WO2014012144A1 - Procédé de diagnostic ou de pronostic d'un trouble neurologique - Google Patents

Procédé de diagnostic ou de pronostic d'un trouble neurologique Download PDF

Info

Publication number
WO2014012144A1
WO2014012144A1 PCT/AU2013/000798 AU2013000798W WO2014012144A1 WO 2014012144 A1 WO2014012144 A1 WO 2014012144A1 AU 2013000798 W AU2013000798 W AU 2013000798W WO 2014012144 A1 WO2014012144 A1 WO 2014012144A1
Authority
WO
WIPO (PCT)
Prior art keywords
asd
snp
subject
snps
gene encoding
Prior art date
Application number
PCT/AU2013/000798
Other languages
English (en)
Inventor
Christos PANTELIS
Efstratios Skafidas
Gursharan Chana
Ian EVERALL
Renee TESTA
Daniela ZANTOMIO
Original Assignee
The University Of Melbourne
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2012903105A external-priority patent/AU2012903105A0/en
Application filed by The University Of Melbourne filed Critical The University Of Melbourne
Publication of WO2014012144A1 publication Critical patent/WO2014012144A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This disclosure relates generally to methods of diagnosing and prognosing a neurological disorder.
  • methods are taught herein for the diagnosis and prognosis of Autism Spectrum Disorder using genetic markers, including single nucleotide polymorphisms.
  • ASD Autism spectrum disorder
  • MZ monozygotic twins
  • the genetic markers are single nucleotide polymorphisms (SNPs) populating molecular pathways that are associated directly or indirectly with the development of or protection from ASD.
  • SNPs single nucleotide polymorphisms
  • the SNPs have been applied to generate a predictive classifier for phenotypes of affected individuals and their parents.
  • ASD Autism Spectrum Disorder
  • a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder comprising collecting a genetic sample from the subject comprising a gene encoding calcium-activated potassium channel subunit ⁇ 4 and screening for a SNP in the gene that is statistically associated with ASD or the absence of ASD, wherein the presence of the SNP is indicative that the subject has ASD, a predisposition to developing ASD, the absence of ASD or is protected from developing ASD.
  • ASSD Autism Spectrum Disorder
  • the genetic marker e.g., SNP
  • the genetic marker is indicative of the presence of ASD or a predisposition to developing ASD in the subject.
  • the SNP is rs968122.
  • Methods taught herein also encompass screening for additional genetic markers, such as SNPs, which are statistically associated with ASD.
  • the genetic marker is a SNP in a gene encoding a protein selected from the list consisting of:
  • the SNP in the gene encoding guanine nucleotide-binding protein G(o) subunit alpha is rs876619; the SNP in the gene encoding metabotropic glutamate receptor 5 is rs 11020772; the SNP in the gene encoding platelet- derived growth factor D is rsl818106; the gene encoding phosphatidylinositol-3,4,5- trisphosphate 5-phosphatase 1 is INPP5D and the SNP is selected from the list consisting of rs9288685 and rs 10193128; the gene encoding adenylate cyclase type 8 is ADCY8 and the SNP is rs7842798; the gene encoding voltage dependent calcium channel ⁇ 2/ ⁇ subunit 3 is CACNA2D3 and the SNP is rs3773540; the gene encoding adenylate cyclase type 3 is ADCY3 and the
  • the method further comprises identifying a profile of two or more SNPs as listed in Table 1.
  • the genetic marker ⁇ e.g., SNP is indicative of the absence of ASD or protection from developing ASD in a subject.
  • the SNP is rs 12317962.
  • Methods taught herein also encompass screening for additional genetic markers, such as SNPs, which are statistically associated with the absence of ASD or protection from developing ASD.
  • the genetic marker is a SNP in a gene encoding a protein selected from the list consisting of:
  • the gene encoding cGMP-dependent protein kinase 1 alpha isozyme is PRKGl and the SNP is rs 17629494; the gene encoding nuclear factor NF-kappa-B pl05 subunit is NFKBl and the SNP is rs4648135; the gene encoding C-terminal binding protein 2 is CTBP2 and the SNP is rs 17643974; the gene encoding olfactory receptor 6S 1 is OR6S1 and the SNP is rs 1243679; the gene encoding olfactory receptor 10H3 is OR10H3 and the SNP is rs2240228; the gene encoding platelet-derived growth factor D is rs260808; the gene encoding Axin-2 is AXIN2 and the SNP is rs4128941; the gene encoding ubiquitin-conjugated enzyme E2D2 is UBE2D2 and the
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD, the method comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD, the method comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD, the method comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD, the method comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD, the method comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD, the method comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD, the method comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD, the method comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder (ASD), the method comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 1, and wherein the suitable number of SNPs is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining the absence of ASD or protection from developing ASD in a subject, the method comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 1, and wherein the suitable number of SNPs is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • the instant disclosure is also instructional for a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder (ASD), the method comprising collecting a genetic sample from the subject and screening for an SNP in the genetic sample that is statistically associated with the presence of ASD or a predisposition to developing ASD, wherein the SNP is selected from the list in Table 5 that is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining the absence of ASD or protection from developing ASD in a subject, the method comprising collecting a genetic sample from the subject and screening for an SNP in the genetic sample that is statistically associated with the absence of ASD or protection from developing ASD, wherein the SNP is selected from the list in Table 5 that is statistically associated with the absence of ASD or protection from developing ASD.
  • the instant disclosure is also instructional for a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder (ASD), the method comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 5, wherein the suitable number of SNPs is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • ASSD Autism Spectrum Disorder
  • the instant disclosure is also instructional for a method for determining the absence of ASD or protection from developing ASD in a subject, the method comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 5 that are statistically associated with the absence of ASD or protection from developing ASD.
  • methods further comprising, where the subject is determined as having or having a predisposition to developing ASD, exposing the subject to a treatment for inhibiting the progression of ASD or for inhibiting the onset of ASD or for ameliorating the symptoms of ASD.
  • the instant disclosure is also instructional for a kit for determining whether a subject has or has a predisposition to develop ASD, the kit comprising a set of primers and/or probes for identifying the genetic marker, such as a SNP, in accordance with the methods disclosed herein.
  • the genetic marker in a SNP.
  • the method disclosed herein may further comprise transferring the data through a firewall.
  • the method further comprises causing the base station to:
  • the instant disclosure is also instructional for a base station for stratifying a subject with respect to ASD in accordance with the methods disclosed herein, the base station comprising:
  • the processing system is adapted to receive data from a remote end station adapted to determine the data.
  • the processing system may also comprise:
  • Figures la and lb show flow charts on the subjects used in the analyses described herein.
  • AGRE Autism Genetic Research Exchange
  • SFARI Simons Foundation Autism Research Initiative
  • WTBC Wellcome Trust 1958 normal birth cohort
  • CEU Central (Western & Northern) European origin
  • TSI - of Tuscan Italian origin
  • Fig la & lb 'red boxes' - samples used in the developing the predictive algorithm; 'blue boxes' - samples used to investigate different ethnic groups; 'green boxes' - validation sets; 'light green boxes' - relatives assessed, including parents and unaffected siblings.
  • Figure 3a shows the Genetic based classification of CEU population (AGRE and Controls) for ASD and Non-ASD individuals, showing Gaussian approximation of distribution of individuals.
  • PPV Test Positive Predictive Value
  • NPV Negative Predictive Value
  • Figure 3b shows Genetic based classification of CEU population, including 1st degree relatives (parents and siblings of ASD children). Note that the distribution of relatives of ASD children maps between the ASD and the control groups, with no difference found between mothers and fathers (see Supplementary material S5). Key: ASD - Autism Spectrum Disorder; Relatives - 1st degree relatives (parents and siblings); Siblings - siblings of ASD cases not meeting criteria for ASD; Autism Classifier Score - scores for each individual derived from the predictive algorithm, with greater values representing greater risk for autism.
  • Figure 4 shows classifier performance. Labels were randomly permuted on the training sample and the resulting classifier was used to determine clinical status in the independent validation samples.
  • the graph indicates the percentage of classifiers versus misclassification rate.
  • the trained classifier on the correctly labelled data had the best performance of all other classifiers.
  • the probability that the other classifier trained on the permuted labeled data has better performance is less than 1x10 " .
  • Figure 5 shows ASD prediction based on the area under ROC.
  • the area under the curve is 0.749.
  • Figure 6 shows the distribution of Relatives and Parents in AGRE.
  • Figure 7 shows a principal components analysis demonstrating separation of HapMap populations on 3 principal components (PC).
  • PC Principal Component
  • ASW African Ancestry South Western USA
  • CEU Central European
  • CHB Han Chinese Beijing
  • CHD Han Chinese Denver
  • GIH Gujurati indian in Houston
  • JPT Japanese Tokyo
  • LWK Luhyan in Webuye
  • MEX Mexican Los Angeles
  • MKK Maasai in Kinawa, Kenya TSI - Sicil Italians
  • Figure 8 shows a principal components analysis demonstrating separation of HapMap populations on 3 principal components (PC). Principal component analysis was performed on the 237 classifier SNPs. Within the assessed populations (White Non- Hispanic AGRE and SFARI) and the 58 WTBC, the two principal components account for 1.86 and 1.69% of the variance Furthermore, a two sample Kolmogorov Smirnov test comparing whether AGRE or SFARI differed from 58-WTBC on either of the two major principal components found that the null hypothesis (i.e., that the data was from the same distribution) could not be rejected (p ⁇ 0.4).
  • Figures 9-11 show training and performance on expanded dataset to include all White Non- Hispanics from the AGRE, SFARI and WTBCC using bootstrapping, where 80% of the sample was used for training and the remaining 20% used as a validations set, 10,000 times in order to estimate the performance of the classifier based on identified SNPs. Note that over all random samples, the trained classifier exceeded 70% performance on both cases and controls (Figure 9). Validation performance on both cases and controls determined for all 10,000 trained classifiers illustrating good performance on both cases and controls for all of the trained samples. Average classifier performance based on 10,000 classifiers on random validation subsamples yielded performance of 70.88% with a standard deviation of 1.67%.
  • Figure 12 shows the distribution of marked language delay (A) and classification performance on minimal and marked language delay within WNH ASD individuals within the AGRE and SFARI cohorts. Marked language delay is defined as individuals with an ADOS score of two or greater. Minimal language delay is defined as individuals with a score of 1 or less. The 90% confidence interval of classifier percentage correct performance had a median of 59.67% [CI: 51.42-67.92] (B).
  • Figure 13 shows a principal component (PC) analysis on SNPs in the classifier mapped to data from the Autism Genome Project (AGP) European cohort, as reported by AGP. Related controls refer to parents.
  • PC principal component
  • Figure 14 shows the distribution of related controls and cases in the AGP cohort demonstrating significant overlap/non overlap between parents and affected children.
  • Figure 15 shows the distribution of training classification performance within the AGP European Cohort (A) and validation of classification performance (B).
  • Training Performance in the AGP cohort demonstrated that over all random samples, the trained classifier showed performance exceeding 61% on both cases and controls (C).
  • Validation Classification Performance on AGP cohorts distinguished pseudo controls from probands. The 90% confidence interval of classifier performance had a median percentage correct performance of 58.27% [CI: 55.20-60.98] (D). WNH - White Non-Hispanic.
  • a SNP may include a single SNP, as well as two or more SNPs; reference to “an agent” includes a agent, as well as two or more agents; reference to “the invention” includes single and multiple aspects of the invention; and so forth. Aspects taught herein are encompassed by the term “invention”. All aspects of the invention are enabled within the width of the claims.
  • the genetic markers are SNPs.
  • Reference to "SNP” includes a single SNP or a panel of SNPs.
  • Machine learning has been applied to the identified SNPs to generate a predictive classifier for ASD diagnosis.
  • an SNP located within the gene encoding calcium-activated potassium channel subunit ⁇ 4 is a significant genetic diagnostic classifier for ASD (i.e., of the presence or a predisposition to developing ASD or of the absence or protection against developing ASD).
  • a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder comprising collecting a genetic sample from the subject comprising a gene encoding calcium-activated potassium channel subunit ⁇ 4 and screening for a genetic marker in the gene that is statistically associated with ASD or the absence of ASD, wherein the presence of the genetic marker is indicative that the subject has ASD, a predisposition to developing ASD, the absence of ASD or is protected from developing ASD.
  • the genetic marker is a SNP.
  • ASD Autism Spectrum Disorder
  • ASD Autism, Asperger's or Pervasive Developmental Disorder- Not Otherwise Specified (PDD-NOS).
  • PPD-NOS Pervasive Developmental Disorder- Not Otherwise Specified
  • ASD does not include RETT syndrome and/or Fragile X.
  • the term "indicative”, as used herein, denotes an association or affiliation of a subject closely to a group or population of subjects who present, or likely to present, with the same or a similar clinical manifestations of ASD or a response to the treatment of ASD.
  • the clinical manifestations of ASD are encompassed by symptoms of ASD.
  • SNPs designate variations of individual base pairs in a DNA strand compared to corresponding wild-type sequence within a population.
  • wild-type refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype and typically refers to an allele in which an SNP of interest is absent. Studies suggest that SNPs represent about 90% of all genetic variants in the human genome, although they may a disproportionate frequency of occurrence in certain regions of the genome.
  • SNPs can occur as a conservative nucleic acid substitution; that is, in which a base (e.g., cytosine) is replaced by another base (e.g., thymine), or as nucleic acid deletions or insertions.
  • a base e.g., cytosine
  • another base e.g., thymine
  • SNPs that are found within a coding region can be silent.
  • a nucleic acid substitution may not alter the translation of the corresponding triplet code into the analogous amino acid and, hence, will have no influence on the translated peptide sequence.
  • differences in the efficiency of translation can arise and, as a consequence, the expression of certain genes can be influenced post-transcriptionally by silent SNPs.
  • biallelic SNPs can occur in three possible genotypes; namely, in one of two homozygotic forms (allele 1/allele 1 or allele 2/allele 2) or in one heterozygotic form (allele 1/allele 2). Since genomic DNA is double-stranded, each SNP can be identified with reference to each of the two strands. Thus, the SNPs may contain one substitution of one nucleotide by another at the polymorphic site of an SNP, or they may have a deletion of a nucleotide from one, or an insertion of a nucleotide into, one of two corresponding sequences.
  • the SNP may be present in a "silent" or non-coding region of the gene, such as in the promoter region or in the 3' untranslated region.
  • the SNP may also be present in the coding region of a particular gene and it may therefore be detectable at the mRNA level.
  • SNPs can be classified by a unique reference SNP ID number ("rs#"), allocated by the SNP database (dbSNP), which is an archive for genetic variations within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI).
  • rs# unique reference SNP ID number
  • NCBI National Center for Biotechnology Information
  • NHGRI National Human Genome Research Institute
  • genetic markers in the Wnt signaling pathway contribute to the diagnosis of ASD in a CEU cohort, but not in a Han Chinese population.
  • the genetic marker is a SNP.
  • the ethnic group is not Han Chinese and method disclosed herein comprises identifying an SNP in a gene encoding a peptide involved in Wnt signalling.
  • the SNP is indicative of the presence of ASD or a predisposition to developing ASD in the subject.
  • the SNP is rs968122.
  • the method further comprises identifying an SNP statistically associated with ASD, or a predisposition to developing ASD, in a gene encoding a protein selected from the list consisting of:
  • the SNP in the gene encoding guanine nucleotide-binding protein G(o) subunit alpha is rs 876619.
  • the SNP in the gene encoding metabotropic glutamate receptor 5 is rsl 1020772.
  • the SNP in the gene encoding platelet-derived growth factor D is rs 1818106.
  • the gene encoding phosphatidylinositol-3,4,5- trisphosphate 5-phosphatase 1 is INPP5D and the SNP is selected from the list consisting of rs9288685 and rsl0193128.
  • the gene encoding adenylate cyclase type 8 is ADCY8 and the SNP is rs7842798.
  • the gene encoding voltage dependent calcium channel ⁇ 2/ ⁇ subunit 3 is CACNA2D3 and the SNP is rs3773540.
  • the gene encoding adenylate cyclase type 3 is ADCY3 and the SNP is rs2384061.
  • the gene encoding phosphatidylinositol-4- phosphate 3-kinase C2 domain-containing ⁇ polypeptide is PIK3C2G and the SNP is rs 12582971.
  • the gene encoding voltage-dependent calcium channel ⁇ subunit is CACNA1A and the SNP is rs 10409541.
  • the gene encoding Calmodulin 1 is CALM1 and the SNP is rs2300497.
  • the gene encoding receptor tyrosine protein kinase erb B4 is ERBB4 and the SNP is rs7562445.
  • the gene encoding tyrosine phosphatase receptor-type R is PTPRR and the SNP is rs7313997.
  • the gene encoding L-type voltage-dependent calcium channel ai C subunit is CACNA1C and the SNP is rs2239118.
  • the methods disclosed herein further comprising identifying a profile of two or more SNPs as listed in Table 1.
  • ASD or a predisposition to developing ASD denotes an SNP that is indicative of the absence of ASD or protection from developing ASD.
  • the SNP in a gene encoding calcium-activated potassium channel subunit ⁇ 4 is indicative of the absence of ASD or protection from developing ASD in the subject.
  • the SNP is rsl2317962.
  • the method further comprising identifying an SNP statistically associated with the absence of ASD, or protection against developing ASD, in a gene encoding a protein selected from the list consisting of:
  • the gene encoding cGMP-dependent protein kinase 1 alpha isozyme is PRKG1 and the SNP is rsl7629494.
  • the gene encoding nuclear factor NF-kappa-B pl05 subunit is NFKB 1 and the SNP is rs4648135.
  • the gene encoding C-terminal binding protein 2 is CTBP2 and the SNP is rs 17643974.
  • the gene encoding olfactory receptor 6S 1 is OR6S 1 and the SNP is rs 1243679.
  • the gene encoding olfactory receptor 10H3 is OR10H3 and the SNP is rs2240228.
  • the SNP is a gene encoding platelet-derived growth factor D is rs260808.
  • the gene encoding Axin-2 is AXIN2 and the SNP is rs4128941.
  • the gene encoding ubiquitin-conjugated enzyme E2D2 is UBE2D2 and the SNP is rs769052.
  • the gene encoding olfactory receptor 5L1 is OR5L1 and the SNP is rs984371.
  • the gene encoding deoxycytidine kinase is DCK and the SNP is rs4308342.
  • the gene encoding guanine nucleotide binding protein, al4 subunit is GNA14 and the SNP is rsl 1145506.
  • the SNP in a gene encoding metabotropic glutamate receptor 5 is selected from the list consisting of rs905646 and rs6483362.
  • the SNP in a gene encoding guanine nucleotide-binding protein G(o) subunit alpha is rs8053370.
  • a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD comprising collecting a genetic sample from the subject and screening the genetic sample for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 1 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with Autism Spectrum Disorder (ASD) or with a risk of developing ASD comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and ASD, or a risk of developing ASD.
  • ASSD Autism Spectrum Disorder
  • a method for determining whether a subject has a genetic profile associated with an absence of Autism Spectrum Disorder (ASD) or with protection from developing ASD comprising collecting a genetic sample from the subject and amplifying genomic DNA or corresponding RNA using primers which are selective for the presence or absence of a selected number of SNPs, wherein the SNPs are selected from the list in Table 5 and wherein the selected number of SNPs provides 50% or greater correlation between the genetic profile and the absence of ASD, or protection from developing ASD.
  • ASD Autism Spectrum Disorder
  • a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 1, and wherein the suitable number of SNPs is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • the method comprises screening for all SNPs listed in Table 1 that are statistically associated with the presence of ASD or a predisposition to developing ASD.
  • a method for determining the absence of ASD or protection from developing ASD in a subject comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 1, and wherein the suitable number of SNPs is statistically associated with the absence of ASD or protection from developing ASD.
  • the method comprises screening for all SNPs listed in Table 1 that are statistically associated with the absence of ASD or protection from developing ASD.
  • a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder comprising collecting a genetic sample from the subject and screening for an SNP in the genetic sample that is statistically associated with the presence of ASD or a predisposition to developing ASD, wherein the SNP is selected from the list in Table 5 that is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • ASSD Autism Spectrum Disorder
  • the SNP is selected from the group consisting of: rsl7618615, rs6650972, rsl0823195, rsl942052, rs9798267, rs4696443, rs4648135, rs243196, rs7145618, rsl013459, rsl0952662, rs7580690, rs7756516, rs3935743, rs7903424, rs8054767, rsl l001056, rs2684777, rs2300497, rsl6931011, rs4324526, rsl 1736177, rs2239118, rs3020827, rs2270838, rsl7643974, rs2036109, rsl2582971, rsl873423, rs3734464, rs7067880,
  • a method for determining the absence of ASD or protection from developing ASD in a subject comprising collecting a genetic sample from the subject and screening for an SNP in the genetic sample that is statistically associated with the absence of ASD or protection from developing ASD, wherein the SNP is selected from the list in Table 5 that is statistically associated with the absence of ASD or protection from developing ASD.
  • the SNP is selected from the group consisting of: rsl 1583646, rs2587891, rsl480645, rsl2462609, rs4651343, rs888817, rsl050395, rs6971999, rsl928168, rs2272197, rsl 1644436, rs8063461, rsl 1602535, rs339408, rsl0762342, rs7536307, rs7512378, rs4947963, rs3904668, rs4128941, rs7870040, rs6679454, rsl2716928, rsl0783235, rs3910363, rs4647992 and rsl6853387.
  • a method for determining whether a subject has or has a predisposition to develop Autism Spectrum Disorder comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 5, wherein the suitable number of SNPs is statistically associated with the presence of ASD or a predisposition to developing ASD.
  • the method comprises screening for the following SNPs: rsl7618615, rs6650972, rsl0823195, rsl942052, rs9798267, rs4696443, rs4648135, rs243196, rs7145618, rsl013459, rsl0952662, rs7580690, rs7756516, rs3935743, rs7903424, rs8054767, rsl 1001056, rs2684777, rs2300497, rsl6931011, rs4324526, rsl l736177, rs2239118, rs3020827, rs2270838, rsl7643974, rs2036109, rsl2582971, rsl873423, rs3734464, rs7067880, rsl02
  • the method comprises screening for all SNPs identified in Table 5 that are statistically associated with the presence of ASD or a predisposition to developing ASD.
  • a method for determining the absence of ASD or protection from developing ASD in a subject comprising collecting a genetic sample from the subject and screening the genetic sample for a suitable number of SNPs selected from the list in Table 5 that are statistically associated with the absence of ASD or protection from developing ASD.
  • the SNPs are selected from the group consisting of: rsl 1583646, rs2587891, rsl480645, rsl2462609, rs4651343, rs888817, rsl050395, rs6971999, rsl928168, rs2272197, rsl 1644436, rs8063461, rsl 1602535, rs339408, rsl0762342, rs7536307, rs7512378, rs4947963, rs3904668, rs4128941, rs7870040, rs6679454, rsl2716928, rsl0783235, rs3910363, rs4647992 and rsl6853387.
  • the method comprises screening for all SNPs listed in Table 5 that are statistically associated with the absence of ASD or protection from developing ASD.
  • Reference to "50% or greater” includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • the correlation is 60% or greater. In an embodiment, the correlation is 70% or greater. In an embodiment disclosed herein, the correlation is 80% or greater. In an embodiment disclosed herein, the correlation is 85% or greater. In an embodiment disclosed herein, the correlation is 90% or greater. In an embodiment disclosed herein, the correlation is 95% or greater.
  • the SNPs have a weighting (weight) of not equal to zero (0), as shown, for example, in Tables 1 and 5. It should be noted that the weight assigned to each SNP is indicative only and may change to reflect, for example, changes in the reference allele (see Table 5 herein where exemplary reference alleles are shown). A change in the reference allele of an SNP may also change the threshold value used to predict the presence or absence of ASD in accordance with the present invention. The threshold may also be changed to alter the sensitivity or specificity of the test depending on its intended use. The present disclosure is instructional for calculating the ASD score following changes in reference alleles.
  • the weights assigned to each SNP may also differ when other SNPs that are in high linkage disequilibrium with the initial set of SNPs are chosen to build the classifier.
  • the methods disclosed herein can use additional, or the substitution SNPs, that are in high linkage disequilibrium with those listed in Tables 1 and 5.
  • any one or more of the SNPs listed in Tables 1 and 5 can be substituted with one or more SNPs that are in high linkage disequilibrium with those listed in Tables 1 and 5.
  • a subject is first selected after a clinical diagnosis indicating the likelihood or otherwise of the subject having ASD.
  • the genetic analysis is included as part of the clinical assessment of ASD.
  • a skilled artisan is familiar with methods of screening for the presence of (i.e., detecting) an SNP within a nucleic acid sample derived from a subject, also referred to herein as a genetic sample.
  • Such methods include direct determination (e.g., sequencing) and indirect determination (e.g. amplification, hybridization and/or detection of proteins encoded by the gene in which the SNP is located, where such proteins vary in sequence, structure or function as compared to the wild-type proteins).
  • a genetic sample in which an SNP of interest is to be detected in accordance with the methods disclosed herein may be derived from any suitable sample derived from a subject.
  • the nucleic acid can be isolated from a biological sample (genetic sample) such as whole blood, cells from oral mucosa, somatic cells from nail, hair and the like, germ cells, sputum, amniotic fluid, urine, gastric juice, gastric lavage fluid and the like, mitochondria and tissue biopsies.
  • the biological sample may be freshly derived from the subject, or it may be a sample that has previously been obtained from the subject and kept in storage (short, medium or long term). Such sample may, for example, have been stored frozen or paraffin-embedded.
  • the biological sample may also be subject to fractionation or processing to remove highly abundant components.
  • Methods of collecting a genetic sample for use in the diagnostic and prognostic methods disclosed herein are known to persons skilled in the art. Such methods may include the use of raw material (e.g., cells or cell lysates) or they may include steps of isolating the nucleic acid from a sample.
  • Commercially available genomic DNA or RNA isolation kits are available for this purpose.
  • Sample nucleic acid can be obtained from any cell type or tissue of a subject.
  • a subject's bodily fluid e.g. blood
  • nucleic acid tests can be performed on dry samples (e.g., hair or skin).
  • Foetal nucleic acid samples can be obtained from maternal blood.
  • the genetic sample may also be an in utero sample (e.g., amniocytes or chorionic villi) that can be used, for example, for prenatal testing.
  • nucleic acid reagents e.g., primers and probes
  • in situ procedures see, for example, Nuovo, G. J. (1992) "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).
  • the nucleic acid may be DNA or RNA (e.g., mRNA), single- stranded or double- stranded. Where the SNP represents a polymorphism found in a non-coding region, then the nucleic acid will comprise genomic DNA. On the other hand, where the SNP represents a polymorphism found in a coding region, the nucleic acid can comprise RNA, such as total RNA or mRNA.
  • nucleic acids would be understood by persons skilled in the art as meaning DNA or RNA molecules that are substantially free of other non-nucleic acid molecules normally present in their natural source, such as proteinaceous and other cellular material. It would also be understood by persons skilled in the art that the nucleic acid need not be completely free of non-nucleic acid material, as long as the non-nucleic acid material does not adversely affect the particular method that is to be employed for screening for an SNP of interest.
  • the nucleic acid that is contained in the sample may be a nucleic acid originally contained in the sample or, alternatively, it may be an amplicon that has been prepared by nucleic acid amplification using the original nucleic acid in a biological sample as a template. Amplifying nucleic acid molecules originally contained in a sample may be required where the number of nucleic acid molecules in the original sample is below the level required for detection using the screening method of choice. It would be understood by persons skilled in the art that different methods of screening for SNPs will have different levels of sensitivity and, as a consequence, the number of copies of a nucleic acid molecule that is required for the identification of an SNP of interest will vary.
  • Amplification may be performed directly, whereby an amplicon is generated by amplifying the nucleic acid originally contained in a biological sample as a template or indirectly by amplifying a template cDNA molecule that has been generated from RNA originally contained in the biological sample by reverse transcription. These amplicons may be used as template nucleic acids for screening for SNPs of interest in accordance with the methods disclosed herein.
  • the length of the amplicon(s) can vary and may be, for example, from 50 bases to 1000 bases, or from 80 bases to 200 bases.
  • nucleic acid amplification methods examples include PCR, NASBA (nucleic acid sequence based amplification), TMA (transcription-mediated amplification) and SDA (strand displacement amplification). Other examples of suitable nucleic acid amplification methods are described below.
  • Allele-specific PCR is a diagnostic or cloning technique used to identify or utilize SNPs. It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.
  • Assembly PCR or Polymerase Cycling Assembly comprises the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments.
  • the oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
  • Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.
  • a modification on this process known as Linear-After- The- Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
  • Helicase-dependent amplification is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.
  • DNA Helicase an enzyme that unwinds DNA, is used in place of thermal denaturation.
  • Hot-start PCR is a technique that reduces non-specific amplification during the initial set up stages of the PCR.
  • the technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase.
  • Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.
  • Hot- start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
  • Ligation-mediated PCR uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
  • Multiplex-PCR uses of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes; that is, their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
  • Multiplex Ligation- dependent Probe Amplification MLPA permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR.
  • Nested PCR increases the specificity of DNA amplification by reducing background due to non-specific amplification of DNA.
  • Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non- specifically amplified DNA fragments.
  • the product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.
  • Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
  • Reverse Transcription PCR is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA.
  • the PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.
  • RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene.
  • the 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named Rapid Amplification of cDNA Ends (RACE-PCR).
  • TAIL-PCR Thermal asymmetric interlaced PCR
  • Touchdown PCR is a variant of PCR that can reduce nonspecific amplification by gradually lowering the annealing temperature as PCR cycling progresses.
  • the annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm.
  • the higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
  • a forward primer i.e., 5' primer
  • a reverse primer i.e., 3' primer
  • Primers used to amplify a target nucleic acid molecule in accordance with the methods disclosed herein typically comprise relatively short nucleic acid sequences that hybridize specifically to a nucleic acid sequence of interest.
  • a primer can be used alone in a detection method, or a primer can be used together with at least one other primer or probe in a detection method.
  • primers comprise a nucleotide sequence which comprises a region having a nucleotide sequence that specifically hybridizes under stringent conditions to about: 6, or alternatively 8, or alternatively 10, or alternatively 12, or alternatively 25, or alternatively 30, or alternatively 40, or alternatively 50, or alternatively 75 consecutive nucleotides of the nucleic acid sequence of interest. Examples include allele specific hybridization using primers overlapping the SNP and having about 5, or alternatively 10, or alternatively 20, or alternatively 25, or alternatively 30 nucleotides around the SNP.
  • Nucleic acids that are obtained by amplification of the nucleic acid molecules originally contained in the genetic sample derived from a subject may be further subjected to detection hybridization using nucleic acid capture probes, a Tm determination and a polymorphism check/assessment.
  • any of a variety of sequencing reactions known in those skilled in the art can also be used to directly sequence at least a portion of the gene of interest and detect the presence of an SNP in that gene in accordance with the methods disclosed herein, by comparing the sequence of the sample sequence with the corresponding wild-type (control) sequence.
  • Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (1997) Proc. Natl. Acad. Sci, USA 74:560) or Sanger et al. (1977) Proc. Nat. Acad. Sci, 74:5463).
  • the presence of an SNP in a gene can be detected by restriction enzyme analysis, whereby the specific SNP results in a nucleotide sequence comprising a restriction site that is absent from the nucleotide sequence of another allelic variant or from the wild-type sequence.
  • Protection from cleavage agents can also be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes.
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agent such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • a sample nucleic acid e.g., RNA or DNA
  • the double- stranded duplexes are treated with an agent that cleaves single- stranded regions of the duplex, such as duplexes formed based on base-pair mismatches between the control and sample strands.
  • an agent that cleaves single- stranded regions of the duplex such as duplexes formed based on base-pair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S I nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.
  • control and sample nucleic acids After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or which nucleotides they are different. In some embodiments, the control or sample nucleic acid is labelled for detection.
  • Alterations in electrophoretic mobility may also be used to identify an SNP of interest. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acid sequences. Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature.
  • SSCP single strand conformation polymorphism
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of a single base difference.
  • the DNA fragments may be labelled or detected with labelled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to sequence differences.
  • RNA rather than DNA
  • Such methods may also utilize heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility.
  • SNPs of interest may also be identified by analyzing the movement of a nucleic acid comprising the SNP in polyacrylamide gels containing a gradient of denaturant, which is assayed using denaturing gradient gel electrophoresis (DGGE).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example, by adding a GC clamp of approximately 40bp of high-melting GC- rich DNA by PCR.
  • a temperature gradient can be used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA.
  • oligonucleotide probes may be prepared in which the known SNP is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found.
  • allele- specific oligonucleotide hybridization techniques may be used for the detection of the nucleotide changes in the polymorphic region of the gene of interest.
  • oligonucleotides having the nucleotide sequence of the specific allelic variant are attached to a hybridizing membrane and this membrane is then hybridized with labelled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.
  • SNPs can also be identified using an oligonucleotide ligation assay.
  • OLA uses two oligonucleotides that are designed to be capable of hybridizing to abutting sequences of a single strand of a target sequence (i.e. , sequence comprising the SNP of interest).
  • One of the oligonucleotides is linked to a separation marker (e.g., biotin, and the other is detectably labelled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut and create a ligation substrate. Ligation then permits the labelled oligonucleotide to be recovered using avidin, or another biotin ligand.
  • Example of methods using OLA would be familiar to persons skilled in the art.
  • SNPs can also be detected by using a specialized exonuclease-resistant nucleotide.
  • a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection.
  • the amplification of nucleic acid molecules in the original genetic sample can be sequence specific, whereby the conditions favour amplification of the nucleic acid sequence comprising the SNP of interest (e.g., using sequence specific primers).
  • amplification of the nucleic acid molecules in the original genetic sample can be non-sequence specific, whereby the conditions favour general amplification of nucleic acid molecules with the expectation that the amplicons will include those comprising the SNP of interest.
  • the screening of an SNP of interest will usually be performed by using an SNP detection probe, also referred to interchangeably herein as an SNP capture probe.
  • probes includes naturally occurring or recombinant single- or double-stranded nucleic acids or chemically synthesized nucleic acids.
  • nucleic acid sequence of the SNP detection probe is not particularly limited, as long as it comprises a base that is complementary to the target nucleic acid sequence (i.e., a nucleic acid sequence comprising the SNP of interest) or a nucleic acid sequence which can hybridize (e.g., under stringent conditions) to a sequence that is complementary to the target nucleic acid sequence.
  • the length of probe may vary, depending, for example, on the nature of the screening method used and the hybridization conditions employed.
  • the probe may be from 5 mer to 50 mer or, more preferably, from 10 mer to 30 mer.
  • the sequence of the SNP detection probe may be one that has the base corresponding to the sequence of interest, more preferably one that is 90- 100% identical to the sequence which is complementary to the sequence of interest, with the exception that it comprises the base corresponding to the SNP of interest. Whilst not essential, it would be understood by persons skilled in the art that, when the sequence of the SNP detection probe corresponds to the mutant type (i.e., the SNP), detection sensitivity may be increased.
  • the detection probe may comprise a base corresponding to the wild-type sequence, wherein the absence of hybridization of the probe to a sample nucleic acid is indicative of the presence of an SNP.
  • Detection probes may be labelled by nick translation, Klenow fill-in reaction, PCR or other methods known to persons skilled in the art. Suitable detectable labels will be known to persons skilled in the art. Examples include a fluorescent dye and a fluorophore, which emit fluorescence by itself (i.e, when it is not hybridized with a complementary sequence) and become quenched through hybridization of the labelled probe with a complementary sequence.
  • the probe may be labelled with a fluorescent dye on its base located in 3' region (e.g., 3' terminus) or 5' region (e.g., 5' terminus) of the probe. Nucleic acid bases that may be used to attach the detectable label include cytosine. Other detection means for detecting a detectable label in accordance with the methods described herein would be well-known to persons skilled in the art.
  • fluorescent dyes examples include fluorescein, phosphor, rhodamine and polymethine dye derivatives, BODIPY FL (Molecular Probes Inc.), FLUOREPRIME (Amersham Pharmacia), FLUOREDITE (Millipore Corporation), FAM (ABI), Cy3 and Cy5 (Amersham Pharmacia) and TAMRA (Molecular Probes Inc.).
  • a combination of detectable labels may also be used, where appropriate, as long as, for example, the detectable labels are detectable under different detection conditions (e.g., at different wavelengths). Suitable combinations of detectable labels would be familiar to persons skilled in the art.
  • the SNP detection probes may also be labelled with two fluorescent dye molecules to form so-called "molecular beacons", which signal binding to a complementary nucleic acid sequence through relief of intramolecular fluorescence quenching between dyes bound to opposing ends on an oligonucleotide probe.
  • molecular beacons for genotyping will be familiar to persons skilled in the art.
  • a quenching molecule is useful with a particular fluorophore if it has sufficient spectral overlap to substantially inhibit fluorescence of the fluorophore when the two are held proximal to one another, such as in a molecular beacon, or when attached to the ends of an oligonucleotide probe from about 1 to about 25 nucleotides.
  • Labelled SNP detection probes can also be used in conjunction with amplification of a nucleic acid molecule comprising the SNP of interest to provide real-time measurements of amplification products during PCR.
  • Such approaches would be familiar to persons skilled in the art and can either employ intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or they can employ probes containing fluorescence-quencher pairs, where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present.
  • the probe is digested by the nuclease activity of a polymerase when hybridized to the target sequence to cause the fluorescent molecule to be separated from the quencher molecule, thereby causing fluorescence from the reporter molecule to appear.
  • the Taq-Man approach uses a probe containing a reporter molecule— quencher molecule pair that specifically anneals to a region of a target polynucleotide containing the polymorphism.
  • SNP detection probes are affixed to a solid support for use as "gene chips” or “microarrays” . Such gene chips can be used to screen for the SNPs of interest by a number of techniques known to one of skill in the art.
  • the SNP detection probes are affixed to an electrode surface for the electrochemical detection of nucleic acid sequences (as described, e.g., U.S. patent 5,952,172).
  • Suitable solid supports for use in gene chips would be known to persons skilled in the art.
  • the support material may have virtually any possible structural configuration, so long as the coupled molecule (e.g., SNP detection probe, antibody) is capable of binding to its target.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the support surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads.
  • SNP detection probes may be attached to the solid support by a variety of processes known to persons skilled in the art, including lithography.
  • a gene chip may hold any number of detection probes of different sequences (specificities).
  • Suitable formats for gene chips or microarrays are known to persons skilled in the art. Examples include LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); a low-density array with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput, automated mass spectrometry systems with liquid-phase expression technology (Gene Trace Systems, Inc.); a thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray (Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughput microarraying system that can dispense from 12 to 64 spots onto multiple glass slides (Intelligent Bio-Instruments); Molecular Biology Workstation and NanoChip (Nanogen, Inc.); a microflui
  • probes and/or primers for use in accordance with the methods disclosed herein may be modified, for example, to improve their stability. Methods of modifying probes and/or primers would be known to persons skilled in the art. Exemplary nucleic acid molecules which are modified include phosphoramidate, phosphothioate and methylphosphonate analogs of DNA. They can also be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule.
  • the probes and/or primers may include other appended groups such as hybridization-triggered cleavage agents or intercalating agents.
  • the nucleic acid probes and/or primers can also include at least one modified sugar moiety, examples of which include arabinose, 2- fluoroarabinose, xylulose, and hexose or, alternatively, comprise at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • Suitable detection probes include both sense and antisense nucleic acid sequences, and may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by persons skilled in the art. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • complementary refers to the nucleic acid sequence that is the complement of a target nucleic acid sequence.
  • complement of a nucleic acid having SEQ ID NO:X refers to the complementary strand of the strand having SEQ ID NO:X or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO:X.
  • the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID NO:X.
  • the nucleotide sequences and complementary sequences thereof are always given in the 5' to 3' direction.
  • the terms “complement” and “reverse complement” can be used interchangeably herein.
  • the means and conditions for facilitation the hybridization of the SNP detection probe and the sample nucleic acid molecule would be known to persons skilled in the art.
  • Conditions for obtaining single- stranded nucleic acids by denaturing double-stranded nucleic acids and conditions for hybridizing the single- stranded nucleic acid sequences with each other are well-known in the art.
  • the heating temperature for dissociating the double- stranded nucleic acid molecules can be in a range of from 85°C to 95°C.
  • the duration of heating will also vary and can be, for example, in a range of from 1 second to 10 minutes, more preferably from 1 second to 5 minutes.
  • the dissociated single- stranded nucleic acid and the SNP detection probe can be hybridized, for example, by lowering the heating temperature after dissociation.
  • the hybridization temperature will also vary, for example, from about 40°C to about 50°C.
  • a signal change can be measured based on the dissociation of the SNP detection probe from the single-stranded nucleic acid molecule by changing temperature of the sample containing the hybrid in order to dissociate the hybrid.
  • the signal value that indicates dissociation of the hybrid of a single-stranded nucleic acid (whether obtained by amplification or not) and the SNP detection probe can be measured with absorbance at a wavelength of 260 nm.
  • the dissociation may be measured by measuring the signal of a detectable label.
  • An example of a suitable SNP detection probe includes a labelled probe showing a signal by itself, but not showing a signal when hybridized. Such a probe will typically not show a signal when hybridized with a target sequence (e.g., when double-strand DNA is formed), but will show a signal when the probe is dissociated by heating. Another example is a labelled SNP detection probe that typically does not show a signal by itself, but shows a signal when hybridized.
  • Such a probe will show a signal when hybridized with a target sequence (e.g., when double- strand DNA is formed), but the signal may be decreased (i.e., quenched) when the SNP detection probe is dissociated by heating.
  • a target sequence e.g., when double- strand DNA is formed
  • the signal may be decreased (i.e., quenched) when the SNP detection probe is dissociated by heating.
  • the progress of dissociation of the hybrid may be monitored and the melting temperature (Tm) for a particular hydrid can be determined, in a similar manner to measurement of absorbance, for example, at 260 nm.
  • Signal changes based on dissociation of the hybrid may be made by changing a temperature of a reaction solution. For example, heating the reaction solution (i.e., a hybrid between the single-strand DNA and the labelled SNP detection probe), and a change of signal value associated with increase of the temperature can be measured. If, for example, a probe comprising a labelled cysteine residue as its terminal base is used, when the probe is hybridized with a single strand DNA, fluorescence is decreased (or quenched), and when the probe is dissociated, fluorescence is emitted. Thus, by gradually heating a hybrid having decreased fluorescence (or quenched), an increase of fluorescent intensity associated with an increase in the reaction temperature can be measured.
  • Tm may be determined by analyzing a signal change obtained in the measuring step, and then assessed. Such measurement will be familiar to persons skilled in the art. For example, an amount of change for fluorescent intensity per unit time may be calculated from the obtained fluorescence for each temperature. When an amount of change is defined as [-d(increased amount of fluorescent intensity)/dt], the temperature showing the lowest value can be determined as a Tm. Also, an amount of change is defined as [d(increased amount of fluorescent intensity )/t], the temperature showing the highest value can be determined as a Tm. Where the labelled probe that used is not a quenching probe, but is a probe which does not show a signal by itself and shows a signal when hybridized, then a decrease of fluorescent intensity can be measured.
  • the SNP may be determined. For example, when a base of the target base site is presumed to be the mutant type, and an SNP detection probe complementary to the target sequence containing the SNP is used, the target base may be identified as the mutant type if the Tm of a formed hybrid is identical to the Tm of the hybrid of full complementary strands.
  • the target base can be identified as a normal type. If both Tms are detected, for example, it can be determined that a nucleic acid of mutation type and a nucleic acid of normal type co-exist in that sample.
  • a labelled SNP detection probe that shows a signal by itself but does not shows a signal when hybridized (e.g., a guanine quenching probe)
  • the probe emits fluorescence when a single- stranded nucleic acid and a labelled SNP detection probe are dissociated, but when the probe is hybridized by lowering the temperature, the fluorescence is decreased (or quenched).
  • a labelled SNP detection probe that shows a signal by itself but does not shows a signal when hybridized
  • the probe emits fluorescence when a single- stranded nucleic acid and a labelled SNP detection probe are dissociated, but when the probe is hybridized by lowering the temperature, the fluorescence is decreased (or quenched).
  • decrease of fluorescent intensity can be measured.
  • the probe does not emit fluorescence when a single strand DNA and a probe are dissociated, but when the probe is hybridized by lowering the temperature, the probe emits fluorescence.
  • increase of fluorescent intensity can be measured.
  • Antibodies directed against wild type or mutant peptides encoded by the allelic variants of the gene of interest may also be used to detect the presence (or absence) of an SNP of interest. Such methods may be used to detect abnormalities in the level of expression of the peptide or abnormalities in the structure and/or tissue, cellular, or subcellular location of the peptide. Proteins from the tissue or cell type to be analyzed may be detected or isolated using techniques which are well known to persons skilled in the art, such as Western blot analysis. Other examples include immunofluorescence techniques employing a fluorescently labelled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
  • the antibodies (or fragments thereof) that are employed in the methods disclosed herein may also be used for histology, as in immunofluorescence or immunoelectron microscopy, for in situ detection of the peptides or their allelic variants.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labelled antibody that specifically binds to a protein encoded by the allelic variant on which the SNP is located or normally located. Through the use of such a procedure, it is possible to determine not only the presence of the peptide, but also its distribution in the examined tissue.
  • histological methods such as staining procedures
  • the screening of a sample for an SNP of interest may also include methods that determine the peptide function.
  • a peptide encoded by an allelic variant of the gene of interest i.e., the genes in which the SNP is located
  • the activity of the peptide encoded by the allelic variant may be greater or less than the activity of the wild-type peptide, or it may have different activity altogether (e.g., where the peptide is an enzyme, it may display activity on a different substrate).
  • the presence of the SNP may be detected by comparing the activity of the calcium-activated potassium channel in a cell derived from a test subject with the activity of the calcium-activated potassium channel in a cell derived from a subject known to express the wild-type protein.
  • Persons skilled in the art would be familiar with the type of methods that can be used to assay a cell for KCNMB4 activity. An example is provided by Wang et al. (2006, J Gen Physiol. 127(4):449-65).
  • encode refers to a nucleic acid molecule that is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to persons skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • the subject is human.
  • the human is one of an ethnic group of humans.
  • the ethnic group is not Han Chinese.
  • the methods for the diagnosis or prognosis of ASD can also be used to monitor the course of treatment or therapy or for determining the most appropriate course of treatment for a subject in need thereof.
  • treatment includes amelioration or at least management of symptoms of ASD.
  • a medical practitioner can recommend a suitable regimen or therapeutic protocol (e.g., behavioural or pharmacological intervention) that is known to be beneficial in subjects with ASD; also referred to as "pharmacogenomics".
  • a suitable regimen or therapeutic protocol e.g., behavioural or pharmacological intervention
  • pharmacogenomics e.g., pharmacogenomics
  • an individual's genetic profile can enable a medical practitioner: 1) to more effectively prescribe a drug that will address the molecular basis of the disease or condition (e.g., where the SNP is located on a gene associated with a known molecular pathway for which intervening agents (agonists or antagonists) are known); and 2) to better determine the appropriate dosage of a particular agent.
  • the SNP expression patterns of a subject can therefore be used to determine the appropriate drug and dose to administer to the subject.
  • the method further comprise, where the subject is determined as having or having a predisposition to developing ASD, exposing the subject to a treatment for inhibiting the progression of ASD or for inhibiting the onset of ASD or for ameliorating the symptoms of ASD.
  • Methods of treating a subject having or predisposed to developing ASD would be known to persons skilled in the art. Examples include (a) determining the presence of an SNP in a target gene in accordance with the methods disclosed herein; and (b) administering to the subject an effective amount of a compound that targets the gene, or the polypeptide encoded by said gene to inhibit or enhance its activity, as required (i.e., an agonist or antagonist).
  • the methods of treatment or prophylaxis may also include gene therapy to replace an SNP that is indicative of the presence of ASD (or a predisposition to developing ASD) with a nucleotide base corresponding to the wild-type sequence, or a conservative nucleic acid substitution thereof.
  • the treatment comprises administering to the subject an agent that is capable of expressing in the subject a nucleic acid molecule comprising an SNP identified as being protective against developing ASD, where such expression counters (or negates) the phenotype associated with the SNP that is indicative of the presence of ASD or a predisposition to developing ASD.
  • the SNP identified as being protective against developing ASD need not be an SNP located on the same gene as the SNP that is indicative of the presence of ASD or a predisposition to developing ASD and can therefore be located on another gene.
  • Methods for the treatment or prophylaxis of ASD also include administering to a subject an agent that targets the expression and/or activity of a peptide encoded by the allelic variant on which the SNP is located.
  • an agent that targets the expression and/or activity of a peptide encoded by the allelic variant on which the SNP is located.
  • the particular agent will depend on the nature of the peptide and persons skilled in the art would be familiar with the type of agent that can be used, having regard to the relevant peptide.
  • Other suitable examples of methods for the treatment of prophylaxis of ASD include the use of a medication for managing high energy levels, inability to focus, depression or seizures that are occasionally seen in patients with ASD.
  • Further examples include the use of anti-psychotic drugs such as Risperidone and Aripiprazole, particularly for the treatment of younger age patients with ASD who experience severe behavioural difficulties, including tantrums, aggression and self injury behaviour.
  • Other methods include early intervention treatment with a focus of improving a child's development.
  • Such early intervention services may include speech and social interaction therapy.
  • Further examples include auditory training, discrete trial training, vitamin therapy, anti- yeast therapy, communication therapy, music therapy, occupational therapy, physical therapy and sensory integration. These may include behavioural and communication approaches, dietary intervention, medication or complementary and alternative medicines.
  • ABA Applied Behaviour Analysis
  • Examples of ABA include discrete trial training, early intensive behavioural intervention, pivotal response training and verbal behaviour intervention.
  • kits for determining whether a subject has or has a predisposition to develop ASD comprising a set of primers and/or probes for identifying a SNP in accordance with the methods disclosed herein.
  • the kit comprises one of more of the components herein described (e.g., primers, probes, detectable labels, reagents for amplication, etc) and, optionally, instructions for use.
  • the kit may comprise at least one probe or primer which is capable of specifically hybridizing to the polymorphic region of the gene of interest and instructions for use.
  • the kits may comprise at least one of the above described nucleic acids.
  • Preferred kits for amplifying at least a portion of the gene of interest comprise two primers, at least one of which is capable of hybridizing to the allelic variant sequence.
  • Such kits are suitable for detection of genotype by, for example, fluorescence detection, by electrochemical detection, or by other detection.
  • Nucleic acid molecule whether used as probes or primers, contained in a kit can be detectably labelled. Labels can be detected either directly, for example for fluorescent labels, or indirectly. Indirect detection can include any detection method known to one of skill in the art, including biotin-avidin interactions, antibody binding and the like. Fluorescently labelled oligonucleotides also can contain a quenching molecule.
  • the nucleic acid molecules may be bound to a solid support, as employed, for example, in gene chips and microarrays.
  • kits may also include reagents for preparing (isolating) nucleic acid molecules from a genetic sample derived from a subject.
  • the kits may also include all or some of the positive controls, negative controls, sequencing markers, and antibodies described herein for screening a subject's genetic sample for an SNP of interest.
  • the genetic marker in a SNP is a SNP.
  • the methods disclosed herein further comprise having the user determine the data using a remote end station; and transferring the data from the end station to the base station via the communications network.
  • the methods disclosed herein further comprise transferring the data through a firewall.
  • the methods disclosed herein further comprise:
  • a base station for stratifying a subject with respect to ASD comprising:
  • the processing system is adapted to receive data from a remote end station adapted to determine the data.
  • the processing system comprises:
  • a prognostic panel of genetic markers comprising one or more primers and/or one or more SNP detection probes, as herein described, for screening a subject for SNPs in accordance with the methods disclosed herein.
  • the one or more primers and/or one or more probes may be attached to a solid support, as hereinbefore described (i.e., as a microarray).
  • Index sample Subject data from 2,609 probands with ASD (including Autism, Asperger's or Pervasive Developmental Disorder-Not Otherwise Specified, but excluding RETT syndrome and Fragile X), and 4,165 relatives of probands, was available from AGRE 1 ; 1,862 probands and 2,587 first-degree relatives had SNP data from the Illumina 550 platform relevant to analyses (see Figure la). Diagnosis of ASD was made by a specialist clinician and confirmed using the Autism Diagnostic Interview Revised (ADI-
  • Control training data was obtained from HapMap 3 instead of relatives, as the latter may possess SNPs that predispose to ASD and skew analysis (see Figures la and lb).
  • HapMap data Phase 3 NCBI build 36 was utilized to allocate individuals to their closest ethnicity. Individuals of mixed ethnicity were excluded; HapMap data has 1,403,896 SNPs available from 11 ethnicities. Any SNPs not included on the AGRE Illumina 550 platform were discarded, resulting in 407,420 SNPs. Mitochondrial SNPs reported in the AGRE, but not available in HapMap were excluded. The 30 most prevalent (>95%) SNPs within each ethnicity were identified and each ASD individual assigned to the group for which they shared the highest number of ethnically specific SNPs. HapMap groups were determined to be appropriate for analysis, as prevalence rates of the 30 SNPs relevant to each ethnicity were similar for each AGRE group assigned to that ethnicity, p ⁇ 0.05.
  • Pathway analysis was selected because it depicts how groups of genes may contribute to ASD etiology (see Example 7) and mitigates the statistical problem of conducting a large number of multiple comparisons required in GWAS studies.
  • the current pathway analysis differs from previous ASD analyses in three unique ways: (1) we divided the cohort into ethnically homogeneous samples with similar SNP rates; (2) both protective and contributory SNPs were accounted for in the analysis, and (3) the pathway test statistic was calculated using permutation analysis. Although this is computationally expensive, benefits include taking account of rare alleles, small sample sizes and familial effects. It also relaxes the Hardy- Weinberg equilibrium assumption, that allele and genotype frequencies remain constant within a population over generations.
  • Example 3 Predicting ASD phenotype based upon candidate SNPs.
  • Each dimension of the vector was assigned a value of 0, 1 or 3, dependent on a SNP having two copies of the dominant allele, heterozygous or two copies of the minor allele.
  • the ⁇ , 1, 3' weighting provided greater classification accuracy over ⁇ , 1, 2'.
  • Such approaches using super- additive models have been used previously to understand genetic interactions 5 .
  • the formulae for the classifier and classifier performance are presented in Example 9.
  • the CEU sample was divided into a training set (732 ASD individuals and 123 controls) and the remainder comprised the validation set.
  • An affected individual was given a value of 10 and an unaffected individual a value of -10, providing a sufficiently large separation to maximize the distance between means (see Example 9).
  • Least squares regression analysis of the training set determined coefficients whose product mapped SNPs to clinical status.
  • Samples included 507 CEU and 18 TSI subjects with ASD from SFARI, and 2,557 CEU and 63 TSI from WTBC (see Figure lb).
  • the most significant pathways were: calcium signaling, gap junction, long term depression (LTD), long term potentiation (LTP), olfactory transduction, and mitogen activated kinase-like protein (MAPK) signaling.
  • Table 2 Statistically significant pathways for the CEU and Han Chinese. -values in bold are statistically significant. The pathways highlighted in 'bold' denote pathways that have reached statistical significance in both populations.
  • KEGG Kyoto Encyclopedia of Genes and Genomes; ftp.kegg.jp); CEU - of Central (Western & Northern) European origin; HAN - of HAN Chinese origin.
  • Substitute Sheet (Rule 26) RO/AU - 58 -
  • Tables 3a and 3b List of 15 most contributory (Table 3a) and 1 5 most protective ⁇ Table 3h) SNPs for ASD diagnosis in the CEU Cohort. Weight indicates the contribution of each SNP to ASD clinical status. 'Weight Lower' indicates the 0.95 lower error bar of the estimate; 'Weight Higher' indicates the 0.95 upper error bar for that SNP. Note that some genes have SNPs that contribute to risk for ASD and SNPs that protect against ASD.
  • GSEA was undertaken to consider all possible genes related to pathways that might contribute to risk for autism. We were interested to examine the contribution of multiple SNPs to risk for autism, each with potentially small effect, rather than seek to identify individual or small numbers of SNPs of large effect. The latter approach, while providing some information about the genes contributing to autism, they have failed to provide any ability to predict which individuals may be at risk.
  • the approach we have taken is to identify which of the known pathways are perturbed in ASD (using KEGG canonical pathways).
  • canonical pathways instead of attempting to identify significance for individual SNPs or genes, we sought to identify canonical pathways that differed compared with control subjects. This has the benefit of taking into account the complex interactions of genes, and since this approach is analyzing a much smaller number of sets it considerably increases the power of the analyses.
  • Illumina 550 platform The other datasets (HAPMAP, SFARI, Wellcome Trust) provided SNP data from the Illumina lM-Duo. The total number of SNPs consistent across the two platforms was 407,420. The number of KEGG Pathway genes examined was 5,936. For each Kegg pathway, we determined the collection of SNPs residing on genes that form part of the pathway. This was performed by firstly identifying all genes that reside on a pathway. NCBI data mapping a SNP to a gene was used to identify all SNPs relevant to a pathway. This included both intronic and exonic genes.
  • Kegg pathways were tested. Only those showing statistical significance were retained at p ⁇ 10 "5 .
  • the significance threshold of p ⁇ 10 "5 was set according to the number of pathways being examined, which was 200. Therefore, significance was less than 0.05/200 [set at less than 1 x 10 ⁇ 5 ].
  • the SNP weights were not assumed to be Gaussian.
  • the distributions of the weights for each of the SNPs were also examined by taking random subsamples of individuals and their genetic data, which were used to train the classifier, providing weights for each SNP with each training set. This was iteratively run 100,000 times and a histogram of the weights for each SNP was plotted. This allowed examination of the distribution of the weights for each SNP, allowing a determination to be made of the confidence interval for each SNP.
  • Example 9 Formula for the classifier & classifier performance
  • the weighting can be -62- negative, so that more deleterious effect is not necessarily assumed to be related to the minor allele. It can be either the least or the most deleterious and the off-set can also change the contribution of those SNPs to the clinical phenotype.
  • each roup can be shown to be equal to
  • the optimal weight vector W which maximizes the distance between the two groups, can be shown to correspond to the eigenvector with the maximum eigenvalue to the matrix
  • W 0 can be chosen such that the two distributions of the two population means are symmetric about the origin. It is also evident that a scale factor can be chosen to place the two means at an arbitrary but symmetric location about the origin. Hence, the choice of the mean value for training is arbitrary, provided that the X values have no physical significance; that is, it does not measure a patient variable.
  • a genetic diagnostic classifier was generated based on a linear function of 237 SNPs (Table 1) that accurately distinguished ASD from controls within a CEU cohort. This same diagnostic classifier was able to correctly predict and identify ASD individuals with accuracy exceeding 85.8% and 84.3% in the unseen CEU and TSI cohorts, respectively. This classifier was able to predict ASD group membership in subjects derived from two independent datasets with an accuracy of 71.7%. However, the classifier was less accurate at predicting ASD in the genetically distinct Han Chinese cohort, which may be explained by differences in allelic prevalence.
  • the SNPs contributing most to diagnosis in our classifier corresponded to genes for KCNMB4, GNAOl, GRM5, INPP5D, and ADCY8.
  • the three SNPs that markedly skewed an individual towards ASD were related to the genes coding for KCNMB4, GNAOl, and GRM5.
  • Homozygosity for KCNMB4 SNP carries a higher risk of ASD than SNPs related to GNAOl and GRM5.
  • a number of SNPs protected against ASD including rs8053370 (GNAOl), rsl2317962 (KCNMB4), rs6483362 and rs905646 (GRM5).
  • KCNMB4 is a potassium channel that is important in neuronal excitability and has been implicated in epilepsy and dyskinesia 6 ' 7 . It is highly expressed within the fusiform gyrus, as well as in superior temporal, cingulate, and orbitofrontal regions (Allen Human Brain Atlas, Allen Institute for Brain Science; ), which are areas implicated in face identification and emotion face processing deficits seen in ASD.
  • GNAOl protein is a subgroup of Ga(o), a g-protein that couples with many neurotransmitter receptors. Ga(o) knockout mice exhibit "autism-like" features, including impaired social interaction, poor motor skills, anxiety and stereotypic turning behaviour 9 . - 64 -
  • GNAOl has also been shown to have a role in nervous development co-localizing with GRIN1 at neuronal dendrites and synapses 10 , and interacting with GAP-43 at neuronal growth cones 11 , with increased levels of GAP-43 demonstrated in the white matter adjacent to the anterior cingulate cortex in brains from ASD patients 12.
  • GRM5 SNPs have both a contributory (rs 11020772) and protective (rs905646, rs6483362) effect on ASD.
  • GRM5 is highly expressed in hippocampus, inferior temporal gyrus, inferior frontal gyrus and putamen
  • GRM5 has a role in synaptic plasticity, modulation of synaptic excitation, innate immune function, and microglial activation 14"17 .
  • GRM5 positive allosteric modulators can reverse the negative behavioral effects of NMDA receptor antagonists, including stereotypies, sensory motor gating deficits, and deficits in working, spatial and recognition memory 18 , features described in ASD 19 ' 20 .
  • this receptor is expressed on microglia 14 ' 21 , with microglial activation demonstrated by us and others in frontal cortex in ASD 22 ' 23.
  • GRM5 signalling is mediated via signalling through GCPRs, a possible interaction between GNAOl and GRM5 is plausible.
  • Genes such as PLCB2, ADCY2, ADCY5 and ADCY8 encode for proteins involved in G-protein signalling.
  • GRM5 may represent a pivotal etiological target for ASD; however, further work is needed in demonstrating these potential interactions and contribution to glutamatergic dysregulation in ASD.
  • our predictive genetic classifier obtained a high level of diagnostic accuracy. This demonstrates that genetic biomarkers can correctly classify ASD from non-ASD individuals.
  • a predictive classifier as described herein is useful tool for screening at birth or during infancy to provide an index of "at-risk status", including probability estimates of - 65 -
  • ASD likelihood Identifying clinical and brain-based developmental trajectories within such a group would provide the opportunity to investigate potential psychological, social and/or pharmacological interventions to prevent or ameliorate the disorder.
  • TDT transmission disequilibrium test
  • Table 4 shows classifier performance in predicting symptoms. Poor performance on Stereotypical Speech and Immediate Echolalia could be due to the fact that there are other SNPs or CNVs that need to be included in our model or that these symptoms may have a large environmental component.
  • AGP DATA 6626 Individuals - 1241 Cases - 5585 Relatives (Mainly Parents).
  • the SNPs identified show ability to distinguish ASD versus controls in 3 independent cohorts of individuals of White Non-Hispanic ancestry. Furthermore, the - 70 - results indicate that the identified SNPs can be used to distinguish individuals with certain features, including marked language delay and gaze avoidance.
  • SNPs in high linkage disequilibrium (LD) across the SFARI and AGRE sets were removed in the original data set. As not all SNPs of interest were genotyped across all validation sets, SNPs were chosen that were in linkage disequilibrium that were available across all data sets, as shows in Table 5, below.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés qui permettent de diagnostiquer et de pronostiquer un trouble du spectre de l'autisme (TSA), en partie en fonction de l'identification de marqueurs génétiques signalant la présence d'un TSA ou une prédisposition à développer un TSA, ou indiquant l'absence de TSA ou la protection contre le développement d'un TSA. Les marqueurs génétiques sont des polymorphismes mononucléotidiques (SNP) qui remplissent les voies moléculaires qui sont associées directement ou indirectement au développement de TSA ou à la protection contre les TSA. Les SNP sont appliqués de façon à générer un classificateur prédictif pour les phénotypes des personnes concernées et de leurs parents. Cette invention concerne également des procédés de traitement, des nécessaires et des stations de base afin d'exécuter les procédés de diagnostic et de pronostic divulgués par les présentes.
PCT/AU2013/000798 2012-07-20 2013-07-19 Procédé de diagnostic ou de pronostic d'un trouble neurologique WO2014012144A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2012903105A AU2012903105A0 (en) 2012-07-20 Method of diagnosing or prognosing a neurological disorder
AU2012903105 2012-07-20

Publications (1)

Publication Number Publication Date
WO2014012144A1 true WO2014012144A1 (fr) 2014-01-23

Family

ID=49948112

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2013/000798 WO2014012144A1 (fr) 2012-07-20 2013-07-19 Procédé de diagnostic ou de pronostic d'un trouble neurologique

Country Status (1)

Country Link
WO (1) WO2014012144A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108823322A (zh) * 2018-07-10 2018-11-16 山东省农业科学院奶牛研究中心 一组用于筛选和/检测种公牛精子畸形率高低的snp分子标记
WO2023235415A1 (fr) * 2022-06-01 2023-12-07 Genentech, Inc. Procédé d'identification d'un patient ayant une probabilité accrue de neuropathie périphérique induite par une chimiothérapie

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042575A2 (fr) * 1998-02-17 1999-08-26 Sanofi-Synthelabo GENE ET PROTEINE DE LA SOUS-UNITE β DU CANAL POTASSIUM ACTIVE PAR LE CALCIUM
WO2000050444A1 (fr) * 1999-02-23 2000-08-31 Icagen, Inc. Sous-unites de bk beta de canaux potassiques de famille slo
WO2005008249A1 (fr) * 2003-07-11 2005-01-27 Universite Francois Rabelais Test de depistage de l'autisme, methode de selection de patients repondant audit test et utilisation des medicaments activateurs de canaux bkca pour le traitement de ladite maladie.
WO2006055927A2 (fr) * 2004-11-17 2006-05-26 The Cleveland Clinic Foundation Gene et proteine pathogenes associes a la dyskinesie paroxystique et a l'epilepsie
US20110207124A1 (en) * 2008-02-20 2011-08-25 Hakon Hakonarson Genetic Alterations Associated with Autism and the Autistic Phenotype and Methods of Use Thereof for the Diagnosis and Treatment of Autism
WO2012079008A2 (fr) * 2010-12-10 2012-06-14 The George Washington University Biomarqueurs de polymorphismes de nucléotides uniques pour le diagnostic de l'autisme

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042575A2 (fr) * 1998-02-17 1999-08-26 Sanofi-Synthelabo GENE ET PROTEINE DE LA SOUS-UNITE β DU CANAL POTASSIUM ACTIVE PAR LE CALCIUM
WO2000050444A1 (fr) * 1999-02-23 2000-08-31 Icagen, Inc. Sous-unites de bk beta de canaux potassiques de famille slo
WO2005008249A1 (fr) * 2003-07-11 2005-01-27 Universite Francois Rabelais Test de depistage de l'autisme, methode de selection de patients repondant audit test et utilisation des medicaments activateurs de canaux bkca pour le traitement de ladite maladie.
WO2006055927A2 (fr) * 2004-11-17 2006-05-26 The Cleveland Clinic Foundation Gene et proteine pathogenes associes a la dyskinesie paroxystique et a l'epilepsie
US20110207124A1 (en) * 2008-02-20 2011-08-25 Hakon Hakonarson Genetic Alterations Associated with Autism and the Autistic Phenotype and Methods of Use Thereof for the Diagnosis and Treatment of Autism
WO2012079008A2 (fr) * 2010-12-10 2012-06-14 The George Washington University Biomarqueurs de polymorphismes de nucléotides uniques pour le diagnostic de l'autisme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SKAFIDAS, E. ET AL.: "Genetic Factors Identifying Risk and Resilience in Autism Spectrum Disorders", BIOLOGICAL PYSCHIATRY, vol. 73, no. 9, 1 May 2013 (2013-05-01), pages 147S *
SKAFIDAS, E. ET AL.: "Predicting the diagnosis of autism spectrum disorder using gene pathway analysis", MOLECULAR PSYCHIATRY, 11 September 2012 (2012-09-11), pages 1 - 7 *
WANG, K. ET AL.: "Common genetic variants on 5p14.1 associate with autism spectrum disorders", NATURE, vol. 459, 28 May 2009 (2009-05-28), pages 528 - 533 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108823322A (zh) * 2018-07-10 2018-11-16 山东省农业科学院奶牛研究中心 一组用于筛选和/检测种公牛精子畸形率高低的snp分子标记
CN108823322B (zh) * 2018-07-10 2021-08-03 山东省农业科学院奶牛研究中心 一组用于筛选和/检测种公牛精子畸形率高低的snp分子标记
WO2023235415A1 (fr) * 2022-06-01 2023-12-07 Genentech, Inc. Procédé d'identification d'un patient ayant une probabilité accrue de neuropathie périphérique induite par une chimiothérapie

Similar Documents

Publication Publication Date Title
JP5262230B2 (ja) 新規多型検出法
KR101646978B1 (ko) 핵산 서열 불균형의 결정
CA2798758C (fr) Procedes de classification de ploidie prenatale non invasive
US20110033862A1 (en) Methods for cell genotyping
US11542556B2 (en) Single nucleotide polymorphism in HLA-B*15:02 and use thereof
KR20100063050A (ko) 디지털 ｐｃｒ에 의한 다양한 길이의 핵산의 분석
KR20100020960A (ko) 자궁내막증과 연관된 유전자 마커 및 이의 용도
Brion et al. New technologies in the genetic approach to sudden cardiac death in the young
US20110269688A1 (en) Genetic Alterations Associated with Schizophrenia and Methods of Use Thereof for the Diagnosis and Treatment of the Same
US20100047798A1 (en) Adenosine a1 and a3 receptor gene sequence variations for predicting disease outcome and treatment outcome
US20200087728A1 (en) Genetic markers associated with endometriosis and use thereof
WO2014012144A1 (fr) Procédé de diagnostic ou de pronostic d'un trouble neurologique
US20100144776A1 (en) Use of snps for the diagnosis of a pain protective haplotype in the gtp cyclohydrolase 1 gene (gch1)
WO2019168971A1 (fr) Méthode d'évaluation du risque de délai accru de conception
WO2008010082A2 (fr) Méthode diagnostique
US10770183B2 (en) Methods of assessing a risk of developing necrotizing meningoencephalitis
US20100003691A1 (en) Genetic Markers Associated with Degenerative Disc Disease and Uses Thereof
TWI674320B (zh) 用以預斷吉特曼症候群的方法及套組
WO2009052559A1 (fr) Essai diagnostique
JP2007510404A (ja) アルツハイマー病の発症年齢に関連するｎｔｒｋ１遺伝子マーカー
US20140045717A1 (en) Single Nucleotide Polymorphism Biomarkers for Diagnosing Autism
KR101167945B1 (ko) Ａｔｇ16ｌ1 유전자로부터 유래된 단일염기다형을 포함하는 폴리뉴클레오티드, 이를 포함하는 마이크로어레이 및 진단키트, 및 이를 이용한 자폐 스펙트럼 장애 분석방법
Belhassan et al. Current approaches to genetic testing in pediatric disease
JP5809388B2 (ja) スティーブンス・ジョンソン症候群の発症リスクの判定方法
KR101167942B1 (ko) Ａｌｇ12 유전자로부터 유래된 단일염기다형을 포함하는 폴리뉴클레오티드, 이를 포함하는 마이크로어레이 및 진단키트, 및 이를 이용한 자폐 스펙트럼 장애 분석방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13820471

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13820471

Country of ref document: EP

Kind code of ref document: A1