Abstract:
Provided are methods and kits to determine whether a subject is predisposed to developing a disease or condition associated with structural polymorphisms in genes CNTNAP2 and CNTNAP4, along with the methods to optimize treatment with various pharmaceutical preparations which modulate ion channels, neurite outgrowth, and myelination signaling.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional application 61/273,512 filed 5 Aug. 2009 which are expressly incorporated by reference herein. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    Not applicable 
       NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
       [0003]    Not applicable 
       SEQUENCE LISTING 
       [0004]    The application includes two sequence listings which are included in the body of the specification as well as in the last section of this application 
       BRIEF SUMMARY OF THE INVENTION 
       [0005]    Among known common structural polymorphisms of genes CNTNAP2 and CNTNAP4 (members of neurexin superfamily), we discovered those that are substantially and significantly associated with neuro-psychiatric diseases known to have abnormal myelination as a component of their pathogenesis. The association with a disease was significant for the non-null allele variant of the polymorphism in CNTNAP2 gene (see below SEQ ID No. 1 and  FIG. 1 ) for both gender (Table 1). The other disease association discovered, namely non-null genotype at the mentioned CNTNAP2 locus in combination with null genotype at the indicated CNTNA4 locus (see below SEQ ID No. 2 and  FIG. 1  and Table 1), was strongly disease-associated in females only (Table 1). 
         [0006]    Since these are genetic markers that could be tested starting from individual&#39;s early ontogenesis, their presence could be used to identify individuals at risk to develop the disease and to start preventive or early therapy. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0007]    A better understanding of the present invention will be realized from the detailed description which follows, taken in conjunction with the accompanying drawings in which: 
           [0008]      FIG. 1  shows the location of amplicons targets in the phase I study for the replication of novel risk factor candidates within known structural polymorphisms. The amplicon in  FIG. 1  shows the amplicon&#39;s location at one of the distal introns of the CNTNAP2 gene, within an overlapping region of known Copy Number Variations ##36645, 22539, 39519, 43538, 6581, 7614. SEQ ID No. 1 is marked as an open circle; and 
           [0009]      FIG. 2  shows the amplicon in one of central introns of the CNTNAP4 gene.  FIG. 2  shows the location within an overlapping region of known Copy Number Variations numbers 35409, 35410, 43580, 39188. SEQ ID No. 2 is marked as an open circle. 
       
    
    
       [0010]    Variation 36644 in  FIG. 1  is a gain. All of the other Variations in  FIG. 1  and all of the Variations in  FIG. 2 , which are shown cross-hatched, are a loss. 
       DETAILED DESCRIPTION OF THE INVENTION 
     Experimental Data 
       [0011]    We tested if any of known structural variations (copy number variations, or CNVs, deletions or insertions) from a group of selected myelination-related genes were disease-associated. Disease-related DNA samples for this testing were from patients with multiple sclerosis (MS), Schizophrenia (Sch), Bipolar disorder (BD) or Alzheimer&#39;s disease (Alz), all known to have myelination abnormalities. The DNA samples were collected from biological samples such as blood, saliva and/or buccal cells. 
         [0012]    From 120 genes belonging to gene ontology category “myelination” (GO:0042552), 26 genes were selected that contained known structural polymorphisms (as determined with the Database of Genomic Variants (http://projects.tcag.ca/variation/). Thirty nine structural polymorphisms within the 26 selected genes were tested for disease-association using TaqMan® qPCR (Applied Biosystems, Foster City, Calif.) The PCR conditions were optimized for precise and reproducible detection of polymorphism copy number present. 
         [0013]    The screening of samples (40 MS, 38 from patients with other above mentioned neuro-psychiatric disturbances, and 40 controls) showed that common structural polymorphisms were present in patients and controls in only ten of the 39 tested CNV-containing loci. Of those ten, two showed a trend to be disease-associated, while only two of these four (within CNTNAP2 and CNTNAP4 genes) did the trend achieve statistical significance (Table 1). 
         [0014]    The first of these two belongs to one of CNTNAP2 gene introns at 7q36.1 location, within the overlapping region of CNVs with Ids ##22539, 36645, 39512, 43538, 6581, 7614 (numbers in accordance with the Database of Genomic Variants) ( FIG. 1 ). The non-null genotype was statistically shown to be disease-associated, (OR=2.98; OR 95% CI=1.19-7.44; p=0.024), and the association remained independent of whether this genotype was homo- or heterozygous. 
         [0015]    The statistical analysis of the combination of the above-mentioned structural polymorphism at gene CNTNAP2 with another structural polymorphism, at gene CNTNAP4 (an intron with 16q23.1 location; the overlapping part of CNVs with IDs ##35409, 35410, 39188, 43580;  FIG. 2 ), resulted in the second statistically significant disease association that additionally appeared to be gender-related (Table 1). The non-null genotype at the corresponding CNTNAP2 locus in combination with null genotype at the indicated CNTNAP4 locus was strongly disease-associated in females (OR=6.65; OR 95% CI=1.35-32.61; p=0.017). 
         [0016]    Another gender dependent association was seen in Alzheimer&#39;s patients for the non-null variant of the studied CNV in CNTNAP2 gene (Table 2). Again, only in female patients the association reached the statistical significance. Gender dependency makes this marker a potential candidate in predicting a) risks or benefits in provoking ALZ by hormone therapy used in patients for other conditions (e.g. cancer), b) individual responses to hormone therapy in ALZ patients. 
         [0017]    The disease association in gender dependent fashion might have a prospect in predicting a) risks or benefits in provoking ALZ by hormone therapy used in patients for other conditions (e.g. cancer), and b) individual responses to hormone therapy in ALZ patients. 
         [0018]    The association of genes from neurexin superfamily with neuro-psychiatric diseases that have myelination problems is not surprising, since at least CNTNAP2 gene is known to be myelination related. Nevertheless the data on association of particular structural polymorphisms of these genes ( FIGS. 1 and 2 ) has never been described. The CNTNAP2 that is located in one of many gene introns has never been described. For example, the CNTNAP2 gene is one of the biggest known genes and occupies a 2.3 MB DNA segment in Chr 7. Since the two genes are also involved in ion channel regulation, neuro-psychiatric diseases are reasonably expected to be associated with the discovered polymorphisms. Epilepsy, which is such a neuro-psychiatric disease, has an imbalanced ion traffic in pathogenesis. 
         [0019]    Though the above disease-associated polymorphisms were discovered using TaqMan qPCR technology, other PCR- or non-PCR-based technologies such as allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, sequencing, 5′ nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and next generation sequencing could be used to identify these polymorphisms. 
         [0020]    The polymorphisms were identified as a presence or absence of relatively small ˜200 bp segments of DNA in introns of the CNTNAP2 and CNTNAP4 genes with the following coordinates: 
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
             
               
               
               
             
           
               
                 CNTNAP2 (Chr7: 147705105 . . . 147705267) 
                   
               
               
                 SEQ ID NO. 1 
                   
               
             
          
           
               
                 gactagaatg gaaaaacgga atcctacccg atcaatgcaa attctatttc tctctctcca 
                 60 
                   
               
               
                   
               
               
                 agaggcccag atgaccagcc ctgacactgg gagccactgg gagcttcagg tgctggccca 
                 120 
               
               
                   
               
               
                 gcagtgcctg gccatgtttt caagtgtaca tcaggtgacc act 
                 163 
               
               
                   
               
             
          
           
               
                 CNTNAP4 (Chr16: 75099169 . . . 75099311) 
                   
               
               
                 SEQ ID NO. 2 
                   
               
             
          
           
               
                 cctactttct tgacacacag caagaagaag tctacagaga ctttgtgcta cttcagtgaa 
                 60 
                   
               
               
                   
               
               
                 tcttgcaaca caaagggagt tcctggacca cgatggcatc tggctctacg tccttgtcac 
                 120 
               
               
                   
               
               
                 tcactctccc ttatctgcaa aat 
                 143 
               
             
          
         
       
     
         [0021]    These polymorphisms could be also identified with the help of surrogate markers, like other nearby polymorphisms—SNPs or structural polymorphisms in which the linkage disequilibrium=1 or less. 
         [0022]    The presence of the identified genetic markers in a particular patient means the presence of a corresponding marker-related disease phenotype. Hence the markers could be used to pre-select among patients for potential responders to therapies that target corresponding mechanisms like myelination, myelination signaling, ion channel function. At the same time, the absence of these markers indicate that other mechanisms in disease pathogenesis should be targeted, like immunity in MS or synaptic transmission in Schizophrenia. The method could be used in both, when selecting an existing therapy for a particular patient, and when selecting patients for testing a pharmaceutical with a specific mechanism of action in clinical trials. 
         [0023]    The usage of the described polymorphisms in combination with known markers for a particular neuro-psychiatric disease or condition might result in better disease assessment than when a single marker is used alone. 
         [0024]    Since both gene products are transmembrane molecules, their extracellular moieties could be potentially targeted with a monoclonal antibody or other ligands to favorably modify the function in case it is causal for the disease. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Frequency (in percent) of novel disease-associated structural 
               
               
                 polymorphisms in CNTNAP2 and CNTNAP4 genes in control 
               
               
                 healthy population and disease (MS, Alzheimer&#39;s, bipolar 
               
               
                 disorder, and schizophrenia. nonMS as used herein refers to 
               
               
                 Alzheimer&#39;s, bipolar disorder and schizophrenia). In parentheses 
               
               
                 are the number of samples with corresponding alleles out of total 
               
               
                 samples number in the group. The Fisher exact test for 2 × 2 
               
               
                 tables was used to calculate p values. 
               
             
          
           
               
                   
                   
                   
                 Male + 
               
               
                   
                 Male 
                 Female 
                 Female 
               
               
                   
                   
               
             
          
           
               
                 CNTNAP2 
                 MS 
                 47 (8/17) 
                 53 (10/19) 
                 50 (18/36) 
               
               
                 non-null allele 
                 MS + nonMS 
                 48 (15/31) 
                 52 (21/40) 
                 51 (36/71) 1   
               
               
                   
                 MS + Schiz 
                 55 (11/20) 
                 57 (12/21) 
                 56 (23/41) 
               
               
                   
                 Controls 
                 35 (7/20) 
                 25 (5/20) 
                 30 (12/40) 
               
               
                 CNTNAP4 
                 MS 
                 41 (10/17) 
                 21 (15/19) 
                 31 (25/36) 
               
               
                 null allele 
                 MS + nonMS 
                 48 (16/31) 
                 30 (28/40) 
                 38 (44/71) 
               
               
                   
                 MS + Schiz 
                 45 (12/20) 
                 19 (17/21) 
                 31 (29/41) 
               
               
                   
                 Controls 
                 35 (13/20) 
                 40 (12/20) 
                 38 (25/40) 
               
               
                 Combination: 
                 MS 
                 18 (3/17) 
                 53 (10/19) 
                 36 (13/36) 
               
               
                 CNTNAP2 
                 MS + nonMS 
                 16 (5/31) 
                 42 (17/40) 2   
                 31 (22/71) 
               
               
                 non-null + 
                 MS + Schiz 
                 25 (5/20) 
                 57 (12/21) 
                 39 (17/41) 
               
               
                 CNTNAP4 
                 Controls 
                 30 (6/20) 
                 10 (2/20) 
                 20 (8/40) 
               
               
                 null 
               
               
                   
               
               
                   1 OR = 2.40; OR 95% CI = 1.05-5.45; p = 0.046; 
               
               
                   2 OR = 6.65; OR 95% CI = 1.35-32.61; p = 0.017; 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The association of the CNV in CNTNAP2 gene with Late Onset 
               
               
                 Alzheimer&#39;s Disease (LOAD). Data were obtained in a Confirmation 
               
               
                 study on 72 LOAD and 90 matched normal control samples, both 
               
               
                 sample sets from National Cell Repository for AD (NCRAD). 
               
             
          
           
               
                   
                 OR 
                 95% CI 
                 p-value 
               
               
                   
                   
               
             
          
           
               
                   
                 Males + Females 
                 1.46 
                 0.82-2.56 
                 0.196 
               
               
                   
                 Males 
                 1.24 
                 0.53-2.87 
                 0.674 
               
               
                   
                 Females 
                 2.44 
                 1.11-5.33 
                 0.029