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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the invention relates to autism and related disorders.
• Autism is a pervasive, behaviourally defined, developmental disorder consisting of a syndrome of delayed or abnormal speech development, impaired social interactions, and severely limited interests and activities. Autism is typically detected by 30 months of age, and is a life-long condition.
• autism-like disorders there is considerable variation in the severity of symptoms or signs, such as mental retardation, which is present in 75% of autistic subjects.
• AD HD attention deficit / hyperactivity disorder
• obsessive and compulsive behavioural disorders neurofibromatosis, developmental coordination disorder, anxiety disorders, schizophrenia, bipolar disorder, depression, Asperger's syndrome, Rett syndrome, Fragile X, Turner's syndrome (XO karyotype), XYY syndrome and tuberous sclerosis (TS).
• chromosome 7q31 which contains the language disorder gene FoxP2 (Newbury & Monaco, 2002; O'Brien etal, 2003).
• ADHD attention deficit / hyperactivity disorder
• AS Asperger's syndrome
• a method of detecting the presence or susceptibility towards autism or related disorders would have major therapeutic and/or prophylactic potential.
• a method of screening for genetic or epigenetic markers associated with autism or related disorders comprising the steps of isolating a biological sample from a mammal; and testing the sample or genetic material isolated from the sample for genetic polymorphisms/mutations and/or epigenetic alterations.
• a genetic marker is defined as a change in DNA sequence that is associated with a behavioural or other disorder.
• a genetic marker may also be understood as a mutation, a polymorphism, or a variant involving a change in DNA sequence associated with a behavioural or other disorder.
• An epigenetic marker is defined as a change in gene expression not involving a change in DNA sequence , that is associated with a behavioural or other disorder.
• An epigenetic marker may comprise a change in chromatic structure or a covalent modification of DNA (such as cytosine methylation) that is associated with a behavioural or other disorder.
• the polymorphism is located in the Xq / Yq pseudoautosomal gene region.
• polymorphism is located in the Xq / Yq pseudoautosomal gene region and extends into the adjacent Xq28 gene region.
• the polymorphism is located in the Xq28 gene region adjacent to the Xq / Yq pseudoautosomal boundary.
• the polymorphism may be a deletion of variable length.
• the screening for deleted nucleic acids is carried out by a method selected from the group consisting of any one or more of enzymatic cleavage and southern hybridisation; in situ hybridisation using probes from the specified region; detection of loss-of-heterozygosity using genetic analysis of polymorphic RFLP and microsatellite markers; gene copy number analysis using real-time or other quantitative PCR technologies or DNA chip or array technologies.
• the polymorphism involves a chromosomal translocation. In another embodiment the polymorphism involves a chromosomal inversion.
• the polymorphism involves a gene conversion event.
• the polymorphism causes a reduction in gene dosage or gene expression, of some or all of the genes that map to the specified region.
• the polymorphism causes an increase in gene dosage or gene expression, of some or all of the genes that map to the specified region.
• the polymorphism causes an alteration in gene dosage, or in the temporal or spatial aspects of gene expression, of some or all of the genes that map to the specified region.
• the polymo ⁇ hism causes an alteration in gene dosage, or in the temporal or spatial aspects of gene expression, of the HSPRY3 gene.
• the polymorphism causes an alteration in gene dosage, or in the temporal or spatial aspects of gene expression, of the SYBL1 gene.
• the polymo ⁇ hism involves a marker of epigenetic deregulation of gene expression.
• the marker of epigenetic deregulation of gene expression may be an alteration in patterns of DNA methylation or an alteration in patterns of nuclease sensitivity of DNA or chromatin.
• the polymo ⁇ hism involves a marker of epigenetic deregulation of gene expression comprising a change in the protein constitution of chromatin.
• the marker of deregulation of gene expression is altered copy number or structure of DNA repeats in the HSPRY3 gene region.
• the marker of deregulation of gene expression is alteration in the DNA sequence of the 'MER31I c' repeat in the HSPRY3 gene promoter.
• the marker of deregulation of gene expression is alteration in the DNA sequence of the 'GTTTT' repeat downstream of the HSPRY3 gene transcriptional start site.
• the marker of deregulation of gene expression is alteration of the DNA sequence downstream of the HSPRY3 gene protein coding region at the site of a recombination hotspot.
• the marker of deregulation of gene expression is alteration of the DNA sequence downstream of the HSPRY3 gene protein coding region at the site of a transcript expressed in the amygdala or other regions of the brain.
• the DNA sequence displaying abnormal levels of CpG methylation is the SYBL1 gene promoter-associated CpG island.
• the marker of epigenetic deregulation of gene expression is loss- of-imprinting (reactivation) of the Y-linked copies of the HSPRY3, SYBL1 and TRPC6-like genes, alone or in combination.
• the marker of epigenetic deregulation of gene expression is loss-of-imprinting (reactivation) of the Y-linked copy of the TRPC6-like gene.
• the marker of epigenetic deregulation of gene expression may be increased or decreased mRNA or protein levels for the specified genes, in the absence of detectable DNA sequence polymo ⁇ hisms.
• the biological sample may be selected from the group consisting of blood (including umbilical cord blood), saliva, semen, urine, amniotic fluid, placental biopsy, hair, tissue.
• the biological sample may be blood, a biopsy from a preimplantation stage embryo, a biopsy from the chorionic villus (extraembryonic tissue) of an implanted embryo (fetus) or fetal DNA or cells obtained from the serum of a pregnant mammal.
• the mammal is a human.
• the biological sample is isolated from developmentally disabled children or the biological sample may be isolated from parents or relatives of developmentally disabled children.
• the invention also provides a method for the treatment of autism and/or related disorders in children having genetic or epigenetic markers associated with autism or related disorders comprising the steps of :- detecting in a biological sample genetic polymo ⁇ hisms/mutations and/or epigenetic alterations; and providing treatment in the form of any one or more of early behaviour training; early dietary interventions or manipulations; or pharmacological interventions.
• the invention also provides a method for the treatment and/or prophylaxis of autism and/or related disorders in children having genetic markers associated with autism or related disorders comprising the steps of :- detecting in a biological sample genetic polymo ⁇ hisms/mutations and/or epigenetic alterations; and providing any one or more of gene therapy; activation or reactivation of epigenetically silenced genes; or silencing or reducing gene expression at the mRNA or protein level.
• the polymo ⁇ hism is located in the Xq Yq pseudoautosomal gene region and extends into the adjacent Xq28 gene region.
• polymo ⁇ hism is located in the Xq28 gene region adjacent to the Xq/Yq pseudoautosomal boundary.
• the invention also provides a method for the treatment and/or prophylaxis of autism and/or related disorders in children having genetic or epigenetic markers associated with autism or related disorders comprising activation or reactivation of epigenetically silenced genes in the Xq/Yq pseudoautosomal gene region.
• the invention further provides a method for the treatment and/or prophylaxis of autism and/or related disorders in children having genetic or epigenetic markers associated with autism or related disorders comprising the step of silencing or reducing gene expression at the mRNA or protein level in the Xq/Yq pseudoautosomal gene region.
• the invention also provides a method for selectively inhibiting or activating HSPRY3, AMD2; SYBL1, TRPC ⁇ -like, IL9R or CXYorfl activity in a human host, comprising administering a compound which selectively inhibits or upregulates the activity of the gene products of HSPRY3, AMD2, SYBL1, TRPC6-like, IL9R or
• the method may be used for the treatment and/or prophylaxis of autism and/or related disorders in children having genetic or epigenetic markers associated with autism or related disorders.
• the invention provides a method for the treatment and/or prophylaxis of tetanus susceptibility, tuberous sclerosis (TS) or attention deficit hyperactivity disorder (ADHD) in patients having genetic or epigenetic markers associated with autism.
• TS tuberous sclerosis
• ADHD attention deficit hyperactivity disorder
• the invention also provides a method for the treatment and/or prophylaxis of tetanus susceptibility, tuberous sclerosis (TS) or attention deficit hyperactivity disorder
• ADHD Alzheimer's disease
• the invention further provides a method for the treatment and/or prophylaxis of tetanus susceptibility, tuberous sclerosis (TS) or ADHD in patients having genetic or epigenetic markers associated with autism or related disorders comprising the step of silencing or reducing gene expression at the mRNA or protein level in the Xq/Yq pseudoautosomal gene region.
• TS tuberous sclerosis
• the invention also provides a method of screening for genetic or epigenetic markers associated with autism and related disorders comprising the steps of : isolating a biological sample from a mammal; isolating the Xq/Yq pseudoautosomal region (PAR) region in the sample; and comparing the isolated Xq/Yq pseudoautosomal region (PAR) region with a control sequence, wherein a deletion, addition or mutation indicates a susceptibility to autism or related disorders.
• the invention further provides a method for screening for genetic or epigenetic markers associated with autism and related disorders comprising the steps of : isolating a biological sample from a mammal; isolating the HSPRY3 gene promoter region in the sample; and comparing the isolated HSPRY3 region with a control sequence, wherein a deletion, addition or mutation indicates a susceptibility to autism or related disorders.
• deletion, addition or mutation is an alteration in any one or more of the alleles listed in Fig. 3
• Another aspect of the invention provides use of the Xq/Yq PAR and adjacent X- chromosome specific region comprising the entire DNA sequence listed in human chromosome X genomic contig NT_025307.15.
• Another aspect of the invention provides use of the Y chromosome region comprising the entire DNA sequence listed in human chromosome Y contig NT_079585.2.
• Another aspect of the invention provides use of the Y chromosome region comprising the entire DNA sequence listed in human chromosome Y WGS clone AADC01160617.1.
• One aspect of the invention provides use of the Xq/Yq PAR and adjacent X- chromosome specific region comprising the entire DNA sequence listed in human chromosome X genomic contig NT_025307.13 in the detection of autism or autism related disorders in patients.
• the invention further provides a DNA sequence comprising a nucleic acid sequence selected from any one or more of SEQ ID Nos. 1 to 13 or SEQ ID Nos. 35 to 41.
• the invention further provides a DNA sequence comprising a nucleic acid sequence selected from any one or more of Seq ID Nos. 14 to 18 or Seq ID Nos. 27 to 34.
• One aspect of the invention provides use of LH1 simple tandem repeat as a genetic marker associated with autism or autism related disorders.
• a further aspect of the invention provides use of Xhol, BsmAI, SYBLI-X ol, Rsal, Styl or Hinfl RFLPs as genetic markers associated with autism or related disorders.
• Another aspect of the invention provides use of polymo ⁇ hisms of the [ MER31I c' repeat in the promoter region of the HSPRY3 gene as genetic markers associated with autism or related disorders.
• Another aspect of the invention provides use of the polymo ⁇ hic A / G diallelic marker in the HSPRY3 gene coding region as a genetic marker associated with autism or related disorders.
• a further embodiment of the invention provides use of polymo ⁇ hisms of the DNA or RNA sequences or encoded protein sequences of transcription factors (transcriptional enhancers or repressors) or chromatin proteins that bind to regulatory regions of genes in the Xq Yq PAR and adjacent X-chromosome region.
• a further embodiment of the invention provides use of polymo ⁇ hisms of regulatory
• RNA sequences (including microRNAs) that bind to the regulatory regions of genes in the Xq/Yq PAR and adjacent X-chromosome region.
• a further embodiment of the invention provides use of polymo ⁇ hisms of DNA, RNA or protein sequences associated with factors that interact with the regulatory regions of the SYBL1 or HSPRY3 genes.
• a further embodiment of the invention provides use of polymo ⁇ hisms of DNA, RNA or protein sequences associated with factors that interact with the 'MER31I c' and ' GTTTT ' repeat regions of the HSPRY3 gene.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the MAZ / PUR1 gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the sex determining region Y (SRY) gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the progesterone receptor gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the vitamin D receptor gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the Retinoid X receptor gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the Fkh-domain factor FKHRL1 (FOXO) gene DNA or protein sequence.
• Fkh-domain factor FKHRL1 Fkh-domain factor FKHRL1
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the Nerve growth factor- induced protein C gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of GAGA-Box binding factor genes DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the v-MYB, variant of AMV v-myb gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the Multifunctional c-Abl src type tyrosine kinase gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the Glucocorticoid receptor C2C2 zinc finger protein gene DNA or protein sequence.
• Another aspect of the invention provides use as genetic markers associated with autism or related disorders of alterative polymo ⁇ hisms of the 'TCFll/MafG heterodimers, binding to subclass of API sites' gene DNA or protein sequence.
• Another aspect of the invention provides a method of assessing the personality of a patient or their susceptibility to autism or related disorders comprising the step of genotyping the ASD locus comprising genes in the Xq/Yq PAR region.
• the method of the invention may be used in early behaviour training, early dietary interventions or manipulations, pharmacological interventions, gene therapy, activation or reactivation of epigenetically silenced genes or silencing or reducing gene expression at the mRNA or protein level in children who have genetic or epigenetic markers associated with autism or related disorders.
• Samples may be isolated from children believed to have autism or related disorders or from clinically normal children.
• the biological sample may also be provided or isolated from parents or relatives of clinically normal children who have genetic markers associated with autism or related disorders.
• Fig. 1 is a schematic representation of the Xq/Yq pseudoautosomal region (PAR) which exhibits an unusual form of genetic/epigenetic regulation.
• the PAR consists of approximately 300 Kb and contains the HSPRY3, AMD2 (S-AdometDC-like), SYBL1, TRPC ⁇ -like, IL9R and CXYorfl genes (AMD2 may be a non-expressed pseudogene).
• X chromosome-specific genes adjacent to the PAR include the TMLHE, CLIC2, RAB39B and VBP1 genes.
• HSPRY3 and SYBL1 undergo random X-inactivation in females, but preferential Y-inactivation in males;
• NT_025307.13 X- chromosome
• the gene size scale is an approximation.
• Horizontal arrows indicate direction of transcription. CXYorfl function unknown but found near several telomeres. AMD2 is a pseudogene - not known if expressed.
• Vertical arrows indicate positions of polymo ⁇ hic small tandem repeats (STRs) and restriction fragment length polymo ⁇ hisms (RFLPs; restriction enzymes in italics).
• STRs polymo ⁇ hic small tandem repeats
• RFLPs restriction fragment length polymo ⁇ hisms
• 'MER31I c' is a DNA repeat upstream of the HSPRY3 transcriptional start site.
• HSPRY3-SNP is an A/G diallelic marker in the HSPRY3 coding region.
• NT_025307.15 is an updated version of NT_025307.13 (released August 2004).
• genes included in the X-specific region as follows: hepatitis C virus core-binding protein 6, mature T-cell proliferation 1, c6.1A, LOC401622, H2AFB. Also, the orientation of the TMLHE gene has been reversed.
• Fig. 2 is a table listing the Coriell autism family collection and the genotype of each individual at genetic loci in the Xq/Yq PAR and adjacent X chromosome-specific region;
• Fig. 3 is a table showing the PCR primer sequences spanning the polymo ⁇ hic sites of restriction enzyme fragment length polymo ⁇ hisms (RFLP) and simple tandem repeats (STRs) identified. All sequences are derived from genomic contig NT_025307.13, or from sources listed under the reference column including Matarazzo et al 2002 & Li and Hamer 1995;
• Fig. 4 is a table showing genotype frequencies at polymo ⁇ hic sites in the Xq/Yq PAR and adjacent X chromosome-specific region in subsets of autistic and control groups;
• Figs. 5, 6 and 7 are tables showing the results of statistical analysis of genotype frequencies for selected polymo ⁇ hic genetic loci in the Xq/Yq PAR. Specifically, the 'within group' distribution of homozygotes and heterozygotes is compared between various affected and control (unaffected) population groups. The analysis shows that there is a statistically significant difference in the distribution of homozygotes and heterozygotes in the affected, compared to the control (unaffected) groups for markers in the SYBL1 gene region. These results indicate a loss-of-heterozygosity (LOH) for the four SYBL1 associated markers: SYBL1 STR#1B, SYBL1 STR#2B, LH1, SYBLl-Rsal.
• LH loss-of-heterozygosity
• Fig. 8 is a multiple alignment of DNA sequences from the promoter region of the HSPRY3 gene spanning the 'MER31I c * and 'GTTTT' repeats.
• the sequences are derived from the public databases and from our single pass sequencing of cloned PCR products from genomic DNA of normal Irish women. The sequences establish that the major polymo ⁇ hisms in the region occur in the 'MER31I c' and 'GTTTT' repeats.
• Fig. 9 shows the evidence for a putative recombination hotspot at the 3' end of the HSPRY3 coding sequence region (CDS) and the 5' end of the
• HSPRY3 3' untranslated region UTR.
• the HSPRY3-SNP (P) and HSPRY3-HinfI (Q) SNPs are separated by 156b ⁇ within a PCR product (Fig. 3).
• PCR and single-pass sequencing was performed on both parents and an autistic individual from thirteen families from the Coriell Autism Resource.
• the linked alleles on each of the two sex chromosomes are displayed in the format P-Q / P-Q. The data suggest that there is a recombination hotspot between the two markers because all our recombination products (A-T, G-G, A-G, G-T) are observed.
• Fig. 10 shows the HSPRY3 promoter region genotypes of normal females of
• Fig. 11 shows PCR products obtained using primers listed in Fig. 3 under
• 'MER31I c' from Coriell Autism Resource family run on an agarose gel. PCR analysis using primers (Fig. 3) spanning 'MER31I c' and 'GTTTT' repeats of HSPRY3 promoter. Samples: genomic DNA from family comprising father (1), mother (2), male proband (3), affected male sib (4) from Coriell Autism Resource. Arrowheads indicate PCR products for clarity. Arrow indicates novel PCR product in affected males, which is not found in parents.
• a genetic alteration is taken to include polymo ⁇ hisms or mutations and/or epigenetic alterations.
• polymo ⁇ hism is intended to include all possible alterative variants of a DNA, RNA or protein sequence. It is analogous to the term 'mutation' and is often, but not exclusively, used to refer to a variant sequence that is present at a frequency of greater than 1% in the population.
• a mutation is taken to include deletions, additions or insertion or substitutions of one or more of the nucleotide or amino acid residues.
• a deletion refers to a change in either nucleotide o amino acid sequence and results in the absence of one or more nucleotides or amino acid residues.
• An insertion or addition refers to a change in a nucleotide or amino acid sequences that results in the addition of one or more nucleotide or amino acid residues as compared with the naturally occurring molecule.
• a substitution refers to the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids.
• Loss-of-imprinting or reactivation is taken to include the pathological, experimental or therapeutic induction of gene expression at a genetic locus that was previously silenced (transcriptionally inactive) due to epigenetic modifications of DNA or chromatin.
• a diallelic marker is taken to include a single nucleotide polymo ⁇ hism where there are two variants present. Transcription factors are taken to include transcriptional enhancers or repressors.
• allelic sequences is an alternative form of a nucleic acid sequence. Alleles may result from at least one mutation in the nucleic acid sequences and may yield altered mRNAs or polypeptides whose structure of function may or may not be altered. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of nucleotides.
• ASD locus comprising genes in the Xq/Yq pseudoautosomal region (PAR).
• PAR pseudoautosomal region
• the locus comprising a number of genes provides an answer to the diverse disturbances and reasons for the failure of standard genetic mapping studies to locate such a locus.
• Deregulation of genes located in the Xq/Yq PAR and adjacent X chromosome-specific (Xq28) region may provide an explanation for many of the features of ASD.
• the region can account for male-biased affected sex ratios due to its unusual genetic / epigenetic regulation.
• Deregulation of the genes in the region might be involved in, for example: structural brain abnormalities (HSPRY3, SYBL1); abnormal neuron function (CLIC2, SYBL1, TRPC6-like); metabolic/mitochondrial disturbances (TMLHE); immune dysfunction (IL9R); or other gene deregulation through effects on chromatin structure (TMLHE).
• the method of the invention provides for screening of subjects for genetic polymo ⁇ hisms and epigenetic markerss associated with autism and related disorders. The method involves isolating DNA from a mammal, specifically a human, and testing the sample for (i) deletions and other structural genomic rearrangements in the Xq / Yq pseudoautosomal gene region, and in the adjacent
• Xq28 gene region polymo ⁇ hic DNA markers in the Xq / Yq pseudoautosomal gene region, and in the adjacent Xq28 gene region, associated with autism and related disorders; (iii) alterations in the epigenetic regulation (including DNA methylation) of genes in the Xq / Yq pseudoautosomal gene region, and adjacent Xq28 gene region; and/or (iv) absence or downregulation, and over-expression (i.e. upregulation) of genes in the Xq / Yq pseudoautosomal gene region, and adjacent Xq28 gene region; altered temporal or spatial patterns of regulation or expression of genes in the Xq /Yq pseudoautosomal gene region, and adjacent Xq28 gene region.
• the presence of such alterations indicates that the subject is afflicted with autism or related disorders, is at greater risk of developing autism or related disorders or is at greater risk of transmitting autism or related disorders to progeny.
• DNA sequences (isolated DNA) have been characterised comprising restriction enzyme fragment length polymo ⁇ hisms (RFLPs), polymo ⁇ hic microsatellite
• RFLPs restriction enzyme fragment length polymo ⁇ hisms
• polymo ⁇ hic microsatellite RFLPs
• sequences are publicly available on the Human Genome Sequence database. Regions were selected that are known to be polymo ⁇ hic (known RFLPs, known simple tandem repeats (STRs)), or STRs were selected which were not known to be polymo ⁇ hic and primers were designed spanning them (Fig. 3).
• STRs 3, 5, 9 & 10; SF5 7-STR#1B, SYBL1-S ⁇ R#ZB; 'MER31I c' repeat; and HSPRY3-SNP was determined. They are polymo ⁇ hic and the alleles occurring are given in Fig. 3.
• LH1 STR marker as well as Xhol, BsmAl, SYBLl-Xhol, Rsal, Styl, and HSPRY3-Hinfl RFLPs may be used to study autism.
• the identification of the ASD locus provides valuable methods of developing therapeutic strategies for autism and related disorders.
• the finding that a specific genetic locus may be implicated in the majority of ASD causation has important application in a number of areas such as for example (i) diagnostic and prognostic tests; (ii) scope for further study in relation to pathogenesis and therapeutics; or (iii) a platform for providing reassurance in relation to public health issues such as vaccination.
• the Xq/Yq PAR as shown in Fig. 1 exhibits an unusual form of genetic/epigenetic regulation (Ciccodicola et al., 2000).
• the PAR consists of approximately 300 Kb and contains the HSPRY3, AMD2 (S-AdometDC-like) , SYBL1, TRPC ⁇ -like, IL9R and CXYorfl genes.
• X chromosome-specific genes adjacent to the PAR include the TMLHE, CLIC2, RAB39B, Histone H2 family, member B homologue (H2AFB),
• LOC401622 (LINE-1 reverse transcriptase homologue); LOC401623 (LINE-1 reverse transcriptase homologue), C6.1A, Mature T-cell proliferation 1 (MTCP1), Hepatitis C virus core-binding protein 6 (HCBP6) and VBP1 genes.
• HSPRY3 and SYBL1 undergo random X-inactivation in females, but preferential Y-inactivation in males.
• IL9R is expressed from both alleles in both males and females i.e. behaves in a standard pseudoautosomal manner.
• the expression patterns of the parental alleles of the AMD2 and TRPC ⁇ -like genes are unknown. AMD2 and TRPC ⁇ -like may be non-expressed pseudogenes.
• Recessive ASD susceptibility alleles, or deregulation of X-linked copies of Y- inactivated, or X-specific, genes would therefore be exposed in males, explaining the increased incidence of the condition in males. Also, for some conditions, loss of imprinting leading to over-expression of Y-inactivated genes may occur.
• ADHD attention deficit hyperactivity disorder
• TS tuberous sclerosis
• CCCK clear cell carcinoma of the kidney
• ADHD also has male biased sex ratios.
• TS is associated with a high rate of autism and increased rate of CCCK.
• the HSPRY3 gene (sequence accession: AJ271735) is a homologue of Drosophila Sprouty, which is involved in specifying forebrain/hindbrain developmental patterning. Chick Sprouty is expressed at the isthmus and rhombomere 1 (which gives rise to the entire cerebellum). Sprouty inhibits FGF8, which is also implicated in hindbrain patterning via inhibition of H x gene expression. Mouse Sprouty genes are also expressed at the isthmus. A key finding in brain scans in autistic subjects is increased cerebral volume coupled with cerebellar abnormalities.
• the SYBL1 gene (sequence accession: AJ271736) encodes a synaptobrevin-like protein (TI-VAMP / VAMP-7), a member of the SNARE protein family that includes synaptobrevin, syntaxin and SNAP-25, and has wide involvement in cellular secretion mechanisms.
• SNARE complexes are integral to synapse function and, in the gastrointestinal tract, in exocytosis and gastric parietal cell function.
• SNAP-25 protein has been associated with attention deficit hyperactivity disorder (ADHD) (Brophy et al., 2002) - a condition which also exhibits a male-biased sex ratio among affected individuals, and which may be classed as part of the wider autistic spectrum.
• ADHD attention deficit hyperactivity disorder
• SYBL1 and SNAP-25 encoded proteins may interact biochemically to influence neurite outgrowth (Martinez- Area et al., 2000). Suggestive similarities between aspects of some autism cases and tetanus, including 4:1 affected sex ratio have been observed (Bolte 1998).
• Bolte proposes that some autism cases are caused by gut infections with Clostridia spp.
• SYBL1 is a susceptibility locus for both tetanus and autistic spectrum disorders.
• Tetanus toxin cleaves the synaptobrevin protein, which is a homologue of the protein encoded by the SYBL1 gene.
• the IL-9R gene encodes the interleukin-9 receptor, which interacts with the gamma chain of the IL-2 receptor for signalling.
• Th2 cytokines There is considerable functional redundancy between various Th2 cytokines, therefore any hypothesis relating aberrant IL-9R regulation to immune abnormalities found in autism must be considered speculative.
• Autism is associated with increased serum IgE.
• IL-9 is strongly implicated in the pathophysiology of allergic diseases, with IgE ove ⁇ roduction (Levitt et al., 1999).
• the epsilon-N-trimethyllysine hydroxylase gene encodes the first enzyme (EC 1.14.11.8) in the camitine biosynthetic pathway. It converts epsilon-N- trimethyllysine to beta-hydroxy-N-epsilon-trimethyllysine.
• the other source of camitine is the diet.
• Camitine is critical for mitochondrial function. Many autistics have reduced camitine and increased lactic acid.
• trimethyllysine is a key modification of histone H3 and marks active genes in Drosophila.
• camitine suppresses position-effect variegation (PEV) in Drosophila
• acetyl-camitine inhibits the cytogenetic expression of the fragile X in vitro. Therefore, this pathway may have general effects on chromatin structure and gene expression/silencing.
• Mutations in t &MECP2 gene, (the product of which binds to methylated DNA), are implicated in Rett Syndrome — a severe neurodevelopmental disorder with autistic features.
• AMD2 S-AdoMetDC-like is related to S-adenosylmethionine decarboxylase proenzyme (AdoMetDC, SamDC).
• AdoMetDC is critical to polyamine biosynthesis and obtains AdoMet (i.e. S-adenosylmethionine) from the same pool as that which provides methyl donors for DNA methyltransferase enzymes.
• AdoMet levels There is abundant evidence that alterations in AdoMet levels affect Drosophila and mouse PEV and gene expression/silencing through effects on DNA methylation and chromatin structure.
• the AMD2 locus may be a non-expressed pseudogene, or might express a non-coding RNA that influences AdoMetDC mRNA processing or translation. Alternatively, this locus might acquire de novo expression patterns following mutations in the region.
• VBPl encodes a von Hipple-Lindau (VHL) binding protein.
• VHL is frequently mutated in clear cell carcinoma of the kidney.
• TS tuberous sclerosis
• CCCK clear cell carcinoma of the kidney
• RAB39B is a member of a large family of GTPases involved in vesicular trafficking.
• CLIC2 encodes a chloride intracellular channel of unknown function.
• IL9R gene has previously been implicated as a susceptibility factor in asthma.
• HSPRY3 is implicated in lung development and might alternatively explain increased susceptibility of some children to asthma and chest infections.
• SYBL1 may be involved in a variety of secretory processes in many cells or tissues and may be the basis for reports of increased susceptibility to gastrointestinal disorders and ear infections in autistic children.
• the SNARE secretory complex (including synaptobrevin) is also implicated in organ of corti function and deregulation of SYBL1 might contribute to poor balance and coordination of movements in autistic individuals.
• secretory processes in immune cells are mediated by SNARE complexes. Deregulation of SYBL1 might therefore explain altered immune responses and cytokine profiles in autism.
• TRPC ⁇ -like gene products may also function in immune cell physiology (Heiner etal., 2003).
• the adjacent cluster of genes on Xq28 may also be deregulated in a subset of autistics.
• TMLHE may have diverse indirect effects on gene regulation via chromatin structure, and also on mitochondrial function via regulation of camitine production. This may explain hypotonia observed in autistics and a variety of other metabolic disorders such as lactic acidosis.
• CLIC2 has an unknown function, but note that synaptic vesicle exostosis is associated with complex interactions between SNARE complexes, RAB proteins and calcium channels (Hibino et al., 2002).
• SYBL1 encodes a tetanus toxin insensitive paralog of synaptobrevin, the principle protein cleaved by tetanus toxin, and therefore SYBL1 may be a susceptibility or resistance (protective) locus for overt clinical tetanus. This suggests the possibility of identifying those genetically susceptible or resistant to tetanus, and may have implications for tetanus vaccination programs.
• the detection and identification of the ASD locus has many applications such as use in lifestyle and education intervention, drug development, gene and cell therapies, animal models and reactivation of epigenetically silenced genes.
• the detection and identification of the ASD locus has potential in the diagnosis, prognosis, prophylaxis, treatment and further research in the area of autism or related disorders.
• the methods described indicate strategies for the development of rational therapies for the clinical spectmm of autism. It will allow early diagnosis and intervention for a large proportion of autistic individuals. It will allow identification of the specific genes that are deregulated in individual patients resulting in more targeted therapeutics. It will indicate a rational basis for testing of other relevant biochemical, metabolic or physiological parameters as an aid to diagnostics, and to develop and monitor novel treatment strategies.
• the genetic material may consist of a gene promoter attached to a gene open reading frame encoding a protein that is missing or mutated in an autistic individual.
• the genetic material may also consist of a gene promoter attached to a DNA sequence that, once transcribed, produces a catalytic RNA molecule e.g. ribozyme, siRNA, microRNA that targets a gene product (mRNA) that is deregulated in an autistic individual.
• the method described herein specifies that the SYBL1 and HSPRY3 genes and their products are primary targets for such therapies.
• some or all of the other genes in the Xq/Yq PAR or adjacent X chromosome-specific region may, in some or all autistic individuals be suitable targets for such therapeutic methods.
• a further aspect to this is the removal of stem cells from autistic individuals, followed by genetic modification of these cells as described above, and their reintroduction into autistic individuals.
• a further aspect is the removal of stem cells from unaffected relatives, or unrelated, tissue-matched individuals, and the introduction of these cells into autistic individuals.
• a deduction from the method described herein is that there are genomically intact, but epigenetically silenced normal copies of some of the genes (SYBL1, HSPRY3, possibly TRPC ⁇ -like) in the region that may be reactivated by (for example) DNA demethylating agents such as 5-azacytidine or other chromatin modifying molecules. Therefore, targeted reactivation of epigenetically silenced genes would be an important application of the invention.
• DNA demethylating agents such as 5-azacytidine or other chromatin modifying molecules. Therefore, targeted reactivation of epigenetically silenced genes would be an important application of the invention.
• the key concept arising from the method described herein is that, for autistic individuals, such technologies should be targeted to genes in and adjacent to the Xq/Yq PAR, particularly SYBL1 and HSPRY3.
• DNA-binding proteins such as transcription factors and chromatin proteins that interact with the regulatory regions of genes in the Xq / Yq PAR and adjacent X-chromosome specific region and affect the expression of genes in the region such as SYBL1 and HSPRY3. These include the factors listed in Tables.
• CTCF sequence accession: AF145477, NM_006565
• BORIS sequence accession: AF336042, AL160176, NM_080618
• other DNA-binding or chromatin proteins that regulate gene expression or imprinting such as the HP1 family (sequence accessions: CBX3: NM_007276; CBX5: NM_012117; CBX1: NM_006807), DNA methyltransferases (sequence accessions: DNMT3A: NM_022552, AB076659,
• DNMT2 and splice variants NM_004412, AJ223333; DNMT3B and splice variants: NM_006892, AL035071; DNMT1: NM_001379, AC010077
• histone acetyltransf erases and deacetylases.
• Variants of the genes encoding these proteins may be considered candidate susceptibility genes for autistic spectmm disorders.
• a variety of methods of assaying the locus of the invention may be envisaged using current state-of-the-art technologies to detect abnormalities in the stmcture and expression of the locus.
• the types of techniques used are those that can distinguish alterations in gene copy number e.g. deletions, duplications, insertions; structural alterations of the locus not involving changes in gene copy number, but affecting gene expression e.g. translocations, inversions, conversions; minor stmctural changes (changes in DNA sequence) that affect gene expression e.g.
• DNA demethylation or hypermethylation DNA demethylation or hypermethylation, post-translational modifications of histones and non-histone proteins bound to DNA in the region, higher order packaging of DNA as euchromatin or heterochromatin, telomere stmcture influencing telomere stability, or spreading of telomeric heterochromatin to genes in the region (telomeric silencing); spreading of heterochromatin from the Y chromosome-specific region to the Y-linked PAR.
• genomic alterations described above may occur in, or influence the function of, any part of the genes in the region, including promoters, introns, exons, or any other regulatory motifs or regions that influence gene expression.
• the biological samples used will typically be a blood sample from a normal, or developmentally (behaviourally) retarded or afflicted, child.
• other samples may appropriately be obtained including saliva, hair, amniotic fluid, biopsy of placental cells or preimplantation embryos, semen (from adult males), or cells from the buccal mucosa (cheek scraping or swabbing), tissue.
• the primary aim is to obtain sufficient cells for the isolation of DNA, RNA, protein or chromatin for analysis.
• the mutational mechanisms may occur by a variety of different mechanisms including (but not exclusively) point mutations in gene regulatory motifs, gene conversions, gene deletions, other gene rearrangements, alterations in chromatin stmcture.
• point mutations in gene regulatory motifs including (but not exclusively) point mutations in gene regulatory motifs, gene conversions, gene deletions, other gene rearrangements, alterations in chromatin stmcture.
• the data showing loss-of-heterozygosity of markers in the SYBL1 gene region indicates that a major mechanism of causation of autistic spectrum disorders is likely to be either gene conversion or gene deletion spanning the SYBL1 locus.
• Preferred diagnostic methods used are those that detect gene conversion or gene deletion events. Such methods include those based on technologies such as cloning and sequencing of DNA from the region; quantitative (e.g. Taqman or real-time) poiymerase chain reaction (PCR); 'long' PCR across deletion boundaries; restriction enzyme cleavage, Southern blotting and hybridisation of DNA probes from the region; in situ hybridisation to DNA, chromosomes, or cells using DNA probes from the region; DNA 'array' or 'chip' technologies containing DNA from the region, and hybridised with sample DNA; DNA methylation analysis using methylation- sensitive restriction enzyme cleavage, Southern blotting and hybridisation of probes from the region; DNA methylation analysis by bisulphite treatment of sample DNA followed by cloning and sequencing of PCR products from the region, or variations of this technique using PCR primers capable of amplifying sequences derived from methylated or unmethylated DNA; analysis of chromatin stmcture using DNA nuclease digestion of
• abnormal regulation of genes in the region may be detected by gene expression studies on tissue samples. These methods require, as a starting point, the isolation of total RNA, mRNA, or protein from samples.
• a variety of standard techniques may be applied including: quantitative northern blotting of RNA followed by hydridisation with DNA or RNA probes from the expressed (exonic) sequences in the region; quantitative reverse transcription-polymerase chain reaction (RT-PCR) using Taqman or real-time platforms; DNA 'array' or 'chip' technologies containing expressed (exonic) DNA sequences from the region, and hybridised with sample RNA or cDNA (complementary DNA); in situ hybridisation to RNA in cells using exonic probes from the region; analysis of gene expression at the protein level: western blotting of homogenised tissue, and quantification of protein using specific antibodies to proteins encoded by genes in the region; use of specific antibodies to quantify proteins encoded by genes in the region in an ELISA or related format; 'array' or 'chip' technologies using specific antibodies to quantify proteins
• the genes in this region are highly conserved amongst mammals.
• the techniques I outlined herein may be relevant to the identification or production of mutants (e.g. mouse mutants) with autism, for further research into mechanisms of pathology, and therapeutics.
• mice models of autistic spectmm disorders may be produced by gene targeting of the genomically dispersed orthologs in the mouse, followed by mouse breeding programs to produce mice with deregulated expression of the relevant genes, or their paralogues.
• Human artificial chromosomes or bacterial artificial chromosomes containing part or all of the human Xq/Yq PAR may also be used to study mutational mechanisms and to produce cellular (cells cultured in vitro) or mouse models of aspects of the disorder.
• Antibodies may be prepared by methods commonly known in the art which specifically bind to an epitope of an altered marker encoded by genes in the Xq/Yq pseudoautosomal (PAR) region and adjacent chromosome-specific (Xq28) region. Antibodies may also be prepared which specifically bind to an epitope of an altered marker encoded by genes (listed in tables 1 and 2) that regulate genes in the Xq/Yq pseudoautosomal (PAR) region and adjacent chromosome-specific (Xq28) region.
• kits based on the identification of the ASD locus.
• the kits may be used for screening for an alteration in the genetic or epigenetic markers associated with autism or related disorders comprising an antibody as described above or a probe or primer selected from any one or more of SEQ ID Nos 1 to 13 and 35 to 41.
• Reagents suitable for western blot, immunohistochemcial assays and ELISA assays are those which are commonly known in the art.
• DNA samples were obtained from the United States Coriell Cell Repository (CCR) Autism Resource comprising a collection of nineteen families, which in addition to probands, includes some or all of the following: affected and non-affected siblings, parents and grandparents. Unrelated controls were obtained from the CCR / National Institute of Aging Longevity Collection, and consisted of healthy young adults, and from the CCR / National Institute of General Medical Sciences Caucasian Panel (HD200CAU).
• Fig. 2 lists the different families examined from the Autism Collection;
• Fig. 4 lists Control samples.
• HSPRY3 promoter region an additional set of thirty two samples from normal young females of Irish origin, collected under the auspices of a Reproductive Tissue Bank, were analysed.
• PCR primers spanning the polymo ⁇ hic site were developed for amplification of short PCR products from genomic DNA, as shown in Fig. 3.
• PCR products were digested with the appropriate restriction enzyme and the resultant digestion products were analysed by agarose gel electrophoresis. Each sample genotype was scored as +/+, +/-, or -/- , depending on whether a digestion product was present (+) or absent (-) (Figs. 2, 3, 4).
• STRs short tandem repeats
• Identified repeats were spanned with PCR primers as described for RFLPs above, and products were analysed using an ABI-310 instmment (Fig. 3).
• the allelic sequence stmcture of STRs were determined in control and affected populations for STR#3, STR#5, STR#9, STR#10, SYBL1-ST ⁇ B and Sr ⁇ ll-STR#2B.
• PCR primers spanning the repeats were designed (Fig. 3) and PCR products were analysed by agarose gel electrophoresis (Fig. 11), ABI-310 capillary electrophoresis (Fig. 10), and cloning and sequencing (Fig. 8).
• the entire CCR Autism Collection was genotyped for the following markers in the Xq/Yq PAR: SYBLl-Xhol, SYBL1 -STR#1B, SYBL1 -STR#2B, LH1, Rsal Styl (Figs. 1, 2, 4).
• the Rsal marker was applied to the Control samples listed in Example 1 (Fig. 4).
• the Rsal marker was applied to the entire HDIOOCAU Caucasian panel comprising two hundred individuals, of which one hundred and ninety nine were successfully genotyped (Fig. 4).
• the SYBLl-Xhol, SYBL1 -STR#1 , SYBL1 -STR#2B, LH1, Rsal, and Styl markers were applied to twenty males and twenty females (forty individuals in total) under forty years of age from the CCR Longevity Panel (see Example 1), of which between nineteen and twenty individuals of each sex were successfully genotyped for each marker.
• CCR Longevity Panel see Example 1
• statistically significant differences between the distribution of homozygotes and heterozygotes were detected between the affected groups and the unaffected groups for markers in the SYBL1 region, suggesting the occurrence either of i. a susceptibility allele; ii. a gene conversion event; or, iii.
• a further control is derived from a comparison of the genotype frequencies of polymo ⁇ hic markers in the various control groups (Longevity and Caucasian panels) with the affected and unaffected groups from the nineteen families from the Autism Resource. In all available comparisons, the distributions of homozygotes and heterozygotes in the two unaffected groups (derived from the Longevity and
• Fig. 8 shows the multialignment (Co ⁇ et, 1988) of genomic DNA sequences encompassing two major repeats within the human (hum) and chimpanzee (chimp) SPRY3 promoter regions (equivalent to nucleotides 510246-510738 in RefSeq chromosome X contig NT_025307.13 and nucleotides 66107-66599 in RefSeq chromosome Y contig NT_079585.2).
• sequences are derived from a combination of sources: National Center for Biotechnology information (NCBI: www.ncbi.nlm.nih.gov) databases including the whole genome shotgun (WGS) database, the high-throughput genomic sequencing (HTGS) database and the reference sequence project (RefSeq) database, plus cloned and sequenced PCR products from genomic DNA derived from five female (Fern) subjects (where two alleles were observed they are represented as Al and A2).
• NCBI National Center for Biotechnology information
• GGS whole genome shotgun
• HGS high-throughput genomic sequencing
• RefSeq reference sequence project
• Source database sequence identifiers are as follows (the suffixes 'X', 'Y' and '4' represent chromosome number, whereas 'U' represents unmapped sequence): AADC01149041.1 (WGS : hum X), AADBOl 164924.1 (WGS : hum U), AADC01160617.1 (WGS : hum Y),
• the 'GTTTT' DNA repeat appears to be not as variable as the 'MER31I c' repeat.
• sample Fem #3 Al has one variant sequence 'GTTT'.
• Sample WGS: hum X has one variant sequence 'GTTCT'.
• Sample WGS: hum Y has the variant sequence 'GTTCT/GTCAT/GCTCT/GTTCT/GTTGT/GTCTT'.
• Sequences from Fem #1, 2, 3, 4, 5 are single pass sequences which may contain minor uncorrected errors. However, these sequences establish the variability of the
• PCR primers were designed to flank the 'MER31I c' and 'GTTTT' repeats of the HSPRY3 gene promoter region (Fig. 3). PCR products were analysed by agarose gel (Fig. 11) and capillary (ABI-310) electrophoresis (Fig. 10), and by cloning and sequencing PCR products (Fig. 8). DNA sequences were obtained from five unaffected young women of Irish origin and single pass sequences are listed in Fig. 8. These sequences indicate that the majority of alleles (PCR product length polymo ⁇ hisms) at this locus are likely to be due to variants of the 'MER31I c' repeat region.
• PCR primers used in this analysis This is because the large number of alleles in the population predicts that homozygotes should be relatively rare. However, fourteen of thirty one females were homozygous, and seventeen of twenty five males were homozygous.
• allele pairs 510 and 511, 550 and 551, 553 and 554 may represent three, not six, different alleles. Full description, validation and discrimination of all alleles will require extensive DNA sequencing. In the normal female population of Irish origin there are therefore potentially between ten and thirteen different alleles: 467, 496,
• proteins or regulatory RNA molecules that regulate chromatin structure, dosage compensation or genomic imprinting e.g. heterochromatin proteins such as HP1 and homologues, and the zinc finger proteins
• CTCF and BORIS may be implicated in regulating different allelic variants such that expression levels, or temporal or spatial patterns of gene expression are altered.
• interactions between DNA repeats on grandmaternally and grandpaternally derived homologues in the germline, or maternally and paternally derived homologues in the embryo may epigenetically modulate HSPRY3 gene silencing or expression.
• An X-linked deleted variant of the HSPRY3 gene promoter may lead to a null phenotype in males (in which the Y-linked homologue is thought to be silenced), or may lead to reactivation of the Y-linked homologue, analogous to the reactivation of the paternally derived X chromosome in the extra-embryonic tissues of XpO (monosomic) mice.
• Table 1 shows the analysis of potential transcription factor binding sites in the promoter region of the HSPRY3 gene spanning the 'Mer31I c' repeat.
• the major factors likely to bind to the repeat are MAZ (Myc-associated zinc finger protein) / Purl / GAGA factor, Vitamin D receptor, RXR (Retinoid X receptor), Forkhead (FOXO).
• MAZ Myc-associated zinc finger protein
• Purl / GAGA factor Vitamin D receptor
• RXR Retinoid X receptor
• Forkhead Forkhead
• Table 2 shows the analysis of potential transcription factor binding sites in the promoter region of the HSPRY3 gene spanning the 'GTTTT' repeat.
• the major factor likely to bind to the repeat is SRY (Sex determining region Y gene product).
• SRY Sex determining region Y gene product
• Table 2 The number of binding sites for the various factors in a particular allele are predicted to vary depending on the number of repeat units and other variations of the HSPRY3 promoter DNA sequence.
• mutations in the WGS: Hum Y sequence (Fig. 8) abolishes the SRY binding sites and adds a Progesterone receptor binding site.
• Different sequences affect the identity, number and location of transcriptional enhancer and suppressor proteins.
• Allelic variants of factors that regulate the HSPRY3 promoter or dosage compensation may be implicated in the causation of autistic spectrum disorders.
• MAZ / GAGA factor homologues regulate gene dosage and X chromosome dosage compensation in Drosophila.
• SRY variants may explain the postulated link between testosterone levels, altered digit lengths and masculinization of the brain as postulated by Baron-Cohen.
• the Progesterone receptor and Vitamin D receptors are expressed in the male brain and variants may influence HSPRY3 gene expression.
• the FOXO gene product predicted to bind to the HSPRY3 gene promoter region is homologous to the FOXP2 gene implicated in autism and language disorders on chromosome 7q31.
• a recombination hotspot is defined as a region of the genome that experiences a relatively high rate of genetic recombination relative to other regions of the genome.
• the HSPRY3-SNP and HSPRY3-BMI markers are separated by 156 bp at the distal end of the HSPRY3 coding sequence / 3' UTR region (Fig. 3). Markers that are physically contiguous are usually found to be in linkage disequilibrium. However, PCR and single pass sequencing of both parents and one affected individual from thirteen families from the Coriell Autism Resource found that all four recombination products are observed (Fig. 9), suggesting the presence of a recombination hotspot in this region.
• This region is also close to the site of origin of a transcript cloned from a human amygdala cDNA library (cDNA FLJ37291, ACCESSION: AK094610).
• This transcript may have a regulatory function in HSPRY3 expression in the brain, or more specifically the amygdala - a brain region strongly implicated in the aetiology of autism - and may, for example, represent the site of a tissue-specific enhancer, silencer or boundary, which may be mutated or deregulated in autistic individuals.
• Fig. 11 shows the PCR analysis using primers (Fig. 3) spanning the 'MER31I c' and 'GTTTT' repeats of the HSPRY3 gene promoter.
• Samples genomic DNA from Coriell Autism Resource Family 104 (samples AU10033, AU10023, AU10021, AU10022) comprising father (1), mother (2), male proband (3), affected male sib (4).
• the extra bands observed in agarose gel electrophoresis may therefore be due to conformational variants of the PCR products possibly generated by the 'MER31I c' or 'GTTTT' repeats. These putative conformational variants were reproduced robustly in different experiments and using different PCR primer sets spanning the region (data not shown). These variants may be analogous to the variant bands detected by single-stranded conformational polymo ⁇ hism (SSCP) gels, which are routinely used to detect novel mutations of unknown sequence.
• SSCP single-stranded conformational polymo ⁇ hism
• the presence of the G-rich 'MER31I c' repeat may be important for generating such stable conformational variants because three bands were never observed using other PCR primers that excluded the 'MER31I c' repeat region.
• CORPET F Multiple sequence alignment with hierarchical clustering, 1988, Nucl. Acids Res., 16 (22), 10881-10890 Croonenberghs J, Bosmans E, Deboutte D, Kenis G, Maes M., Activation of the inflammatory response system in autism. Neuropsychobiology. 2002;45(l):l-6.
• RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage- gated Ca(2+) channels. Neuron. 2002 Apr 25;34(3):411-23.
• NLGN3 and NLGN4 are associated with autism. Nat Genet. 2003 May;34(l):27-9.
• Newbury DF Monaco AP. Molecular genetics of speech and language disorders.
• SEQ ID No. 2 >STR#2 GATAAAACATAAGTAAAGACGAAGGAATTCGAATAAAACATAATAAATA
• SEQ ID No. 12 >SYBL-STR#lb CGAAATGCTTCCCTTTTATCCATGAAACTATTTGAGTATAAAGACGTAAG
• the numbers refer to the position on human chromosome X genomic contig NT 025307.13

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