6i shows a brain section posterior with respect to the brain section shown at Figure 6h, each highlighting PCDHl 9 mRNA expression.
• Figures 6j, 6k and 61 show higher magnification images of the boxed regions in Figures 6g and 6h, respectively.
• PCDHl IY gene is thought to have arisen by transposition from Xq after the divergence of chimpanzees and humans (Lambson B et al, 1992; Page DC et al, 1984) and therefore, the PCDHl IY gene is only found in humans and in males.
• Figure 8 also shows expression profiles of PCDH19, PCDHl IX and PCDHl IY in the adult human Frontal lobe, temporal lobe, cerebellum, hippocampus, substantial nigra, caudate nucleus, amygdale, thalamus, hypothalamus, pons, medulla, and spinal cord.
• PCDHl 9 PCDHl IY and PCDHl IX are expressed in human brain.
• PCDHl IX and PCDHIlY show high sequence similarity, being 98.1% identical at the nucleotide level and 98.3% identical at the amino acid level (Blanco P et ⁇ l., 2000) and show similar expression profiles in brain regions (Blanco P et ⁇ l., 2000 and Figure 8).
• PCDHl IX and PCDHl /7 have undergone sequence divergence at the 5' and 3' ends of their ORF sequences, are regulated differently and show slight differences in their regions of expression in the brain (Blanco P et ⁇ l., 2000). It is therefore possible that PCDHl IX and PCDHIl Y have evolved different functions.
• PCDHl IX compensates for PCDHl 9 loss of function mutations in females and that both PCDHl IX and PCDHl IY compensate for PCDH19 mutations in males.
• a uniquely evolved function of PCDHl IY may enable the protein to provide greater rescue of PCDHl 9 mutations than PCDHl IX, which provides rationale for the greater frequency of spared male carriers than females presenting with EFMR.
• PCDHl 9 gene is located at Xq22.1 and is now known to harbour EFMR mutations.
• PCDHl IX on the X chromosome
• PCDHl IY on the Y chromosome.
• the results provided herein indicate that PCDHl IY may functionally rescue PCDHl 9 mutations in transmitting males, while in females PCDHl IX is unable to carry out rescue, explaining the EFMR phenotype being limited to females.
• PCDH19 mutations are causative of EFMR.
• the identification of nucleotide and amino acid sequences corresponding to a complete PCDHl 9 ORF provide for the development of diagnostic and therapeutic agents for EFMR and similar disorders associated with deficiencies in functional PCDHl 9 protein. Further, the elucidation of the suspected mechanism of PCDH19 rescue by PCDHl IY provides for the possibility of identifying and developing alternative therapeutic agents for the treatment of illnesses associated with PCDH 19 protein- deficiency.
• Gaitan Y Bouchard M. Expression of the ⁇ -protocadherin gene Pcdhl9 in the developing mouse embryo. Gene Express Patterns 6, 893-899 (2006).

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