JP2006025744A - Method for genetical examination of panic disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the examination of episodic aporioneurosis called also as panic aporioneurosis to outbreak severe anxiety without reason and forget himself by the overwhelming horror of death, known as panic seizure or panic disease which is the most grave neuropathy to develop symptoms difficult to understand by ordinary persons. <P>SOLUTION: The method for the examination of anxiety disorder and related diseases including familial and sporadic syndrome, mainly agoraphobia, sociophobia, panic seizure and panic disease comprises the use of the duplication of an arbitrary genom sequence of 15q24-25 limited by D15S925 (near end) and D15S736 (far end) as boundaries or the use of a protein encoded by an arbitrary genom sequence in 15q24-25 which is a duplicated region of human chromosome or an excessively expressed polypeptide expressed from the other region of the genom as a result of the duplication of 15q24-25. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention mainly relates to diagnosis of panic disease and panic attack, and to a genetic test method thereof.

  Some scenes, places, people and things are scary, but if you avoid them, you don't have to worry too much. The most common are phobias for outings, social phobias, and other terrible subjects such as snakes, lightnings, airplanes, and blood. “Fear of going out” is because of tremendous anxiety just by going into the crowd, going to the plaza of the city where people gather, – people traveling, traveling abroad, etc. This is a case of running away. This anxiety is always accompanied by physical symptoms. Palpitation, tachycardia, sweating, trembling, dry mouth, dizziness, suffocation, etc. These people know well that their anxiety is not (reasonable. So they try to do it, but it's hard to do.

“Interpersonal phobia” is a case where you feel very anxious about acting in front of them, even if you are in a small group of familiar relationships. I usually worry about my blush, stiffness of the face, voice and hand trembling, and the way I see my eyes, which gives me an unpleasant feeling and makes me feel light. Unlike people with fear of going out, entering a stranger doesn't feel unnecessary. As long as you live without a weak interpersonal scene, you don't have to worry. However, social phobia, which is called outing phobia, is socially important for human social life. It is not good to leave it, especially when you are in the age of fundamentals for enriching human relationships, such as youth.

Paroxysmal anxiety is also called panic anxiety. As you can imagine from its name, intense anxiety suddenly breaks out for no reason, is overwhelmed by the fear of death, and loses everything. It is the most dramatic type of neurosis. The troubles of people with neurosis are often not known to third parties, but in this case they are often seen from the outside. It may be accompanied by tachycardia, tremor, dry mouth, chest pain, respiratory distress, and dizziness. Soon, the seizure will be repeated once a week, even if it is mild, and repeated three or four times a week if it is severe. What is different from phobia (neuropathy) in the previous section is that it does not occur in connection with a specific scene or thing, and the degree of anxiety is intense.

After the panic disorder has healed, I often get worried about "Is there a seizure again?" For this reason, there are people who can only act around their homes or within a range where they can immediately hit the doctor's gate. It becomes the “going-out phobia” mentioned earlier. Contrary to panic anxiety, there is another type of anxiety that is not very strong and shallow. In this type of symptoms, the anxiety is mild, but the expressions are diverse, including palpitations, hot flashes, tremors, thirst, chest pain, suffocation, dizziness, use of the larynx, tension, irritability, etc. There are even symptoms of the heart. This situation lasts for at least six months. It is said that this type rarely shifts to the panic type. Instead, if anxiety persists for a long time, the energy reserves in the mind will gradually disappear, and will become chronically “Yu-Utsu” and “Okuu”.

Several kinds of drugs for treating such panic symptoms are already known, but drug discovery from a genetic point of view has not been studied much. To help understand what panic is, it is shown in FIG.

The JP-A 5-213965 (Patent Document 1), a therapeutically effective amount of virginiamycin M ₁ compound and pharmaceutical compositions useful for the treatment of panic disorder or anxiety disorder consisting of a pharmaceutically acceptable carrier. A pharmaceutical composition useful for the treatment of panic symptoms or anxiety comprising a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier. Disclosed is a pharmaceutical composition useful for the treatment of panic symptoms or anxiety comprising a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier.

JP 2003-512363 A (Patent Document 2) includes a general formula (where Z is a sulfur atom or an oxygen atom, R 1 and R 2 are the same or different alkyl groups or alkenyl groups, and X is CO, CO ₂ , SO or SO ₂ , R is an alkyl, allyl, alkenyl or aralkyl group, and when Z is an oxygen atom, X is a group represented by SO ₂ or R, R 1 and R 2 Are not methyl groups.) The use of neurological disorders characterized by epilepsy, anxiety, depressive symptoms, sleep disorders, panic symptoms, muscle contraction, pain, alcohol or benzodiazepine dependence or psychotic behavior is disclosed.

JP-T-2002-507884 (Patent Document 3) discloses an in vitro diagnostic method for measuring dopamine-β-hydroxylase specific activity for evaluation of a patient's sympathetic nervous system function from a serum sample. A) filtering the serum sample to substantially remove the drug and endogenous inhibitors of dopamine-β-hydroxylase; b) converting the filtered sample of step a) to a detectable compound. Incubating with a substrate of dopamine-β-hydroxylase, c) measuring the amount of detectable compound to assess dopamine-β-hydroxylase activity and comparing to a normal sample, thereby A lower amount than normal samples indicates reduced sympathetic nervous system function and a higher amount than normal samples indicates increased sympathetic nervous system function. Features, a method. An evaluation method is disclosed in which when the sympathetic nervous system function is reduced, it indicates neurodegenerative disease, neuroendocrine disease, emotional psychosis and cardiovascular disease, burnout, chronic fatigue, stress and panic syndrome.

In JP-T-8-502027 (Patent Document 4),
A compound represented by the formula: wherein R is hydrogen, CH ₃ CH ₂ —, CH ₂ ═CHCH ₂ —, (CH ₃ ) ₂ CH—, or a pharmaceutically acceptable salt thereof . 2.
A compound wherein the compound is 6- (2-bromophenyl) -8-fluoro-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxamide. A method for treating a host panic disease, wherein R is hydrogen, CH ₃ CH ₂ —, CH ₂ ═CHCH ₂ —, (CH ₃ ) ₂ CH—. A method comprising administering an anti-panic effective amount of a compound or a pharmaceutically acceptable salt thereof to a host in need of such treatment.
JP-A-5-213965 Japanese translation of PCT publication No. 2003-512363 Japanese translation of PCT publication No. 2002-507884 Japanese National Patent Publication No. 8-502027 Lesch, KP, Bengel, D., Heil, s A., Sabol, SZ, Greenberg, BD, Petri, S., Benjamin, J., Muller, CR, Hamer, DH, Murphy, DL Association ofanxiety-related traits with a polymorphism in the serotonin transporter generegulatory region.Science 1996; 274: 1527-1530. Bulbena, A., Duro, JC, Mateo, A., Porta, M., Vallejo, J. Jointhypermobility syndrome and anxiety disorders. Lancet 1988; 2: 694. Bulbena, A. Duro JC, Porta M, Martin-Santos R, Mateo A, Molina LL. Anxietydisorders in the Joint hypermobility syndrome. Psychiatr Res 1993; 46: 59-68. Nadal, M., Moreno, S., Pritchard, M., Preciado, MA, Estivill, X., Ramos-Arroyo, MA Down syndrome: characterisation of a case with partial trisomy of chromosome 21 owing to apaternal balanced translocation (15; 21) (q26; q22.1) by FISH. J Med Genet 1997; 34: 50-54. Celi, FS, Cohen, MM, Antonorakis, SE, Wetheimer, E., Roth, J., Shuldiner, AR Determination of gene dosage by quantitative adaptation of thepolimerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences Genomics 1994; 21: 304-310. Lamballe, F., Klein, R., Barbacid, M. TrKC, a new member of the trkfamily of tyrosine protein kinases, is a receptor for neurotrophin-3.Cell 1991; 66: 967-979. Mcgregor, LM, Baylin, SB, Griffin, CA, Hawkins, AL, Nelkin, BD Molecular cloning of the cDNA for human TrKC (NTRK3), chromosomalassignment, and evidence for a splice variant.Genomics 1994; 22: 267-272. Kim, Y., Boyd, CD, Csiszar, KA highly polymorphic (CA) repeatsequence in the human lysyl oxidase-like gene.Clin Genet 1997; 51: 131-132. Cottingham, RW, Idury, RM, Schafer, AAFaster sequential genetic linkage computations. Am J Hum Genet 1993; 53: 252-263. Ott, J. Analysis of human Genetic Linkage. 2nd ed. Baltimore, John Hopkins University Press, 1991. 11. Anastasis, J., LeBeau, MM, Vardiman, JW, Westbrook, CADetection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situhybridization with a chromosome-specific probe.Am J Pathol 1990; 136: 131-139

The present inventor has detected intervening duplication of chromosome 15q24-25 in patients with panic disease, depression and joint hyperkinetics from familial cases of panic disease and overt sporadic cases. The overlap of 15q24-25 is present in 7% of the general population. This cytogenetic variation is responsible for common and complex anxiety as well as mood sickness. There are at least 10 genes in this overlapping region, of which NTRK3 and LOXL1 may be involved in anxiety disease and joint hypermotility, respectively. The inventor has developed cytological and cytogenetic methods based on fluorescence in situ hybridization to detect 15q24-25 duplication and the associated somatic mosaicism. The inventor has developed a molecular method based on DNA or RNA analysis to quantify genes (including NTRK3 and LOXL1) or other nucleic acid sequences within the overlapping region 15q24-25.

The inventor has clearly demonstrated, by linkage analysis and affected-only analysis, that duplication of chromosome 15q24-25 is involved in several familial anxiety cases, The genetic conditions of those pathological changes were identified and a common biological background for panic disease, agoraphobia and social phobia was defined. This chromosomal change is a major susceptibility mutation for the anxiety phenotype due to the fact that the duplication is detected in all independent and unrelated patients with panic disease and / or agoraphobia The idea arises. Although the psychiatric clinical scope involving 15q24-25 duplication mutations has not yet been fully defined, the inventor has identified several psychiatric clinical taxa such as agoraphobia, social In panic with and without phobia and simple phobia, this mutation was shown to occur simultaneously.

Clearly from this study, we cannot claim pure diseases such as panic disease and agoraphobia, but rather from a genetic point of view, models ranging from panic disease to agoraphobia, or general nerves It can be seen that the sex syndrome would be more realistic. The overlapping region 15q24-25 contains more than 50 genes, of which only 10 genes are accurately located. The inventor has developed cytological, cytogenetic and molecular methods for diagnosing 15q24-25 duplication. Currently, NTRK3 and LOXL1 are good candidate genes to explain the anxiety and joint hyperkinetic syndromes indicated in the text.

A family with patients with panic disease and / or agoraphobia was identified.
DSM-III-R (American
Psychiatric Association, Diagnositic and Statistical Manual of Mental Disorders
(DSM-III-R), 3rd ed., Revised, Washington DC, 1987), 19% of patients were panic and / or agoraphobia and 22% were social Phobia, 34% had simple phobia, and 18% had severe depression. Their incidence is significantly higher than the prevalence of each disease in the general population, which means the presence of a common genetic cause, and the same molecular changes that cause these diseases This suggests the possibility of appearing clinically differently.

Hyperarthritis with a Beighton score [3] of 4 or more was detected in 65 (44%) of the family. Panic disease and / or agoraphobia were isolated in 64% patients with joint hyperkinetic syndrome, 59% with social phobia, and 46% with simple phobia. Overall, 54% of patients segregated one of these anxiety disorders with joint hyperkinetic syndrome.

The separation of anxiety disease along with joint hyperkinetic syndromes suggested that these may be continuous genetic syndromes. At the same time, the present inventor analyzed the segregation analysis with a set of microsatellite markers present in the vicinity of or within the candidate genes for joint hyperkinetic syndrome (collagen, fibrillin, elastin, etc.), and the chromosomes in which these candidate genes exist. Cytogenetic studies focused on detecting rearrangement of the contained chromosomes. A sample of 10 patients in the seven families showed a putative cytogenetic change on the long arm of chromosome 15 (15q24-25), which was subsequently identified as an intervening duplication It was done. The putative cytogenetic abnormality was examined by fluorescence in situ hybridization (FISH) using several YAC clones derived from this region. Clearly from YAC 802-b-4, 929-c-7, 750-b-7, 891-e-7 and 753-h-11, an intervening overlap of 15q24-25 in the patient (referred to as dup15q24-25) Proved.

In order to determine the degree of overlap, a YAC / PAC contig covering the 15q24-25 region was created (Fig. 2). In creating the map, a total of 41 YACs, 3 PACs and 2 BACs were used. Information regarding the chimerism of the YAC clone and the content of the STS has been deposited with CEPH. This overlapping region spans about 10 cM from the D15S739 marker (proximal) to the D15S930 marker (distal) in human chromosome 15. This region contains 10 known genes (LOXL1, CRABP1, IREB1, NIC3A, NIC5A, NIC4B, FAH, IL16, NMB and NTRK3), which are within the overlapping region by PCR based on their STS. It was decided to be located. Although it is known that there are many ESTs in this overlapping region, they may contain 50 to 200 genes.

The centromere end of this overlapping region is
Located between non-overlapping YAC 753-h-11 and 875-a-3 (within 33iF3), and the telomeric ends are all (within YAC 964-f-8 with deletions) Located between non-overlapping YAC 802-b-4 and 966-a-2. Several attempts were made to obtain YAC, PAC and BAC to fill these gaps. The inventor acquired PAC 252-a-23 and 216-i-14 for the telomeric end and PAC 251-c-23 for the centromeric end, but both clones had the gap. I did not fill it. However, since these PAC clones were located at the end of the 15q24-25 overlapping region, the present inventor used the cosmid probe (t216) for the FISH analysis of the overlap and the segregation analysis with the anxiety phenotype in the family. -1 and c251-3) were prepared.

Three forms of 15q24-25 overlap were detected from analysis of another sample from a patient in the family. The most common form is called the “telomere type” and was found in 79% of patients. This telomeric duplication was observed as a tandem (tandem) or inverted type depending on the relative distance of the signals observed by FISH. Finally, “centromere-type” duplications were detected in 21% of the families with signals located far away from the origin and close to the centromere. This form of duplication appears to result from differences in the rearrangement of the same region of 15q24-25. The overlapping regions and markers in the centromeric and telomeric forms are identical, but exclude the addition of centromeric or telomeric loci to the duplication and the presence of breakpoint clusters within a few kb interval It is not possible.

In order to confirm that this duplication was a characteristic of the patient, 81 anonymous amniotic fluid samples from unrelated pregnant women were examined using probes t216-1 and c251-3. This duplication was detected in the samples (6.2%). Furthermore, examination of 54 irrelevant 54 persons who did not have panic disease and / or agoraphobia and joint hypermotility, 15 duplication of 15q24-25 was detected in 5 persons (9.2%). From a total of 135 unrelated control samples, telomeric and centromeric duplications were found at a rate of 70 and 20%, respectively.

The strength of the association between 15q24-25 duplication and several anxiety disorders and joint hyperkinetic syndrome was analyzed by linkage analysis in the seven families. The anxiety phenotype was examined based on the following five models. 1) Panic disease and / or agoraphobia, 2) Social phobia, 3) Simple phobia, 4) Panic disease and / or agoraphobia and / or social phobia, and 5) Panic disease and / or agoraphobia And / or social phobia and / or simple phobia. The five models with the addition of joint hyperkinetic syndrome (Beighton decision 4 or higher) were also examined, and this phenotype was also examined as the only trait.

The linkage between the 15q24-25 duplication and 11 models when the penetration rate of this duplication is 78% or analyzed under conditions of almost perfect permeability is considered. In a model containing 4 traits (panic and / or agoraphobia and / or social phobia and / or joint hyperkinetic syndrome), the highest rod score of 4.94 was obtained with a recombination rate of 0. Except for the case of simple phobia (rod score 2.33), significant linkage values were obtained in all remaining models including joint hyperactivity syndrome. The rod score for the joint hyperkinetic syndrome itself was 3.82 under the condition of zero recombination rate. The rod scores for models with only psychotic traits did not all exceed 3, whereas the rod scores for models with 3 or 4 anxiety phenotypes exceeded 2.

The eleven models were examined by 15-affected-only analysis for 15q24-25 duplication under the two penetration conditions described above. The rod score for the phenotype defined by panic disease and / or agoraphobia and / or social phobia was 3.36 and the rod score for the four anxiety phenotypes was 2.96. The rod score increased to 3.66 when hyperjoint syndrome was added to the model of panic and / or agoraphobia and / or social phobia. In the case of panic and / or agoraphobia and social phobia, the addition of hyper joint syndrome resulted in a rod score of 3.1 and 3.28, respectively. Positive rod scores were also obtained in other models, but did not reach significant levels.

We examined the phenotypic characteristics of 93 patients in the family and their association with 15q24-25 duplication. The percentage of patients in the family with duplication (72%) is higher than in the general population (7%). Notably, all patients with social phobia had this duplication. Of the 45 patients with panic disease and / or agoraphobia and / or social phobia 44 (98%) had 15q24-25 overlap. Similarly, 87% of patients with joint hyperkinetic syndrome had a 15q24-25 duplication. If one or several psychotic traits and joint hyperkinetic syndrome were present simultaneously by clinical judgment, all of the patients had this duplication. Finally, 53 (79%) of patients with duplication were diagnosed with one or several anxiety diseases, and only one (2%) of patients without duplication had panic or Diagnosed with agoraphobia (patients with agoraphobia only). 88% of the family members who did not have either the anxiety or the joint hyperkinetic syndrome were negative for the dup15q24-25 mutation.

In order to repeat this test in another group of patients, the present inventor identified 50 unrelated patients with panic and / or agoraphobia from among those who regularly visit Anxiety Clinic. I chose. A possible relationship between the patient and the village patient was excluded. Surprisingly, dup15q24-25 was detected in all 50 patients, but was detected in only 10 of 135 unrelated controls selected from the general population (chi-square value (Yates correction) Yes) 138.5; Fisher's direct test P <0.0001; odds ratio 1207.2).

Some origins and mosaicism of 15q24-25 overlap 6 microsatellite markers, 4 located within 15q24-25 overlap region (LOXL1, D15S154, D15S201, NTRK3-BP2) and 2 outside this overlap region Analysis of the family using individual (D15S1040 and D14S158) markers revealed no linkage or a linkage value of less than 1 in all the phenotypic models examined. These markers are spread over a region of 20 cM on the human chromosome 15 genetic map (4 markers in the overlapping region span about 10 cM). Interestingly, in the 67 family patients with 15q24-25 duplication, the three alleles could not be detected even when the duplication was present over three generations in the family. A total of 28 different haplotypes were generated based on the 6 markers from a total of 71 duplicated chromosomes in which mutant haplotypes could be determined. In each family, the founder's dup15q24-25 haplotype was easily identified among other duplicated haplotypes of the new members of the family.

Such a large number of haplotypes (42% of duplicated chromosomes have different haplotypes) implies the existence of an independent source and suggests that this mutation occurs easily. At least 7 couples in the family had duplicates. This is equivalent to 44% of couples who analyzed both for duplication and revealed a tendency to mate more frequently between individuals due to the phenotype associated with the 15q24-25 mutation Shown in Several anomalous cases of 15q24-25 overlap were found in the family. This means the generation of new forms, the conversion from overlapping chromosomes to non-overlapping chromosomes, and the conversion of one form of duplication from another.

Mosaic duplication of 15q24-25 was first observed in metaphase chromosomes of lymphocytes by FISH using probes t216-1 and c251-3. When 100 individuals of 100 interphase nuclei were evaluated for dup15q24-25 mosaicism, the median of duplicated cells (40.4-70.6%) was found to be 55%. The hybridization efficiency with the probe in the interphase nucleus of controls without duplication was 85%.

The inventor examined 15q24-25 duplication in spermatozoa obtained from controls without duplication and patients who were homozygous for duplication (15% homozygous for duplication, 85% heterozygous). As expected, the inventor detected duplication only in the gamete of the patient, but only 40% of the sperm had duplication. This result therefore indicates that there is a 15q24-25 duplication in germ cells, meaning that it is propagated from one generation to the next.

Cytological detection of 15q24-25 duplication in peripheral lymphocytes Several DNA probes were used to detect 15q24-25 duplication in prepared peripheral lymphocytes. Fluorescence in situ hybridization (FISH) of interphase cells was performed as in the conventional non-patent document 4, using probes t216-1 and c251-3 located proximal and distal to the overlapping region of 15q24-25. .

Cytogenetic detection of 15q24-25 duplication in peripheral lymphocytes Several DNA probes were used to detect 15q24-25 duplication in lymphocyte cells at mid-fission. Using probes t216-1 and c251-3, FISH was performed as in the conventional non-patent literature.

Molecular detection method for 15q24-25 duplication in peripheral lymphocytes In order to molecularly detect the 15q24-25 mutation associated with anxiety, several genes or nucleic acid sequences derived from the 15q24-25 duplication region were used. The sequences described herein are merely examples of sequences that can be used to detect 15q24-25 duplication.

Quantification of RNA of IL16 by RT-PCR
In order to evaluate the expression level of the gene existing in the overlapping region of 15q24-25, IL16 that is sufficiently expressed in blood lymphocytes was selected. IL16 overexpression was examined by quantitative PCR. As expected, homozygous patients had approximately twice the cDNA concentration in peripheral blood lymphocytes compared to controls. The analysis of the expression of other genes in this region excludes the possibility that the duplicated genes are not expressed in patients with the dup15q24-25 mutation. Total RNA obtained from cultured cells from patients and controls was amplified by competitive PCR [5]. IL16 gene cDNA sequence (GenBank
G06653) corresponding to the 326 bp fragment (STS WI-7689) (front primer: 5'-TCC CAT AAC CGC TGA TTC TC-3 'and rear primer: 5'-AAT AAA TGT CAC TGT TTG GGG G-3 ') Using endogenous IL16
mRNA was amplified. An internal standard was created using a 42 nucleotide primer (20 nucleotides at the 5 'end corresponding to 76 nucleotides upstream). These primers amplified the 228 bp product, which was further subcloned into the pMOS Blue-T vector (Amersham) and used as an internal standard. Since the PCR product corresponding to IL16 mRNA may be due to remaining genomic DNA, each sample was subjected to control PCR amplification after pretreatment with RNase A.

Quantitative Southern blot analysis of the NYRK3 gene
The lysyl oxidase analog (LOXL1) and neurotrophin 3 receptor (NTRK3) genes are physically located at opposite ends within the 15q24-25 overlap region (Figure 2), and the phenotype found in this patient Therefore, Southern blot analysis was performed using a probe corresponding to the gene. 5 μg of genomic DNA from patients and normals was digested with 40 units of EcoRI and the DNA was transferred to a nylon membrane (Hybond-N +, Amersham). By comparison with a control gene located outside this overlapping region or on another chromosome, the amount of genes in this overlapping region is 1.5 times in heterozygous patients and twice in homozygous patients compared to normal subjects. Expected to be.

For this purpose, the NTRK3 gene (GenBank
The EcoRI / SstI fragment of plasmid pNIJ3-SR0.4 containing the 5 ′ cDNA portion of G913721) and the control marker of chromosome 2 (exon 7 of rBAT gene) were labeled with α ³² P-dATP. Gene dosage was assessed by quantification of band density on art radiographs. From the quantification of the band density of the rBAT gene on chromosome 2 of the NTRK3 gene, the peak height, and comparison with the control marker, it was found that the band intensity increased in individuals with duplication.

DNA analysis by quantitative PCR of NTRK3 The neurotrophin 3 receptor (NTRK3) gene is located in the 15q24-25 overlapping region and is considered to be a good candidate gene for the anxiety phenotype. That is, the amount of gene analysis was evaluated through simultaneous PCR amplification of the NTRK3 gene fragment and the PROC gene (exon 1) control fragment. Primer corresponding to the 134bp fragment of NTRK3 gene is TRK3U2 ': 5'-TAT GAA GAT GTT CGC with 5' end fluorescently labeled
TTC AG-3 ′ and TRK3U2F: 5′-TCT ACC TGG ACA TTC TTG GCT-3 ′. The primer corresponding to 222 bp of the PROC gene is a fluorescently labeled PC111: 5'-GTG CTA GTG CCA CTG TTT
GT-3 'and PC112: 5'-ATC ACC ACC TAG CTC TCT TC-3'. Samples were run on an ABI PRISM 373 DNA sequencer and the results were processed by the programs GENESCAN and GENOTYPERa. Simultaneous PCR amplification of the NTRK3 fragment and the control fragment of the PROC gene revealed a difference in DNA amount due to 15q24-25 duplication.

The percentage of cells with duplication in an individual's blood lymphocytes (assessed by measuring the percentage of duplication and normal by interphase nuclei FISH) is sufficient to obtain a clear and reproducible difference between peaks Southern blot analysis and PCR analysis were useful only when they were high. In fact, the presence of mosaicism has considerably complicated the assessment of duplication by Southern or PCR analysis.

Methods for practicing the invention Clinical evaluation of patients With a structured clinical interview based on DSM-III-R, with regard to panic disease with or without agoraphobia, social phobia, simple phobia and severe depression All patients were evaluated.

Cytogenetic fluorescence in situ hybridization analysis Metaphase chromosome slides were prepared after 72 hours of culture of amniotic cells and patient's peripheral blood lymphocytes by standard methods. In all cases the GTL band method was performed. In order to obtain 900 G-band level highly degradable chromosomes, methotrexate was used to synchronize peripheral blood culture cells. Unstained slides were stored at −20 ° C. until hybridization. Prior to hybridization, the slides were baked at 55 ° C. for 30 minutes. Slides were covered with 40 μl of antifade solution (Vector Laboratories) containing 0.5 mg / ml propidium iodide or 150 ng / ml DAPI.

Inter Alu-PCR was performed using primers A33 and A44 of Non-Patent Document 4 to prepare probes from YAC, BAC and PAC clones. The PCR product was ethanol precipitated and resuspended in 40 μl distilled water before labeling. All probes (2 mg each) used for FISH (cDNA, cosmid, YAC and PAC) were labeled with biotin-16dUTP or digoxigenin-11dUTP (Boehringer-Mannheim) according to standard nick translation methods and gel filtered. Then 400 ng of each probe was ethanol precipitated with 1 mg cot1-DNA (GIBCO-BRL) and 1 mg salmon sperm DNA (Sigma), and 50% formamide and 50% 12 ×
Resuspended in hybridization mixture containing SSC and dextran sulfate.

FISH was performed as described in Non-Patent Document 4. In summary, 70 ng of each probe (7 ml of hybridization mixture) was added to each slide and sealed with rubber cement (5 ml of each probe was used when hybridizing with more than one probe at a time). After denaturing the slides by heating them at 80 ° C. for 8 minutes, they were incubated overnight at 37 ° C. in a moisturizing chamber. The post-hybridization washes were performed 3 times with a mixture of 50% formamide and 50% 2x SSC, then 3 times with 2x SSC, all at 42 ° C. The slide was then incubated for 10 minutes in blocking solution (Boehringer Mannheim). This slide was incubated with avidin-FITC (for biotin-labeled probe) or anti-digoxigenin-TRITC (for digoxigenin-labeled probe) for 20 minutes at 37 ° C. in a moisturizing chamber and then with 4x SSC / 0.1% Tween-20 Wash twice.

When detecting cDNA probes and cosmid probes, the biotin signal was amplified once. To that end, the slides were incubated with biotinylated anti-avidin (Vector Laboratories) for 20 minutes, washed as before, and again incubated with avidin-FITC for an additional 20 minutes and washed again. Slides with 0.1 mg / ml DAPI (Vectashield, Vector
Covered with anti-fading solution containing Laboratories). Fluorescent microscope (VANOX, with appropriate filter)
Olympus) was used to observe the slide. Microscopic images were analyzed by Cytovision system (Applied Imaging Ltd., UK). For each hybridization, at least 20 metaphase nuclei were examined.

Interphase nuclear analysis
Mosaic detection by FISH was performed using known diploid controls, taking into account the efficiency of the method. A patient was considered to have an overlap if the percentage of nuclei showing three hybridization signals exceeded two standard deviations of the mean (30%) of false trisomy signals in the control sample. 100 nuclei were analyzed in each of the control and test samples.

Sperm fish
The collected sperm was first washed 3 times with PBS. Two drops of the suspension were added to a clean slide and air dried. Slides were stored at −20 ° C. until FISH analysis. Prior to hybridization, sperm heads were decondensed by rinsing slides with 2x SSC for 3 minutes, dehydrated by continuous ethanol treatment (70, 80, 95%), and 37 ° C for up to 10 minutes in DTT 5 mM. Incubated with The slides were then rinsed in 2x SSC for 3 minutes and again dehydrated by continuous ethanol treatment. And FISH was performed as described above.

Macromolecular DNA was isolated from peripheral blood by salt blotting and Southern blot analysis and quantitative DNA evaluation by PCR. 5 mg of patient and normal genomic DNA was cut with 40 units of EcoRI and electrophoresed in 0.5% TBE buffer in a 1% agarose gel for 12 hours. DNA in nylon membrane (Hybond-N +, 20x SSC)
Amersham). By random hexamer primer method, NTRK3 (GenBank
G913721) EcoRI / SstI fragment of plasmid pNIJ3-SR0.4 containing 400 bp cDNA on the 5 ′ side and a control marker (rBAT gene fragment) derived from chromosome 2 (GenBank G349705) are highly specific activity by α32P-dATP Labeled (5x 10 ^-9 cpm). Perform overnight hybridization at 65 ° C, stringent conditions 0.1x SSC,
Washed with 0.1% SDS for 15 minutes. The nylon membrane was exposed to X-ray film (Curix, Agfa) at −80 ° C. overnight. Using a densitometer (Phoretix), the band intensity and peak height on the autoradiograph were quantified for each band and the amount of DNA was assessed by comparison with a control marker.

Alternatively, two fragments, namely the NTRK3 gene (GenBank
The 3 ′ cDNA fragment of G913721) and the control fragment of the PROC gene (exon 1) were quantitatively analyzed by PCR amplification in the same reaction tube. In a 10 ml reaction containing 10 ng genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl ₂ PCR buffer, 0.1 unit Taq polymerase (Boehringer Mannheim), and 0.8 μM NTRK3 primer pair and 1.2 μM PROC primer pair PCR was performed. The PCR conditions were 94 ° C for 3 minutes denaturation followed by 22 cycles of 94 ° C for 30 seconds, 56 ° C for 50 seconds and 74 ° C for 30 seconds. Reaction samples were mixed with loading buffer, heated at 94 ° C. for 5 minutes, and run on an ABI PRISM 373 DNA sequencer (Perkin-Elmer). The results were processed with the GENESCAN program, and using the GENEOTYPEa program, allele designation, area and peak height were determined.

Quantitative analysis of mRNA in cultured cells Total RNA was prepared from cultured cells obtained from patients and controls by the guanidinium chloride method and the cesium chloride gradient method. Competitive PCR was performed according to Celi Non-Patent Document 5. RT-PCR was performed with Superscript and random primers (Gibco, BRL) using 5 μg of total RNA. Target and internal standard were amplified simultaneously as follows. 1 μl of reverse transcript was added to a mixture containing 0.2 mM dNTPs, 1.5 mM MgCl ₂ PCR buffer, 0.1 unit Taq polymerase, and 0.5 μM target sequence primer pair and 1 μl of various concentrations of internal standard. PCR conditions were 94 ° C for 4 minutes denaturation followed by 30 cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds and 74 ° C for 30 seconds.

Creating a map
Whitehead
Based on YAC contig of chromosome 15 and STS information obtained from Institute for Biomedical Research / MIT Center for Genome Research
(http://www-genome.wi.mit.edu/cgi-bin/contig/phys
map), 15q24-25 narrowed the end of the overlapping region. YAC clone, Fondation Jean
Obtained from Dausset-CEPH (www.cephb.fr), mapped onto chromosome 15q24-25 by FISH analysis, and then precisely mapped by STS PCR amplification. CEPH human (mega) YAC was screened by PCR amplification of a fragment of the LOXL1 gene, but no positive clone was obtained. The ICI human YAC library (whose filter is supplied by HGMP-Resource Center, UK, www.hgmp.mrc.ac.uk.) was imported into the LOXL1 gene (GenBank
When a primer for exon 7 of G307145) was used as a probe, a positive YAC clone (33i-F3) containing LOXL1 was detected.

Using various probes, HGMP-Resource
By screening human PAC library filters obtained from the Center (UK) (www.hgmp.mrc.ac.uk) and human BAC library filters obtained from Research Genetics, Inc. (Huntsville, USA). , Extended the contig, and accurately determined the end of the overlap. A total of 57 STSs were used to place PAC, BAC and YAC clones. The primers were obtained from Genset (France) or synthesized using a 392 DNA / RNA synthesizer (Applied Biosystems).

PCR amplification was performed in a 25 μl reaction solution containing 100 ng template DNA, 0.5 μM each oligonucleotide primer, 0.2 mM dNTPs, 1.5 mM MgCl ₂ PCR buffer, and 0.1 unit Taq polymerase. The reaction conditions were 94 ° C for 5 minutes denaturation followed by 35 cycles of 94 ° C for 30 seconds, 50-65 ° C for 30 seconds (depending on the primer) and 74 ° C for 4 seconds, and finally an extension reaction at 72 ° C for 5 minutes. there were.

The location of the gene that was previously cloned and placed within the overlap region was determined by PCR amplification of the STS containing the gene. Alternatively, for genes that appear to be within this region from linkage analysis, somatic cell hybrid analysis, or cytogenetic studies, synthesize primers for the 3 'region of those cDNAs and PCR from the YAC containing that region The position of the gene was determined by amplification.

To examine the segregation pattern within the family, the following 15q microsatellite markers were amplified: dinucleotide repeats present in intron 1 of the LOXL1 gene [8], D15S154, D15S201, D15S158, D15S1040 and NTRK3- BP2. The last is a novel microsatellite marker identified from the sequence of the 1.5 kb BamHI fragment of PAC252 A23 (isolated from the human PAC library at the HGMP-Resource Center (UK) using the 3 'end probe of the NTRK3 gene) Was). The repetitive sequence is (CA) ₂₄ , and the primers used to amplify this marker are BP2F: 5′-TTG CTT GAA GGG CAC CTG-3 ′ and BP2R:
5′-AAC ATC CTG GGT ACA TGC-3 ′.

For PCR amplification, in the case of LOXL1, D15S154, D15S201 and D15S158, the GT strand oligonucleotide primer flanking the (GT) _n sequence was end-labeled with polynucleotide kinase. PCR was performed under standard conditions in a 25 μl reaction containing 100 ng genomic DNA, 1 μM of each oligonucleotide primer, 0.2 mM dNTPs, 1.5 mM MgCl ₂ PCR buffer, and 0.1 unit Taq polymerase. . The amplification conditions consisted of a denaturation reaction at 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 58-65 ° C for 30 seconds (depending on the primer) and 74 ° C for 40 seconds, and finally 72 ° C for 5 minutes. It was an elongation reaction. The reaction product (2 μl) was mixed with 2 μl formamide stop solution and electrophoresed in a 6% polyacrylamide DNA sequencing gel at 40 W for 3.5-4 hours. The gel was dried and exposed to X-ray film (Curix, Agfa) at −80 ° C. for 4-12 hours to obtain an autoradiograph. Markers D15S822, D15S1040 and NTRK3-BP2 were amplified under the same conditions as described above. However, the reverse primer was not end-labeled. Instead, 2 μl reaction product was mixed with 2 μl formamide stop solution, electrophoresed in a 5% polyacrylamide DNA sequencing gel at 40 W for 4-5 hours, and the gel was silver stained and dried.

Linkage and association studies We considered autosomal inheritance patterns with incomplete penetrance due to traits of psychosis and hyperkinetic syndrome. The final penetrance (P) of the trait of the locus by different genotypes is given by Ott
1991), P = fc + (1-f) (1-c), where f is the penetrance in a given genotype, and c is the probability of correct diagnosis (affected versus non-affected) ) Is considering misdiagnosis of psychosis (c). Thus, the penetrance is P = 0.05 for genotype + / + taking into account phenotypic replication or misdiagnosis, P = 0.736 for genotype + / D, and genotype D / D Case P = 0.9. Where + means wild type allele and D means deleterious allele.

15q24-25 duplications were analyzed as afflicted or dichotomous loci. In order to include all observed traits due to this cytogenetic phenomenon, the genotype inherited from each parent and present in the zygote as a true genotype, ie DUP / DUP, DUP / + or + Considered / +. However, DUP is a “deficient allele with duplication” that retains the 15q24-25 mutation, and + is a wild-type allele with no duplication. The observed phenotype is derived from this zygote genotype, which contains mosaicism and is observed by FISH. The trait penetration of this locus is the probability that the original genotype of the zygote that confers the final phenotype will be observed by FISH. Furthermore, the probability of cytogenetic diagnosis error (c) based on probe efficiency or misinterpretation was considered.

In this way, the present inventor has considered three liability categories. LC1 was set up for unaffected or non-overlapping cases and was labeled “1 1” in the standard format of the linkage family tree. Permeability (1-P) in LC1 is 0.9 (+ / +), 0.05
(+ / DUP) and 0.01 (DUP / DUP). LC2 was set up for “heterotypic duplication” and was entered as “2 2” in the form of a linked family tree. Permeability (P) in LC2 is 0.05 (+ / +), 0.90
(+ / DUP) and 0.05 (DUP / DUP). LC3 was set up for “homotype duplication” and was entered as “2 3”. The penetration rates in this case were 0.01 (+ / +), 0.05 (+ / DUP) and 0.90 (DUP / DUP).

Furthermore, the present inventor considered a model having “almost perfect permeability”. In this case, the permeability (1-P) at LC1 is 0.99 (+ / +), 0.01
(+ / DUP) and 0.001 (DUP / DUP), and the permeability (P) at LC2 is 0.001 (+ / +), 0.99
(+ / DUP) and 0.01 (DUP / DUP), and the penetration rate (P) at LC3 is 0.001 (+ / +), 0.01
(+ / DUP) and 0.99 (DUP / DUP).

The inventor assumes that at both trait loci (D or DUP), the frequency of the disease allele or wild type allele (q) is the same, and their values are psychotic phenotypes in the general population. And a similar value: q≈0.075 was obtained. The effect of this was negligible in our analysis. This is because most members in the pedigree were diagnosed (typing) and therefore their genotype could be estimated in this way.

The present inventor performed a two-point linkage analysis between two loci traits using functional MLINK in the FASTLINK V2.2 program. Data was entered according to the above considerations. As pointed out in the results section, eleven models based on the phenotypes of psychosis and hyper joint syndrome were examined. In addition, in order to investigate the effects of penetrance and diagnostic criteria, an affected-only analysis was performed on the 11 models.

Furthermore, the present inventor considered four markers (LOXL1, D15S154, D15S201 and NTRK3-BP2) located within the 15q24-25 overlapping region and two markers (D15S1040 and D14S158) outside this overlap. Their map location and genetic distance between markers are publicly available (http://www.cephb.fr). In each analysis, the same allele frequency was considered for each marker locus. The inventor performed a two-point calculation between the marker and the locus trait. In addition, the genetic distance between markers was recalculated. In this way, the inventor can determine the recombination differences that occur due to the effects of 15q24-25 duplication.

Illustration of panic disease. Physical map of the 15q24-25 region where duplication exists in patients with anxiety and joint hyperkinetic syndrome. The order of the markers is based on public information (http://www-genome.wi.mit.edu/cgi-bin/contig/phys map). The position of each marker and clone was examined. The 57 STSs that span this region are shown on the horizontal bar that represents this chromosomal region. Arrows indicate both ends on the centromere side (CEN) and the telomere side (TEL). Markers were displayed at regular intervals. The genetic distance shown below the map is based on the published data. YAC 33iF3 was obtained from ICI and all other YAC clones were obtained from CEPH. PAC and BAC are preceded by P or B, respectively. Black circles in YAC, PAC and BAC represent STS in the clone, and white circles represent STS not present in the clone. A combination of a vertical arrow and a horizontal bar indicates the position of a gene present in a YAC, PAC or BAC clone. Only RBP1 is located outside the 15q24-25 region.

Claims (1)

Use any duplication of 15q24-25 arbitrary genomic sequences limited by D15S925 (proximal end) and D15S736 (distal end) as boundaries, or any within 15q24-25, an overlapping region of the human chromosome Familial and sporadic onset, characterized by the use of overexpressed proteins of polypeptides encoded by the genomic sequence of or expressed from other regions of the genome as a result of 15q24-25 duplication Methods for testing for anxiety and related diseases, mainly including agoraphobia, social phobia, panic attacks and panic diseases.