METHOD AND APPARATUS FOR PREDICTING SUSCEPTIBILITY TO A DEVELOPMENTAL DISORDER

Abstract

A nucleotide sequence signal amplification composition that includes an isolated, synthetic nucleotide sequence of greater than 7 nucleotides. The sequence is a fragment of SEQ ID NO:3 and further comprising a T nucleotide at position 1438 of SEQ ID NO:3 and one or more primers that bind to the synthetic nucleotide sequence, a thermostable DNA polymerase, a restriction enzyme, or a combination thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No. 13/258,929 filed on Nov. 17, 2011, which is the 371 filing of International application no. PCT/CA2010/000448 filed on Mar. 26, 2010, which claims the benefit of U.S. application No. 61/164,200 filed on Mar. 27, 2009.

FIELD OF THE INVENTION

[0002] The present invention relates to a method and apparatus for predicting susceptibility to a developmental disorder. In particular, the present invention relates to a method and apparatus for predicting susceptibility to non-syndromic autosomal recessive mental retardation and autism by detecting the presence of genetic mutation in the TRAPPC9 gene (Trafficking Protein Particle Complex, Subunit 9) and its encoded protein, also known as NIBP (NIK- AND IKK-β-Binding Protein), and KIAA1882.

BACKGROUND OF THE INVENTION

[0003] Mental retardation (MR) is believed to occur with a prevalence of ˜2% within the population. MR is significantly more frequent in males than in females, and for that reason it had been assumed that ˜25% of severe cases were X-linked, however recent review of data suggests that X-linked mutations contribute to no more than 10% of cases (Ropers & Hamel, 2005). Very little, however, is currently known about autosomal non-syndromic forms of MR. Autosomal dominant MR tends to occur only sporadically, due to the decreased likelihood of patients to procreate. Autosomal recessive forms of non-syndromic MR (NS-ARMR) are believed to be more common, yet only 5 genes have been identified so far, including PRSS12 (MRT1 on 4q25-q26; Molinari et al., 2002), CRBN (MRT2A on 3p26.2; Higgins et al., 2004), and CC2D1A (MRT3 on 19q13.12; Basel-Vanagaite et al., 2006). A recent study, using homozygosity mapping in large consanguineous families from Iran, has identified a further 8 loci (MRT4-12; Najmabadi et al., 2007). From this study, the discovery of GRIK2 as the cause of MRT6 on 6q21 (Motzacker et al., 2007), and TUSC3 on 8q12 as the cause of MRT7 have recently been made (Garshasbi et al., 2008).

[0004] The contribution of genetic factors to autism is also well established, but the mode of genetic transmission in unclear. It is apparent, however, that autism is a complex non-Mendelian disorder, and it is anticipated that genetic heterogeneity and oligo/plolygenic inheritance are involved. Several genome-wide linkage studies have been performed, implicating a number of chromosomes, including 7q, 16p, 19q and 11 p (IMGSAC, 1998 & 2001; CLSA, 1999; Liu et al, 2001; AGP 2007), however no genes have been identified so far. Evidence from studies of overlap between autism and mental retardation syndromes, as well as a number of studies using cytogenetic aberrations, also genomic copy number variants inferred from microarray analysis, have now implicated a number of specific genes such as SHANK3, NLGN3 & 4, NRXN1, CNTNAP2, UBE3A, FMR1, MECP2 and others (see reviews by Abrahams & Geschwind, 2008; Sutcliffe 2008). However, only very recently, several groups have started exploring the hypothesis that at least a small proportion of autism may be inherited in an autosomal recessive mode. The recent paper by Morrow et al (2008), where several genes such as PCDH10 and DIA1 were mapped through the identification of large homozygous deletions in consanguineous families with autism from the Arabian peninsula, Turkey and Pakistan, is an example of some of the potential of such an approach. Identification of autosomal recessive genes for autism may lead to the identification of relevant etiological biological pathways, and potentially the identification of other genes from the same pathway that may contribute to autism, and possibly inherited in a non-Mendelian fashion.

[0005] There is a need in the art to identify genetic markers associated with mental retardation and autism. Further there is a need in the art to identify nucleotide sequences associated with mental retardation and autism. There is also a need in the art for new diagnostic assays for mental retardation and autism.

SUMMARY OF THE INVENTION

[0006] The present invention relates to gene mutations. More specifically, the present invention relates to gene mutations associated with mental retardation.

[0007] According to an embodiment of the present invention, there is provided a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of:

[0008] obtaining a genomic DNA sample from the human subject;

[0009] determining a) if the DNA sample from the subject encodes a mutated NIBP protein relative to SEQ ID NO: 2, wherein said mutated NIBP protein is truncated, or b) if the DNA sample from the subject encodes a mutated NIBP protein relative to SEQ ID NO: 2, said DNA comprising a deletion, insertion, translocation, point mutation, frameshift mutation, or combination thereof that results in a mutated NIBP protein defined by truncation of NIBP, a non-functional NIBP protein or one that comprises nonsense mutations relative to SEQ ID NO:2;

[0010] wherein the presence of the mutated NIBP protein or a nucleotide sequence encoding the mutated NIBP protein identifies the subject as susceptible to the developmental disability.

[0011] As can be appreciated, a person of skill in the art could practice a substantially similar method by screening the mRNA from a human subject. However, such a screening method is less preferred.

[0012] According to a further embodiment of the present invention, there is provided a method as described above and further comprising,

[0013] amplifying a nucleic acid sequence corresponding to the nucleotide sequence containing position 1438 of SEQ ID NO:1 using a first primer that binds upstream of said position and a second primer that binds downstream of said position;

[0014] detecting the presence or absence of a T nucleotide at the position corresponding to 1438 in SEQ ID NO:1; and

[0015] determining the genotype of the human subject at the position corresponding to 1438 in SEQ ID NO:1,

[0016] wherein a homozygote for the T nucleotide is predictive of the developmental disability and a heterozygote for the T nucleotide is a carrier of the developmental disability.

[0017] Similar methodology may be employed to screen subjects for any point mutation, missense mutation, deletion, insertion, translocation, frameshift mutation or the like which results in truncation of the NIBP protein as compared to SEQ ID NO:2. Similarly, nonsense mutations in TRAPPC9 gene which result in extraneous or unrelated addition of amino acid sequences can easily be determined by a person of skill in the art, with or without the need of programs to align a mutated NIBP protein to the wild-type NIBP protein as provided in SEQ ID NO:2. Typically, in such an alignment, a first portion of the NIBP protein will exhibit high identity (greater than 95% identity, preferably 99% or higher identity) to a portion of SEQ ID NO:2, whereas a second portion of the NIBP protein will exhibit little (for example, less than 20% identity, preferably less than 10% identity, more preferably less than 5% identity) or no identity when aligned using a basic alignment program as known in the art using default parameters. In separate embodiments, a mutation causing truncation or addition of extraneous or unrelated addition of amino acids occurs in exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of the TRAPPC9 encoding gene. As we have described mutations in exons 14 and 7 which result in truncated NIBP proteins and correlate with developmental disorder phenotype, it is reasonable to conclude that any mutation in exons 1-14 that results in truncation or the addition of extraneous or unrelated amino acids in NIBP could serve as a diagnostic marker to identify subjects that are susceptible to a developmental disorder as described below.

[0018] In a further embodiment, there is provided a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of

[0019] obtaining a biological sample from the human subject that comprises NIBP protein or a mutant protein thereof;

[0020] optionally isolating protein from the sample;

[0021] exposing the protein to an antibody that recognizes and binds a portion of a polypeptide sequence that corresponds to position 476 to 1246 of SEQ ID NO. 2; and

[0022] detecting the presence of the antibody/polypeptide complex,

[0023] wherein the absence of binding between the antibody and the polypeptide is predictive of the developmental disability.

A similar method may be employed for any NIBP truncation protein or mutant NIBP protein as described herein.

[0024] In still a further embodiment, there is provided a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of:

[0025] obtaining a biological sample from the human subject that comprises NIBP protein or a mutant protein thereof;

[0026] optionally isolating protein from the sample;

[0027] exposing the protein to an antibody that recognizes and binds a portion of a polypeptide sequence that corresponds to positions 1 to 1246 of SEQ ID NO. 2, for example, but not limited to the N-terminal region; and

[0028] detecting the presence of the antibody/polypeptide complex,

[0029] determining if the antibody has bound to wild-type, truncated or mutant NIBP protein based on one or more characteristics thereof, for example, size, known or available epitopes, amino acid sequence, isoelectric point, hydrophilicity, hydrophobicity, ability to interact with known binding partners, activity or any other method known in the art. For example, but not wishing to be considered limiting, a truncated NIBP protein may migrate further during electrophoresis than its counterpart wild-type protein. Such differences can easily be identified by a person of skill in the art.

[0030] The present invention also contemplates combinations of the methods as described throughout the disclosure herein.

[0031] The present invention also contemplates a nucleic acid sequence or complement thereof which hybridizes to a nucleotide sequence encoding a mutant NIBP protein defined by truncation of TRAPPC9, a non-functional NIBP protein, one of reduced function, or one that comprises nonsense mutations relative to SEQ ID NO:2.

[0032] In an embodiment, the nucleic acid as described above, or complement thereof does not hybridize to SEQ ID NO:1 or a complement thereof. In a further embodiment, the nucleic acid comprises a contiguous nucleotide sequence of SEQ ID NO:3 and comprises position 1438 thereof, or a contiguous nucleotide sequence complementary thereto.

[0033] The nucleic acid sequence as described above may be any length, for example from about 7 nucleotides to 100 or more nucleotides, for example, but not limited to, a 7-mer, 10-mer, 15-mer, 20-mer, 25-mer-, 30-mer, 35-mer, 40-mer, 45-mer, 50-mer, 55-mer, 60-mer, 65-mer, 70-mer, 75-mer, 80-mer, 85-mer, 90-mer, 95-mer, and 100-mer nucleic acid sequence or any size therein between.

[0034] The present invention also provides a physical support or substrate comprising the nucleic acid as described above attached thereto. The physical support or substrate may be, for example, but not limited to, a DNA array, microarray, bead, plastic well, carrier protein, non-proteinaceous macromolecule or the like. In a preferred embodiment the nucleic acid is covalently attached to the physical support or substrate optionally via a linker. Any linker known in the art may be used.

[0035] According to an aspect of the present invention there is provided a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of: amplifying a nucleic acid sequence containing position 1438 of SEQ ID NO:1 using a first primer that binds upstream of said position and a second primer that binds downstream of said position; detecting the presence of a T nucleotide at position 1438 in SEQ ID NO:1; and determining the genotype of the human subject at position 1438 in SEQ ID NO:1 (which is position 1423 from first translated nucleotide), wherein a homozygote for the T nucleotide is predictive of the developmental disability and a heterozygote for the T nucleotide is a carrier of the developmental disability.

[0036] In one embodiment, the developmental disability is mental retardation or autism.

[0037] In another embodiment, the step of amplifying involves polymerase chain reaction.

[0038] According to another aspect of the invention, there is provided an oligonucleotide comprising a contiguous nucleic acid containing position 1438 of SEQ ID NO. 3 and complements thereof.

[0039] In one embodiment the oligonucleotide is selected from a group consisting of 7-mer, 10-mer, 15-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, 50-mer, 55-mer, 60-mer, 65-mer, 70-mer, 75-mer, 80-mer, 85-mer, 90-mer, 95-mer, and 100-mer nucleic acid sequence or complements thereof. It is also contemplated that an oligonucleotide of a length in between any one of the values or more than 100 nucleotides is encompassed within the scope of the present invention.

[0040] According to a further aspect of the present invention, there is provided an oligonucleotide that is 90% identical, more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to a contiguous nucleotide sequence containing position 1438 of SEQ ID NO:3, or any other mutation in TRAPPC9 that results in a truncated or mutated NIBP protein.

[0041] According to another aspect of the invention, there is provided a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of: obtaining a sample from the human subject; isolating protein from the sample; exposing the protein to an antibody that recognizes a portion of a polypeptide sequence that corresponds to position 476 to 1246 of SEQ ID NO. 2; and detecting the presence of the antibody/polypeptide complex, wherein the absence of binding between the antibody and the polypeptide is predictive of the developmental disability. Further, the method may also employ a positive control step of exposing the protein to an antibody that recognizes a portion of a polypeptide that corresponds to positions 1 to 475, for example in exon 1, 2, 3, 4, 5 or 6.

[0042] In an embodiment, the developmental disability is mental retardation or autism.

[0043] According to further aspect of the present invention, there is provided an apparatus for detecting a nucleotide in a nucleic acid sequence. The apparatus comprising: a substrate; a first oligonucleotide bound to the substrate, the first oligonucleotide comprising a contiguous nucleic acid sequence complementary to SEQ ID NO. 3 containing position 1438 of the sequence, or a nucleic acid sequence at least 90% identical thereto.

[0044] In one embodiment, the apparatus further comprises a second oligonucleotide bound to the substrate, the second oligonucleotide comprising a contiguous nucleic acid sequence complementary to SEQ ID NO. 1 containing position 1438 of the sequence.

[0045] In an embodiment, the first oligonucleotide comprises a 25-mer contiguous nucleic acid sequence.

[0046] In another embodiment, the second oligonucleotide comprises a 25-mer contiguous nucleic acid sequence.

[0047] In a further embodiment, the first oligonucleotide comprises a 60-mer contiguous nucleic acid sequence.

[0048] In yet a further embodiment, the second oligonucleotide comprises a 60-mer contiguous nucleic acid sequence.

[0049] According to a further aspect of the present invention, there is provided a nucleic acid comprising a sequence selected from the group consisting of: a) a nucleic acid sequence comprising SEQ ID NO. 4; b) a complement of a nucleic acid sequence comprising SEQ ID NO. 4; c) a fragment of either a) or b); d) a nucleic acid sequence capable of hybridizing to any one of a), b) or c); and e) a nucleic acid sequence that exhibits greater than about 70% sequence identity with the nucleic acid defined in a), b) or c).

[0050] According to another aspect of the present invention, a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of isolating RNA from the subject; hybridizing an oligonucleotide comprising a contiguous nucleic acid of SEQ ID NO. 1 to the RNA; wherein the absence of RNA is predictive of the developmental disability.

[0051] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0053] FIG. 1 shows a representative wild-type TRAPPC9 nucleic acid sequence (SEQ ID NO. 1) encoding NIBP;

[0054] FIG. 2 shows a representative wild-type NIBP protein sequence (SEQ ID NO. 2) encoded by TRAPPC9;

[0055] FIG. 3 shows a representative TRAPPC9 nucleic acid sequence (SEQ ID NO. 3) encoding a truncated NIBP protein; and

[0056] FIG. 4 shows a representative nucleic acid sequence (SEQ ID NO. 4) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The following description is of a preferred embodiment.

[0058] A truncating mutation in the TRAPPC9 gene on chromosome 8q24 has been identified that associates with the developmental disabilities: non-syndromic autosomal recessive mental retardation (NS-ARMR) and autism.

[0059] TRAPPC9 is a 23 exon gene that encodes a 1246 amino acid protein, NIK and IKKβ binding protein (NIBP). This protein is expressed at high levels in the muscle and kidney, and to a lesser extent in the brain, heart and placenta (Hu et al, 2005). Only isoform 1 of the gene is present in the brain (Hu et al, 2005). NIBP is involved in the NF-Kappa-β signaling pathway, and directly interacts with IKKβ and MAP3K14 (Hu et al, 2005). It is likely involved in both classical and alternative activation of the NF-Kappa-β signalling pathway (Hu et al, 2005). It potentially plays a role in neuronal differentiation, but this requires further investigation (Hu et al, 2005). It is expressed in the cell bodies and processes of neurons (Hu et al, 2005). NIBP contains one known conserved region originally identified in Saccharomyces cerevisiae called Trs120 (pfam08626; Sacher et al, 2000). It is known to function in ER to Golgi traffic (Sacher et al, 2000).

[0060] One mutation that causes the truncation of the NIBP protein resides at position 1438 of the nucleic acid sequence shown in SEQ ID NO. 1 (FIG. 1). The single nucleotide polymorphism at this position results in a thymidine (T), as shown in SEQ ID NO. 3 (FIG. 3) instead of the wild-type cytosine (C) ("the C allele"). For the purposes of present invention, this polymorphism will be referred to as the C1438T polymorphism. However, it is to be noted that the polymorphism is also termed C1423T in Mir et al., 2009 (The American Journal of Human Genetics 85, 1-7, December 11 which is hereby incorporated by reference) due to differences in numbering conventions when counting from the coding sequence of GenBank Accession number NM_--031466. The presence of T at position 1438 of SEQ ID NO. 3 ("the T allele"), results in a termination codon, instead of the wild-type arginine in the amino acid sequence shown by SEQ ID NO. 2 (FIG. 2). The resulting truncated protein comprises 475 amino acids compared to the full-length protein, which is made up of 1246 amino acids.

[0061] It has been found that the T allele is inherited in an autosomal recessive manner. As a result, individuals heterozygous for the allele may be carriers of the allele without having the phenotype of the disorder. Identifying and counselling these individuals may limit or prevent the possible transmission of the recessive genotype onto offspring.

[0062] The sample obtained from a subject may comprise any biological sample from which genomic DNA may be isolated, for example, but not to be limited to a tissue sample, a sample of saliva, a cheek swab sample, blood, or other biological fluids that contain genomic DNA. In a preferred embodiment, which is not meant to be limiting in any manner, the sample is a blood sample. In another embodiment, RNA or mRNA is isolated from the subject.

[0063] The method of obtaining and analyzing DNA or RNA is not critical to the present invention and any method or methods may be used (e.g. Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3, or Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, p. 387-389). For example, which is not to be considered limiting in any manner, DNA may be extracted using a non-enzymatic high-salt procedure (Lahiri and Nurnberger 1991). Alternatively, the DNA may be analyzed in situ. RNA can isolated, for example, by phenol chloroform extraction and analyzed using RT-PCR.

[0064] Genotyping of the C1438T marker (or any of the other markers as described herein) may be performed by any method known in the art, for example PCR, sequencing, ligation chain reaction (LCR) or any other standard method known in the art that may be used to determine SNPs (single nucleic acid polymorphisms). In an embodiment, which is not meant to be limiting in any manner, amplifying the nucleic acid sequence containing the C1438T marker and genotyping the same is performed by PCR analysis using appropriate primers, probes and PCR conditions.

[0065] In one embodiment, the step of amplifying the sequence containing the C1438T marker involves subjecting the nucleic acid sample to PCR, wherein the program for denaturing, annealing, amplifying is stored on a computer readable medium for execution by a microprocessor. The program causes a machine containing the samples to cycle through various temperatures for set periods of time. A similar or different machine comprising one or more programs may be employed to convert physical information, for example, but not limited to binding of nucleic acids or probes to target sequences, amplification or the like to a different state, such as electronic or otherwise, for example a signal that can be printed, displayed pictorially or digitized.

[0066] In a further embodiment, the restriction enzyme Taq I is used to detect the presence of the T allele at C1438T marker. Taq I recognizes the consensus sequence, T C G A, which corresponds to the wild-type sequence of the TRAPPC9 gene in the area of the polymorphism. The T allele will disrupt this consensus sequence and Taq I will not be able to cut the sequence. As such Taq I (or an isoschizomer of TaqI, or other restriction enzyme recognizing this or the complementary wild-type or mutated sequence) can be used to easily determine the genotype of the subject. Other methods also may be used.

[0067] An apparatus, such as microarray or DNA chip, can be used to detect the presence or absence of the C1438T marker or any other nucleic acid which results in a truncated NIBP protein or mutated NIBP protein as described herein. In this case, but without wishing to be limiting in any manner, an oligonucleotide may be bound to a substrate, which is suitable for this type of application. In an embodiment the oligonucleotide preferably comprises a contiguous nucleic acid, for example, the sequence from SEQ ID NO. 3 (FIG. 3) containing position 1438 of the sequence or a sequence substantially identical thereto. Another oligonucleotide can also be bound to the substrate. For example, but not wishing to be limiting, a nucleotide sequence comprising a complement of the nucleic acid sequence from SEQ ID NO:3 containing position 1438 of the sequence may be employed. In a further embodiment, the oligonucleotide comprises a contiguous nucleic acid sequence from SEQ ID NO. 1 containing position 1438 of the sequence, or a complement thereof or a sequence substantially identical thereto. In one embodiment the oligonucleotides are 7, 10, 12, 15, 16, 17, 19, 21, 23, 25 or more nucleotides in length. In another embodiment, the oligonucleotides are 60 nucleotides in length or more. Alternatively, the oligonucleotides may be defined by a range of any two of the values noted above or any two values therein between. A person skilled in the art will recognize that the length of the oligonucleotides can be altered based on the parameters of the assay. It is envisaged that the apparatus can contain other oligonucleotide sequences to confirm the subject's susceptibility to the developmental disability or to test for the susceptibility of additional diseases or disorders, comorbid or otherwise.

[0068] As noted above, the C1438T marker in TRAPPC9 gene, results in a truncation of the NIBP protein. In a separate study, sequence analysis also identified a 4 base pair deletion resulting in a frameshift and premature truncation: pLeu772TrpfsX7 in exon 14. This observation provides a unique opportunity to use the difference in protein length, between the wildtype and the truncation proteins, to predicted the susceptibility of a subject to a developmental disability.

[0069] As such, the present invention also contemplates screening methods which identify and/or characterize the proteins as defined above within biological samples from subjects. Such samples may or may not comprise DNA or RNA. For example, such screening or testing methods may employ immunological methods, for example, but not limited to antibody binding assays such as ELISAs or the like, protein sequencing, electrophoretic separations to identify the proteins as described above in a sample. As will be evident to a person of skill in the art, the screening methods allow for the differentiation of the proteins as defined herein from wild type proteins known in the art.

[0070] Also contemplated by the present invention is a nucleic acid comprising or consisting of a sequence selected from the group consisting of: a) a nucleic acid sequence comprising SEQ ID NO. 4 (FIG. 4); b) a complement of a nucleic acid sequence comprising SEQ ID NO. 4; c) a fragment of either a) or b); d) a nucleic acid sequence capable of hybridizing to any one of a), b) or c); and e) a nucleic acid sequence that exhibits greater than about 70% sequence identity with the nucleic acid defined in a), b) or c).

[0071] A nucleic acid sequence exhibiting at least 70% identity thereto is understood to include sequences that exhibit 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identity, or an value therein between to SEQ ID NO. 4. Further, the nucleic acid may be defined as comprising a range of sequence identity as defined by any two of the values listed or any values therein between.

[0072] Any method known in the art may be used for determining the degree of identity between nucleic acid sequences. For example, but without wishing to be limiting, a sequence search method such as BLAST (Basic Local Alignment Search Tool: (Altschul S F, Gish W, Miller W, Myers E W, Lipman D J (1990) J Mol Biol 215: 403-410) can be used according to default parameters as described by Tatiana et al., FEMS Microbiol Lett. 174:247-250 (1990), or on the National Center for Biotechnology Information web page at ncbi.nlm.gov/BLAST/, for searching closely related sequences. BLAST is widely used in routine sequence alignment; modified BLAST algorithms such as Gapped BLAST, which allows gaps (either insertions or deletions) to be introduced into alignments, PSI-BLAST, a sensitive search for sequence homologs (Altschul et al., (1997) Nucleic Acid Res. 25:3389-3402); or FASTA, which is available on the world wide web at ExPASy (EMBL-European Bioinformatics Institute). Similar methods known in the art may be employed to compare DNA or RNA sequences to determine the degree of sequence identity.

[0073] Stringent hybridization conditions may be, for example but not limited to hybridization overnight (from about 16-20 hours) hybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours); or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO₄ buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each for unique sequence regions.

[0074] Also contemplated by the present invention is a method of predicting susceptibility to a developmental disability in a human subject, comprising the steps of:isolating RNA from the subject;hybridizing an oligonucleotide comprising a contiguous nucleic acid of SEQ ID NO. 1 to the RNA; wherein the absence of RNA complementary to the oligonucleotide is predictive of the developmental disability.

[0075] The presence of the T allele at position 1438 of SEQ ID NO. 4 is believed to lead to nonsense-mediated RNA decay, and hence reduction or total loss of the mRNA. This presents a unique opportunity to detect the presence of TRAPPC9 mRNA in a subject for the purposes of predicting the susceptibility of the subject to a developmental disorder.

[0076] The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

EXAMPLE 1

[0077] The family in which the C1438T mutation was identified, MR-2019, is a large family with multiple incidences of consanguinity between first cousins. We obtained the DNA of 8 affected and 12 unaffected family members.

[0078] Affymetrix 5.0 SNP microarray analysis was conducted to identify genetic differences between the affected and unaffected family members. The results were analyzed with dChip, using homozygosity mapping as a basis for identifying regions of susceptibility. A 3.2 Mb region of autozygosity was identified in the family at locus 8q24, from 139,465,102-142,726,810 (UCSC March 2006 Build), consisting of a run of 606 consecutive homozygous SNPs. This region overlaps with a 6.8 Mb locus identified in an Iranian NS-ARMR pedigree (Najmabadi et al, 2007).

[0079] The 3.2 Mb region contains 12 genes, only one of which has been previously implicated in MR. This gene, KCNK9 (MIM 605874), has been shown to be causal in recently identified Birk-Barel Syndrome (Barel et al, 2008) (MIM 612292). KCNK9 was sequenced and found to be normal in the family used in this example.

[0080] Additional genes in the region were sequenced that appeared to be good candidates for MR based on functional and expression data obtained from the UCSC database. An expression-based algorithm was used to identify genes in our region that co-expressed with known causal MR genes (genome.ucla.edu/projects/UGET). A mutation in TRAPPC9 was identified. The mutation, at C1438T causes the gene to be truncated at the end of its 7th exon. Each of the 8 affected individuals were homozygous for the T allele, whereas all of the 12 unaffected individuals were either homozygous for the C allele or carriers of the T allele.

[0081] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

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[0094] Molinari, F.; Rio, M.; Meskenaite, V.; Encha-Razavi, F.; Auge, J.; Bacq, D.; Briault, S.; Vekemans, M.; Munnich, A.; Attie-Bitach, T.; Sonderegger, P.; Colleaux, L. (2002). Truncating neurotrypsin mutation in autosomal recessive nonsyndromic mental retardation. Science 298:1779-1781.

[0095] Morrow E M, Yoo S Y, Flavell S W, Kim T K, Lin Y, Hill R S, Mukaddes N M, Balkhy S, Gascon G, Hashmi A, Al-Saad S, Ware J, Joseph R M, Greenblatt R, Gleason D, Ertelt J A, Apse K A, Bodell A, Partlow J N, Barry B, Yao H, Markianos K, Ferland R J, Greenberg M E, Walsh C A (2008) Identifying autism loci and genes by tracing recent shared ancestry. Science.321:218-223.

[0096] Motazacker M M, Rost B R, Hucho T, Garshasbi M, Kahrizi K, Ullmann R, Abedini S S, Nieh S E, Amini S H, Goswami C, Tzschach A, Jensen L R, Schmitz D, Ropers H H, Najmabadi H, Kuss A W. (2007) A defect in the ionotropic glutamate receptor 6 gene (GRIK2) is associated with autosomal recessive mental retardation. Am J Hum Genet 81:792-798.

[0097] Najmabadi H, Motazacker M M, Garshasbi M, Kahrizi K, Tzschach A, Chen W, Behjati F, Hadavi V, Nieh S E, Abedini S S, Vazifehmand R, Firouzabadi S G, Jamali P, Falah M, Seifati S M, Gruters A, Lenzner S, Jensen L R, Ruschendorf F, Kuss A W, Ropers H H: (2007) Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci. Hum Genet 121:43-48

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[0100] Sutcliffe J S (2008) Genetics. Insights into the pathogenesis of autism. Science 321:208-209

Sequence CWU 1

1

414472DNAHomo Sapiens 1aaagtcggga gtgccatggt gccagctggg gatcaagacc gcgcgccaca cagggggaag 60ccggcccagg ctggggctcg cacctcacgt gcctcccggg ccctgcgatc ctggaggcgc 120tcccaggccg cgcgcgccac ggtcacccac ccacgtgggg ggcacgaccg tgggagtcac 180ggggggtacc gtgagggtca cagggggtgc cgcagggatc cacagtgggc ttccgcgggg 240cctccacccc tgagcttcac agaggaagtg aaatttgagc tgcgcgccct gaaggactgg 300gacttcaaaa tgagcgtccc tgactacatg cagtgtgctg aggaccacca gacgctgctc 360gtggtggtcc agcctgtggg catcgtctcc gaggagaact tcttcaggat ctataagagg 420atttgctctg tgagtcagat cagcgtgcgg gactcccagc gagtcctcta catccgctac 480aggcaccact acccacccga gaacaacgag tggggtgact tccagaccca ccgcaaagtc 540gtgggcctca tcaccatcac agactgcttc tcggccaagg actggccaca gacctttgag 600aagttccacg tgcagaagga gatctacggc tccacactgt atgactcccg gctctttgtc 660ttcgggctgc agggggagat cgtggagcag ccgcgcaccg acgtggcttt ctaccccaac 720tacgaggact gccagacggt ggagaagaga atcgaggact tcatcgagtc actgttcatc 780gtgctggagt ccaagcgtct ggacagagcc acagacaagt ctggggataa gatccccctt 840ctctgtgtcc cgtttgagaa aaaggacttt gtaggactgg acacagacag cagacattac 900aagaagcggt gccaaggccg catgcggaag cacgtggggg acctgtgcct gcaggcaggg 960atgctgcagg actccctggt gcattaccac atgtcggtgg agctgctgcg ttctgtgaat 1020gactttctgt ggcttggagc tgccctggaa ggattgtgtt cagcttctgt catctatcac 1080tatcctggtg gaactggtgg gaagagtgga gctcggaggt tccagggcag cacccttcct 1140gctgaagcag ccaatagaca ccggccaggg gcacaggaag ttctcattga tccaggtgcc 1200ctcaccacca atggcatcaa ccctgacacc agtactgaga tcggacgtgc taagaactgc 1260cttagccctg aagacataat tgacaagtat aaagaggcga tttcctatta cagcaagtat 1320aagaatgcgg gagtgattga gttggaagcg tgcatcaagg ctgtacgtgt ccttgcaatt 1380cagaaacgga gcatggaagc atcagaattt cttcagaatg cagtttacat taaccttcga 1440cagctttctg aggaagagaa aattcagcgc tacagcatcc tctccgagct ctatgagctg 1500atcggcttcc atcgcaagtc tgcgttcttc aagcgcgtgg ccgccatgca gtgcgtggcc 1560ccaagcatcg cggagcctgg gtggagggcc tgctacaaac tcctcctgga aacgctgccc 1620ggctacagtc tgtcgctgga tcccaaagat ttcagcagag gcacgcacag aggctgggct 1680gcggtccaga tgcgtttgct ccatgaattg gtctacgcct cccgaaggat ggggaaccct 1740gccctctctg tcagacacct gtccttcctt ctacagacca tgctggactt cttgtcggat 1800caggaaaaga aagatgtggc ccaaagccta gagaactata cgtccaagtg tcctgggacc 1860atggagccca tcgccctccc tggcggcctc accctgccac cggtgccctt caccaagctt 1920cccgtcgtca ggcatgtgaa actattgaac cttcctgcta gcctccggcc acacaaaatg 1980aaaagcttgc tgggtcagaa cgtgtcaacc aaaagtcctt tcatctattc accaattatc 2040gcacacaacc gtggagaaga gcggaacaag aaaatagatt tccagtgggt tcaaggagat 2100gtgtgtgaag ttcagctgat ggtatataac ccaatgccgt ttgaacttcg agttgaaaac 2160atggggctgc tcaccagcgg agtggagttc gagtctctcc ctgcggcgct ttctcttccg 2220gctgaatctg gtctgtaccc agtgacgctc gtcggggtcc cgcagacgac tggaacgatt 2280actgtgaacg gttaccatac cacggtcttc ggtgtgttca gtgactgttt gctggataac 2340ctgccgggaa taaaaaccag tggctccaca gtggaagtca ttcccgcgtt gccaagactg 2400cagatcagca cctctctgcc cagatctgca cattcattgc aaccttcttc tggtgatgaa 2460atatctacta atgtatctgt ccagctttac aatggagaaa gtcagcaact aatcattaaa 2520ttggaaaata ttggaatgga accattggag aaactggagg tcacctcgaa agttctcacc 2580actaaagaaa aattgtatgg cgacttcttg agctggaagc tagaggaaac ccttgcccag 2640ttccctttgc agcctgggaa ggtggccacg ttcacaatca acatcaaagt gaagctggat 2700ttctcctgcc aggagaatct cctgcaggat ctcagtgatg atggaatcag tgtgagtggc 2760tttcccctgt ccagtccttt tcggcaggtc gttcggcccc gagtggaggg caaacctgtg 2820aacccacccg agagcaacaa agcaggcgac tacagccacg tgaagaccct ggaagctgtc 2880ctgaatttca aatactctgg aggcccgggc cacactgaag gatattacag gaatctctcc 2940ctggggctgc atgtagaagt cgagccgtct gtatttttca cccgagtcag caccctccca 3000gcaaccagta cccggcagtg tcacctgctc ctggatgtct tcaactccac cgagcatgag 3060ctgaccgtca gcaccaggag cagcgaggca ctcatcctgc acgccggcga gtgccagcga 3120atggctattc aagtggacaa gttcaacttt gagagtttcc cggagtcccc tggggagaag 3180gggcaatttg caaaccccaa gcagctggag gaagagcggc gggaagcccg aggcctggag 3240atccacagca agctgggcat ctgctggaga atcccctccc tgaagcgcag tggcgaggcg 3300agtgtggaag gactcctgaa ccagctcgtc ctggagcacc tgcagctggc gcctctgcag 3360tgggatgtgc tggtggacgg acagccatgt gaccgcgagg ctgtggcggc ctgccaggtg 3420ggcgaccccg tgcgcctgga ggtgcggctg accaaccgga gcccgcgcag cgtagggccc 3480ttcgccctca ctgtggtccc cttccaggac caccagaacg gcgtgcacaa ctacgacctg 3540cacgacaccg tctccttcgt gggctccagc accttctacc tcgacgcggt gcagccgtcc 3600ggccagtcgg cctgcctcgg ggccctcctc ttcctctaca cgggagactt cttcctccac 3660atccggttcc acgaggacag caccagcaag gagctgccac cctcttggtt ctgcctgccc 3720agtgtgcacg tgtgtgccct ggaggcgcag gcctgagccc gcctacttcc gtccctcttt 3780ctgcagggcc agaggtgacc ctgcctggcc tcccacaccc cctgcaatga gcaaggcctt 3840cactgcagcc ccatctcctc ctcctccccc agacccctcc cagccctctc ctcctgttcc 3900tcctgtagca tctttgctgg gctacgcaga agccccggac atggcagccc caccccatgc 3960cacgcccctt cctacactgt tccctggacc atacacaggc tgaagcagag gaaatcccaa 4020agcgggtgcc catccagccc aggtcccagg atccctgcac ccatttctgt gacctggggc 4080cccagccgtg ctgtgctgct catcccagca gagggacctc cctcgtccag cgacttccct 4140ttggccatag aaagaaatgg tgagcatgag actgggcaca gcctgagggc gtgggcagct 4200tcccaccctc cctgggcctt ggaatccccc aaggctggtt ttcttcctgg agacccccat 4260gggcaacttg gcaggagaga tggtgccgta ggaggtcgtg gatggttgat gccaagagag 4320gccctccacc cgtggtgggc aaatgtccag gcctgggctg gcagcccagg gctgtttctg 4380ggtgctccct ggccccaggg tggcgtctgg ttaccatggc tgtgtgtgtc catgtctgca 4440agcagttctt caataaatgg cctgcctccc cc 447221246PRTHomo Sapiens 2Met Val Pro Ala Gly Asp Gln Asp Arg Ala Pro His Arg Gly Lys Pro 1 5 10 15 Ala Gln Ala Gly Ala Arg Thr Ser Arg Ala Ser Arg Ala Leu Arg Ser 20 25 30 Trp Arg Arg Ser Gln Ala Ala Arg Ala Thr Val Thr His Pro Arg Gly 35 40 45 Gly His Asp Arg Gly Ser His Gly Gly Tyr Arg Glu Gly His Arg Gly 50 55 60 Cys Arg Arg Asp Pro Gln Trp Ala Ser Ala Gly Pro Pro Pro Leu Ser 65 70 75 80 Phe Thr Glu Glu Val Lys Phe Glu Leu Arg Ala Leu Lys Asp Trp Asp 85 90 95 Phe Lys Met Ser Val Pro Asp Tyr Met Gln Cys Ala Glu Asp His Gln 100 105 110 Thr Leu Leu Val Val Val Gln Pro Val Gly Ile Val Ser Glu Glu Asn 115 120 125 Phe Phe Arg Ile Tyr Lys Arg Ile Cys Ser Val Ser Gln Ile Ser Val 130 135 140 Arg Asp Ser Gln Arg Val Leu Tyr Ile Arg Tyr Arg His His Tyr Pro 145 150 155 160 Pro Glu Asn Asn Glu Trp Gly Asp Phe Gln Thr His Arg Lys Val Val 165 170 175 Gly Leu Ile Thr Ile Thr Asp Cys Phe Ser Ala Lys Asp Trp Pro Gln 180 185 190 Thr Phe Glu Lys Phe His Val Gln Lys Glu Ile Tyr Gly Ser Thr Leu 195 200 205 Tyr Asp Ser Arg Leu Phe Val Phe Gly Leu Gln Gly Glu Ile Val Glu 210 215 220 Gln Pro Arg Thr Asp Val Ala Phe Tyr Pro Asn Tyr Glu Asp Cys Gln 225 230 235 240 Thr Val Glu Lys Arg Ile Glu Asp Phe Ile Glu Ser Leu Phe Ile Val 245 250 255 Leu Glu Ser Lys Arg Leu Asp Arg Ala Thr Asp Lys Ser Gly Asp Lys 260 265 270 Ile Pro Leu Leu Cys Val Pro Phe Glu Lys Lys Asp Phe Val Gly Leu 275 280 285 Asp Thr Asp Ser Arg His Tyr Lys Lys Arg Cys Gln Gly Arg Met Arg 290 295 300 Lys His Val Gly Asp Leu Cys Leu Gln Ala Gly Met Leu Gln Asp Ser 305 310 315 320 Leu Val His Tyr His Met Ser Val Glu Leu Leu Arg Ser Val Asn Asp 325 330 335 Phe Leu Trp Leu Gly Ala Ala Leu Glu Gly Leu Cys Ser Ala Ser Val 340 345 350 Ile Tyr His Tyr Pro Gly Gly Thr Gly Gly Lys Ser Gly Ala Arg Arg 355 360 365 Phe Gln Gly Ser Thr Leu Pro Ala Glu Ala Ala Asn Arg His Arg Pro 370 375 380 Gly Ala Gln Glu Val Leu Ile Asp Pro Gly Ala Leu Thr Thr Asn Gly 385 390 395 400 Ile Asn Pro Asp Thr Ser Thr Glu Ile Gly Arg Ala Lys Asn Cys Leu 405 410 415 Ser Pro Glu Asp Ile Ile Asp Lys Tyr Lys Glu Ala Ile Ser Tyr Tyr 420 425 430 Ser Lys Tyr Lys Asn Ala Gly Val Ile Glu Leu Glu Ala Cys Ile Lys 435 440 445 Ala Val Arg Val Leu Ala Ile Gln Lys Arg Ser Met Glu Ala Ser Glu 450 455 460 Phe Leu Gln Asn Ala Val Tyr Ile Asn Leu Arg Gln Leu Ser Glu Glu 465 470 475 480 Glu Lys Ile Gln Arg Tyr Ser Ile Leu Ser Glu Leu Tyr Glu Leu Ile 485 490 495 Gly Phe His Arg Lys Ser Ala Phe Phe Lys Arg Val Ala Ala Met Gln 500 505 510 Cys Val Ala Pro Ser Ile Ala Glu Pro Gly Trp Arg Ala Cys Tyr Lys 515 520 525 Leu Leu Leu Glu Thr Leu Pro Gly Tyr Ser Leu Ser Leu Asp Pro Lys 530 535 540 Asp Phe Ser Arg Gly Thr His Arg Gly Trp Ala Ala Val Gln Met Arg 545 550 555 560 Leu Leu His Glu Leu Val Tyr Ala Ser Arg Arg Met Gly Asn Pro Ala 565 570 575 Leu Ser Val Arg His Leu Ser Phe Leu Leu Gln Thr Met Leu Asp Phe 580 585 590 Leu Ser Asp Gln Glu Lys Lys Asp Val Ala Gln Ser Leu Glu Asn Tyr 595 600 605 Thr Ser Lys Cys Pro Gly Thr Met Glu Pro Ile Ala Leu Pro Gly Gly 610 615 620 Leu Thr Leu Pro Pro Val Pro Phe Thr Lys Leu Pro Val Val Arg His 625 630 635 640 Val Lys Leu Leu Asn Leu Pro Ala Ser Leu Arg Pro His Lys Met Lys 645 650 655 Ser Leu Leu Gly Gln Asn Val Ser Thr Lys Ser Pro Phe Ile Tyr Ser 660 665 670 Pro Ile Ile Ala His Asn Arg Gly Glu Glu Arg Asn Lys Lys Ile Asp 675 680 685 Phe Gln Trp Val Gln Gly Asp Val Cys Glu Val Gln Leu Met Val Tyr 690 695 700 Asn Pro Met Pro Phe Glu Leu Arg Val Glu Asn Met Gly Leu Leu Thr 705 710 715 720 Ser Gly Val Glu Phe Glu Ser Leu Pro Ala Ala Leu Ser Leu Pro Ala 725 730 735 Glu Ser Gly Leu Tyr Pro Val Thr Leu Val Gly Val Pro Gln Thr Thr 740 745 750 Gly Thr Ile Thr Val Asn Gly Tyr His Thr Thr Val Phe Gly Val Phe 755 760 765 Ser Asp Cys Leu Leu Asp Asn Leu Pro Gly Ile Lys Thr Ser Gly Ser 770 775 780 Thr Val Glu Val Ile Pro Ala Leu Pro Arg Leu Gln Ile Ser Thr Ser 785 790 795 800 Leu Pro Arg Ser Ala His Ser Leu Gln Pro Ser Ser Gly Asp Glu Ile 805 810 815 Ser Thr Asn Val Ser Val Gln Leu Tyr Asn Gly Glu Ser Gln Gln Leu 820 825 830 Ile Ile Lys Leu Glu Asn Ile Gly Met Glu Pro Leu Glu Lys Leu Glu 835 840 845 Val Thr Ser Lys Val Leu Thr Thr Lys Glu Lys Leu Tyr Gly Asp Phe 850 855 860 Leu Ser Trp Lys Leu Glu Glu Thr Leu Ala Gln Phe Pro Leu Gln Pro 865 870 875 880 Gly Lys Val Ala Thr Phe Thr Ile Asn Ile Lys Val Lys Leu Asp Phe 885 890 895 Ser Cys Gln Glu Asn Leu Leu Gln Asp Leu Ser Asp Asp Gly Ile Ser 900 905 910 Val Ser Gly Phe Pro Leu Ser Ser Pro Phe Arg Gln Val Val Arg Pro 915 920 925 Arg Val Glu Gly Lys Pro Val Asn Pro Pro Glu Ser Asn Lys Ala Gly 930 935 940 Asp Tyr Ser His Val Lys Thr Leu Glu Ala Val Leu Asn Phe Lys Tyr 945 950 955 960 Ser Gly Gly Pro Gly His Thr Glu Gly Tyr Tyr Arg Asn Leu Ser Leu 965 970 975 Gly Leu His Val Glu Val Glu Pro Ser Val Phe Phe Thr Arg Val Ser 980 985 990 Thr Leu Pro Ala Thr Ser Thr Arg Gln Cys His Leu Leu Leu Asp Val 995 1000 1005 Phe Asn Ser Thr Glu His Glu Leu Thr Val Ser Thr Arg Ser Ser 1010 1015 1020 Glu Ala Leu Ile Leu His Ala Gly Glu Cys Gln Arg Met Ala Ile 1025 1030 1035 Gln Val Asp Lys Phe Asn Phe Glu Ser Phe Pro Glu Ser Pro Gly 1040 1045 1050 Glu Lys Gly Gln Phe Ala Asn Pro Lys Gln Leu Glu Glu Glu Arg 1055 1060 1065 Arg Glu Ala Arg Gly Leu Glu Ile His Ser Lys Leu Gly Ile Cys 1070 1075 1080 Trp Arg Ile Pro Ser Leu Lys Arg Ser Gly Glu Ala Ser Val Glu 1085 1090 1095 Gly Leu Leu Asn Gln Leu Val Leu Glu His Leu Gln Leu Ala Pro 1100 1105 1110 Leu Gln Trp Asp Val Leu Val Asp Gly Gln Pro Cys Asp Arg Glu 1115 1120 1125 Ala Val Ala Ala Cys Gln Val Gly Asp Pro Val Arg Leu Glu Val 1130 1135 1140 Arg Leu Thr Asn Arg Ser Pro Arg Ser Val Gly Pro Phe Ala Leu 1145 1150 1155 Thr Val Val Pro Phe Gln Asp His Gln Asn Gly Val His Asn Tyr 1160 1165 1170 Asp Leu His Asp Thr Val Ser Phe Val Gly Ser Ser Thr Phe Tyr 1175 1180 1185 Leu Asp Ala Val Gln Pro Ser Gly Gln Ser Ala Cys Leu Gly Ala 1190 1195 1200 Leu Leu Phe Leu Tyr Thr Gly Asp Phe Phe Leu His Ile Arg Phe 1205 1210 1215 His Glu Asp Ser Thr Ser Lys Glu Leu Pro Pro Ser Trp Phe Cys 1220 1225 1230 Leu Pro Ser Val His Val Cys Ala Leu Glu Ala Gln Ala 1235 1240 1245 34472DNAArtificial SequenceRepresentative TRAPPC9 nucleic acid sequence encoding a truncated NIBP protein 3aaagtcggga gtgccatggt gccagctggg gatcaagacc gcgcgccaca cagggggaag 60ccggcccagg ctggggctcg cacctcacgt gcctcccggg ccctgcgatc ctggaggcgc 120tcccaggccg cgcgcgccac ggtcacccac ccacgtgggg ggcacgaccg tgggagtcac 180ggggggtacc gtgagggtca cagggggtgc cgcagggatc cacagtgggc ttccgcgggg 240cctccacccc tgagcttcac agaggaagtg aaatttgagc tgcgcgccct gaaggactgg 300gacttcaaaa tgagcgtccc tgactacatg cagtgtgctg aggaccacca gacgctgctc 360gtggtggtcc agcctgtggg catcgtctcc gaggagaact tcttcaggat ctataagagg 420atttgctctg tgagtcagat cagcgtgcgg gactcccagc gagtcctcta catccgctac 480aggcaccact acccacccga gaacaacgag tggggtgact tccagaccca ccgcaaagtc 540gtgggcctca tcaccatcac agactgcttc tcggccaagg actggccaca gacctttgag 600aagttccacg tgcagaagga gatctacggc tccacactgt atgactcccg gctctttgtc 660ttcgggctgc agggggagat cgtggagcag ccgcgcaccg acgtggcttt ctaccccaac 720tacgaggact gccagacggt ggagaagaga atcgaggact tcatcgagtc actgttcatc 780gtgctggagt ccaagcgtct ggacagagcc acagacaagt ctggggataa gatccccctt 840ctctgtgtcc cgtttgagaa aaaggacttt gtaggactgg acacagacag cagacattac 900aagaagcggt gccaaggccg catgcggaag cacgtggggg acctgtgcct gcaggcaggg 960atgctgcagg actccctggt gcattaccac atgtcggtgg agctgctgcg ttctgtgaat 1020gactttctgt ggcttggagc tgccctggaa ggattgtgtt cagcttctgt catctatcac 1080tatcctggtg gaactggtgg gaagagtgga gctcggaggt tccagggcag cacccttcct 1140gctgaagcag ccaatagaca ccggccaggg gcacaggaag ttctcattga tccaggtgcc 1200ctcaccacca atggcatcaa ccctgacacc agtactgaga tcggacgtgc taagaactgc 1260cttagccctg aagacataat tgacaagtat aaagaggcga tttcctatta cagcaagtat 1320aagaatgcgg gagtgattga gttggaagcg tgcatcaagg ctgtacgtgt ccttgcaatt 1380cagaaacgga gcatggaagc atcagaattt cttcagaatg cagtttacat taacctttga 1440cagctttctg aggaagagaa aattcagcgc tacagcatcc tctccgagct ctatgagctg 1500atcggcttcc atcgcaagtc tgcgttcttc aagcgcgtgg ccgccatgca gtgcgtggcc 1560ccaagcatcg cggagcctgg gtggagggcc tgctacaaac tcctcctgga aacgctgccc 1620ggctacagtc tgtcgctgga tcccaaagat ttcagcagag gcacgcacag aggctgggct 1680gcggtccaga tgcgtttgct ccatgaattg gtctacgcct cccgaaggat ggggaaccct 1740gccctctctg tcagacacct gtccttcctt ctacagacca tgctggactt cttgtcggat 1800caggaaaaga aagatgtggc ccaaagccta gagaactata cgtccaagtg tcctgggacc 1860atggagccca tcgccctccc tggcggcctc accctgccac cggtgccctt caccaagctt 1920cccgtcgtca ggcatgtgaa actattgaac cttcctgcta gcctccggcc acacaaaatg 1980aaaagcttgc tgggtcagaa cgtgtcaacc aaaagtcctt tcatctattc accaattatc 2040gcacacaacc gtggagaaga gcggaacaag aaaatagatt tccagtgggt tcaaggagat 2100gtgtgtgaag ttcagctgat ggtatataac ccaatgccgt ttgaacttcg agttgaaaac 2160atggggctgc tcaccagcgg agtggagttc gagtctctcc

ctgcggcgct ttctcttccg 2220gctgaatctg gtctgtaccc agtgacgctc gtcggggtcc cgcagacgac tggaacgatt 2280actgtgaacg gttaccatac cacggtcttc ggtgtgttca gtgactgttt gctggataac 2340ctgccgggaa taaaaaccag tggctccaca gtggaagtca ttcccgcgtt gccaagactg 2400cagatcagca cctctctgcc cagatctgca cattcattgc aaccttcttc tggtgatgaa 2460atatctacta atgtatctgt ccagctttac aatggagaaa gtcagcaact aatcattaaa 2520ttggaaaata ttggaatgga accattggag aaactggagg tcacctcgaa agttctcacc 2580actaaagaaa aattgtatgg cgacttcttg agctggaagc tagaggaaac ccttgcccag 2640ttccctttgc agcctgggaa ggtggccacg ttcacaatca acatcaaagt gaagctggat 2700ttctcctgcc aggagaatct cctgcaggat ctcagtgatg atggaatcag tgtgagtggc 2760tttcccctgt ccagtccttt tcggcaggtc gttcggcccc gagtggaggg caaacctgtg 2820aacccacccg agagcaacaa agcaggcgac tacagccacg tgaagaccct ggaagctgtc 2880ctgaatttca aatactctgg aggcccgggc cacactgaag gatattacag gaatctctcc 2940ctggggctgc atgtagaagt cgagccgtct gtatttttca cccgagtcag caccctccca 3000gcaaccagta cccggcagtg tcacctgctc ctggatgtct tcaactccac cgagcatgag 3060ctgaccgtca gcaccaggag cagcgaggca ctcatcctgc acgccggcga gtgccagcga 3120atggctattc aagtggacaa gttcaacttt gagagtttcc cggagtcccc tggggagaag 3180gggcaatttg caaaccccaa gcagctggag gaagagcggc gggaagcccg aggcctggag 3240atccacagca agctgggcat ctgctggaga atcccctccc tgaagcgcag tggcgaggcg 3300agtgtggaag gactcctgaa ccagctcgtc ctggagcacc tgcagctggc gcctctgcag 3360tgggatgtgc tggtggacgg acagccatgt gaccgcgagg ctgtggcggc ctgccaggtg 3420ggcgaccccg tgcgcctgga ggtgcggctg accaaccgga gcccgcgcag cgtagggccc 3480ttcgccctca ctgtggtccc cttccaggac caccagaacg gcgtgcacaa ctacgacctg 3540cacgacaccg tctccttcgt gggctccagc accttctacc tcgacgcggt gcagccgtcc 3600ggccagtcgg cctgcctcgg ggccctcctc ttcctctaca cgggagactt cttcctccac 3660atccggttcc acgaggacag caccagcaag gagctgccac cctcttggtt ctgcctgccc 3720agtgtgcacg tgtgtgccct ggaggcgcag gcctgagccc gcctacttcc gtccctcttt 3780ctgcagggcc agaggtgacc ctgcctggcc tcccacaccc cctgcaatga gcaaggcctt 3840cactgcagcc ccatctcctc ctcctccccc agacccctcc cagccctctc ctcctgttcc 3900tcctgtagca tctttgctgg gctacgcaga agccccggac atggcagccc caccccatgc 3960cacgcccctt cctacactgt tccctggacc atacacaggc tgaagcagag gaaatcccaa 4020agcgggtgcc catccagccc aggtcccagg atccctgcac ccatttctgt gacctggggc 4080cccagccgtg ctgtgctgct catcccagca gagggacctc cctcgtccag cgacttccct 4140ttggccatag aaagaaatgg tgagcatgag actgggcaca gcctgagggc gtgggcagct 4200tcccaccctc cctgggcctt ggaatccccc aaggctggtt ttcttcctgg agacccccat 4260gggcaacttg gcaggagaga tggtgccgta ggaggtcgtg gatggttgat gccaagagag 4320gccctccacc cgtggtgggc aaatgtccag gcctgggctg gcagcccagg gctgtttctg 4380ggtgctccct ggccccaggg tggcgtctgg ttaccatggc tgtgtgtgtc catgtctgca 4440agcagttctt caataaatgg cctgcctccc cc 4472427DNAArtificial SequenceRepresentative nucleic acid sequence 4aatgcagttt acattaacct tngacag 27

Claims

1. A nucleotide sequence signal amplification composition comprising an isolated, synthetic nucleotide sequence of greater than 7 nucleotides comprising a fragment of SEQ ID NO:3 and further comprising a T nucleotide at position 1438 of SEQ ID NO:3 and one or more primers that bind to the synthetic nucleotide sequence, a thermostable DNA polymerase, a restriction enzyme, or a combination thereof.

2. The nucleotide sequence signal amplification composition of claim 1, wherein the isolated, synthetic nucleotide sequence comprises SEQ ID NO:4 wherein X is a thymidine base (T).

3. The nucleotide sequence signal amplification composition of claim 1, wherein the isolated, synthetic nucleotide sequence comprises-greater than 10 nucleotides.

4. The nucleotide sequence signal amplification composition of claim 1, wherein the isolated synthetic nucleotide sequence comprises greater than 25 nucleotides.

5. The nucleotide sequence signal amplification composition of claim 1, wherein the isolated synthetic nucleotide sequence of comprises greater than 60 nucleotides.

6. The nucleotide sequence signal amplification composition of claim 1, wherein the isolated, synthetic nucleotide sequence is SEQ ID NO:4 wherein X is a thymidine base (T).

Images