WO2000005407A2 - DUPLICATIONS OF HUMAN CHROMOSOME 15q24-25 AND ANXIETY DISORDERS, DIAGNOSTIC METHODS FOR THEIR DETECTION - Google Patents
DUPLICATIONS OF HUMAN CHROMOSOME 15q24-25 AND ANXIETY DISORDERS, DIAGNOSTIC METHODS FOR THEIR DETECTION Download PDFInfo
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Definitions
- Panic disorder, agoraphobia, social phobia and other anxiety disorders affect 5-10% of the general population. There are no biochemical, cytological or molecular tools for the diagnosis of anxiety disorders. Moreover, the gene or genes predisposing to anxiety disorders have not yet been localised. We have studied the clinical association between panic/agoraphobia and joint hypermobility syndrome, and have identified several pedigrees in which these disorders cosegregate. We have detected a 10 centiMorgan (cM) duplication of human chromosome 15 (15q24-25) in the affected subjects of families with several members suffering from anxiety and depression disorders. The 15q24-25 duplication segregates with panic disorder, agoraphobia, social phobia, depression and joint hypermobility syndrome.
- cM centiMorgan
- the 15q24-25 duplication is strongly linked to panic disorder, agoraphobia, social phobia and joint hypermobility syndrome (lod score 4.9).
- Affected-only analysis for the phenotype defined only by the anxiety disorders gave a lod score of 3.36. All but one of the 45 subjects of these families with these anxiety disorders had the 15q24-25 duplication.
- Mosaicism was detected in 80% of the affected subjects, with 40-70% of their lymphocytes having the 15q24- 25 duplication.
- the duplicated region contains 10 known genes of which NTRK3 and LOXLl are likely to be involved in anxiety and joint hypermobility.
- this genomic mutation which is present in 7% of the general population, is the major susceptibility mutation for panic disorder, agoraphobia, major depression and social phobia in familial and sporadic cases.
- Anxiety disorders are neurotic alterations that include generalised anxiety disorder, phobic disorders, panic disorders (panic attacks, panic disorder and agoraphobia) and obsessive-compulsive disorders.
- the prevalence of this group of alterations is estimated in about 10% in the adult population and up to 5% in infantile patients.
- Several million people worldwide are affected by anxiety disorders, but the actual prevalence rates of these alterations are probably higher.
- Anxiety and panic disorders aggregate in families.
- the familial transmission of anxiety disorders has often been explained by common familial environmental factors. Twin studies of anxiety disorders have shown a high concordance among monozygotic twins.
- the mode of familial transmission of panic disorder is unclear, but it has been suggested that anxiety, panic attacks and agoraphobia have an autosomal dominant pattern of inheritance with incomplete penetrance.
- a major gene is supposed to be involved in panic disorder, multifactorial/polygenic inheritance has also been postulated.
- joint hypermobility has been further been clarified by the identification of features of anxiety in about 70% of patients with joint hypermobility 3 .
- Joint hypermobility is about 17 times higher in patients with panic disorders than in control subjects.
- the investigation of these associations may lead to the identification of susceptibility mutations for anxiety disorders.
- the 15q24-25 mutation is the major genetic alteration of susceptibility for these anxiety disorders .
- NTRK3 as a candidate for anxiety disorders
- NTRK3 neurotrophin-3 receptor
- TRKC neurotrophin-3 receptor 6 ' 1
- neurotrophic factors have potential importance in neurodegenerative diseases, they have other roles and it has been suggested that they participate in psychiatric disorders. Thus, neurotrophic factors are involved in plasticity of the nervous system and may mediate the changes in neural connections associated with learning and memory.
- anti- depressant drugs cause modifications of the levels of expression of neurotrophins and their receptors, in particular neurotrophin-3 (NT-3) .
- NTRK3 is abundantly expressed in the noradrenergic neurons of the forebrain, including the cerebral cortex, hippocampus, thalamus and hypothalamus, and is the only neurotrophin receptor detected in the locus coeruleus (LC) .
- the LC is the principal norepinephrine (NE) containing nucleus in the CNS, playing a major role in behavioural arousal in response to novel or stressful stimuli.
- NE norepinephrine
- a significant increase in central noradrenergic function is relevant for the pathogenesis of panic disorder because the NE system integrates and coordinates fear responses to threatening stimuli.
- NTRK3 The specific expression of NTRK3 in noradrenergic neurons and its location in the duplicated chromosomal region in patients with panic disorders, which might cause its over- expression, argues for NTRK3 as an excellent candidate for anxiety disorders.
- the over-expression of NTRK3 in the LC might induce an excessive trophic and proliferative effect on NE neurones.
- stress and antidepressants regulate NT-3 expression in the LC.
- the major source of NE in the forebrain is the LC, the final consequence of the over-expression of NTRK3 would be an over-activity and enhanced efficacy of the NE synapses, resulting in a dysregulation of the NE response. This would decrease the emotional arousal threshold of the individual and alter the alarm/fear regulatory system.
- the 15q24-25 duplicated region may contain more than 50 genes, of which only 10 have been precisely mapped.
- NTRK3 and LOXLl are good candidates to explain the anxiety disorders presented here and joint hypermobility syndrome.
- the development of transgenic mice that over-express these genes might provide models for pharmacological and clinical studies of these common alterations.
- Joint hypermobility with Beighton scores 3 equal or higher than 4 were detected in 65 (44%) subjects of these families.
- Panic disorder and/or agoraphobia co-segregated with joint hypermobility syndrome in 64% of patients, social phobia in 59%, and simple phobia in 46%.
- YACs 802-b-4, 929-C-7, 750-b-10, 891- e-7 and 753-h-ll clearly demonstrated an interstitial duplication at 15q24-25 (named dupl5q24-25) in affected subjects ( Figure 1) .
- Figure 2 To determine the extent of the duplication, a YAC/PAC contig covering the 15q24-25 region was constructed ( Figure 2) .
- a total of 41 YACs, 3 PACs and 2 BACs were used in the construction of the map. The information on the chimaerism and the STS content of the YAC clones has been deposited at CEPH.
- the duplicated region spans about 10 cM of human chromosome 15 between markers D15S739 (proximal) and D15S930 (distal).
- the region contains 10 known genes (LOXLl , CRABP1 , IREE2 , NIC3A, NIC5A, NIC4B, FAH, IL1 6, NMB , and NTRK3) , which were localised to the duplicated region by PCR of their STSs.
- a large number of ESTs are known to be in the duplicated region, which might contain between 50 and 200 genes .
- the centromeric limit of the duplication was located between YACs 753-h-ll and 875-a-3, which do not overlap (within the deleted YAC 33iF3) , and the telomeric limit between YACs 802-b-4 and 966-a-2, which do not overlap either (within the deleted YAC 964-f-8) .
- telomeric duplication was observed as a direct (tandem) form or an inverted form, depending on the relative distance of the signals seen by FISH.
- the strength of the relationship between the 15q24-25 duplication and several anxiety disorders and joint hypermobility syndrome was analysed in the seven families by linkage analysis.
- the anxiety phenotypes were studied under five models: 1) panic disorder and/or agoraphobia, 2) social phobia, 3) simple phobia, 4) panic disorder and/or agoraphobia and/or social phobia, and 5) same as 4 and/or simple phobia. These five models were also studied adding the joint hypermobility syndrome (> 4 Beighton criteria) , and this phenotype was also tested as a unique trait.
- Table 1 shows the lod score values for linkage between the 15q24-25 duplication and the 11 models analysed under 78% and almost complete penetrance of the duplication.
- the phenotypic characteristics of 93 subjects of .these seven families and their association with the 15q24-25 duplication are shown in Table 2. The proportion of duplicated subjects (72%) in these families is higher than in the general population (7%) . Remarkably, all patients with social phobia had the duplication. Forty-four of the 45 patients (98%) with panic disorder and/or agoraphobia and/or social phobia had the 15q24-25 duplication. Similarly, 87% of subjects with joint hypermobility syndrome had the 15q24- 25 duplication. When the clinical criteria was the coexistence of one or several psychiatric traits and joint hypermobility syndrome, all subjects had the duplication.
- dupl5q24-25 was detected in all 50 affected patients and in only 10 of 135 unrelated controls from the general population (Chi-square 138.5, with Yate's correction; P ⁇ 0.0001, by Fisher's exact test; Odds ratio 1,207.2) .
- haplotypes A total of 28 different haplotypes was generated with these 6 markers from a total of 71 duplicated chromosomes in which it was possible to unambiguously determine the mutated haplotype.
- the founder dupl5q24-25 haplotypes were easily identified among the other haplotypes with the duplication carried by new members of the family.
- This high number of haplotypes (42% of the chromosomes with the duplication have different haplotypes) indicates independent origins and suggests that this mutation is easily generated.
- both members carried the duplication. This represents 44% of the couples in which both members were analysed for the duplication and is a clear evidence of assortative matting for the phenotype (s) associated with the 15q24-25 mutation.
- FISH Fluorescent in situ hybridisation
- FISH 15q24-25 duplication in lymphocyte metaphase cells. FISH was performed as described previously 4 using probes t216-l and c251-3 ( Figure 1) .
- IL1 6 which is sufficiently expressed in blood lymphocytes.
- Over-expression of IL1 6 was studied by quantitative PCR.
- the homozygous individual used against a control patient showed an approximately double concentration of cDNA in the peripheral blood lymphocytes ( Figure 4a).
- the analysis of the expression of other genes in the region should exclude the possibility that the duplicated genes are silenced in patients with mutation dupl5q24-25.
- Total RNA from cultured cell lines of affected and control individuals was amplified by competitive PCR 5 .
- the endogenous IL16 mRNA was amplified using primers corresponding to a 326bp fragment (STS WI-7689) of the cDNA sequence of the gene (GenBank G06653) (forward primer: 5'-TCC CAT AAC CGC TGA TTC TC-3' and reverse primer : 5'-AAT AAA TGT CAC TGT TTG GGG G- 3' ) .
- An internal standard was constructed with a 42 nucleotides primer in which 20 nucleotides at the 5' end correspond to 76 nucleotides upstream. Amplification with these primers results in a 228bp product that was further subcloned in a pMOS Blue-T vector (Amersham) and used as internal standard.
- the IL16 mRNA PCR product could be due to residual genomic DNA
- control PCR amplifications of each sample were performed with previous treatment with RNAase A. Quanti ta tive Southern blot ting analysis of the NTRK3
- NTRK3 neurotrophin 3 receptor
- Primers corresponding to a 222bp fragment of PROC gene were: PC111, fluorecently labelled at 5' end: 5'-GTG CTA GTG CCA CTG TTT GT-3' and PC112 5'-ATC ACC ACC TAG CTC TCT TC-3' .
- Samples were run in an ABI PRISM 373 DNA sequencer and the results were processed by GENESCAN and GENOTYPERa softwares.
- a PCR co-amplification of a fragment of NTRK3 and a control fragment of the PROC gene showed dosage differences due to the 15q24-25 duplication ( Figure 4c) .
- Metaphase chromosome spreads were prepared after harvesting 72 h amniocyte cultures and lymphocyte cultures from peripheral blood of the patients according to standard methods. GTL banding techniques were performed in all cases. Peripheral blood cultures were synchronised with methotrexate to obtain high resolution chromosomes at a level of 900 G bands. Non stained slides were kept at -20°C until hybridisation. Prior to hybridisation, slides were baked at 55°C for 30 min. Slides were mounted with 40 ⁇ l of antifade solution (Vector Laboratories) containing 0.5 mg/ml of propidium iodide or 150 ng/ml of DAPI .
- each probe 400 ng of each probe were then ethanol precipitated with 1 mg of cotl- DNA (GIBCO-BRL) and 1 mg of salmon sperm DNA (Sigma) and resuspended in a hybridisation mix containing 50% formamide and 50% of 12xSSC and dextran sulphate.
- GEBCO-BRL cotl- DNA
- salmon sperm DNA Sigma
- FISH FISH was performed as described elsewhere 4 . Briefly, 70 ng of each probe (7 ml of the hybridisation mix) were applied to each slide and sealed with rubber cement (when hybridising more than one probe at a time, 5 ml of each probe were applied instead) . After heat denaturation of the slides at 80 °C for 8 min, they were incubated overnight at 37 °C in a humid chamber. Post hybridisation washes were performed in three changes of 50% formamide and 50% 2xSSC followed by three changes of 2xSSC all at 42°C Then slides were incubated in blocking solution (Boehringer Mannheim) for 10 min.
- Detection was performed by incubating the slides with either avidin-FITC (for probes labelled with biotin) or with anti-digoxigenin-TRITC (for probes labelled with digoxigenin) (both antibodies from Vector Laboratories) for 20 min at 37 °C in a humid chamber and then washed in two changes of 4xSSC/0.1% Tween-20.
- biotin signals were amplified once by incubating the slides with biotinylated anti-Avidin (Vector Laboratories) for 20 min, washed as mentioned above and the incubated again with Avidin-FITC for another 20 minutes and washed again.
- Slides were mounted with an antifade solution (Vectashield, Vector Laboratories) containing 0.1 mg/ml of DAPI . Slides were studied under a fluorescence microscope (VANOX, Olympus) equipped with the appropriate filter set. Images were analysed with the Cytovision system (Applied Imaging Ltd. , UK) . For each hybridisation, at least 20 metaphases were studied.
- the detection of mosaicism by FISH was made taking into account the technique efficiency in known diploid controls. Patients were considered duplicated when the percentage of nuclei displaying three hybridisation signals exceeded two standard deviations the mean of false trisomic signals in control samples (30%) n . One hundred nuclei were analyzed in each control and test sample.
- dosage analysis was assessed trough PCR amplification of two fragments in the same reaction tube, one corresponding to a 3' cDNA fragment of the NTRK3 gene (GenBank G913721) and the other to a control fragment (exon 1) of PROC gene.
- PCR reactions were carried out in a 10 ml volume containing 10 ng of genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl2 PCR buffer, 0.1 U of Taq Polymerase (Boehringer
- RNA from cultured cell lines of affected and control individuals was prepared using the guanidium chloride method and cesium chloride gradient.
- Competitive PCR was according to Celi 5 .
- RT-PCR was attempted with 5 ⁇ g of total RNA with Superscript and random primers (Gibco, BRL) .
- Co-amplification of target and internal standard was performed as follows: 1 ⁇ l of RT product was included in a mixture containing 0.2 mM dNTPs, 1.5 mM MgCl2 PCR buffer, 0.1 U of Taq Polymerase and 0.5 ⁇ M target sequence primer pair and 1 ⁇ l of varying amounts of internal standard.
- PCR conditions were as follows: 94 °C for 4 min to denature followed by 30 cycles of 94°C for 30 sees, 56°C for 30 sec, and 74°C for 30 sec.
- YACs were obtained from Fondation Jean Dausset-CEPH (www. cephb.fr) and clones were confirmed to map to chromosome 15q24-25 using FISH analysis and further refined by PCR amplification of STSs content.
- the CEPH human ("mega") YAC was also screened by PCR amplification of a fragment of the LOXLl gene but no positive clones were obtained.
- the ICI humanYAC library (filters maintained and distributed by HGMP-Resource Center, UK, www.hgmp.mrc.ac.uk.) was screened using primers of exon 7 of LOXLl gene (Gen Bank G307145) as a probe and we found a positive YAC containing LOXLl (33i-F3) .
- PCR amplifications were carried out in 25 ⁇ l reactions with 100 ng of template DNA, 0.5 ⁇ M of each oligonucleotide primer, 0.2 mM dNTPs, 1.5 mM MgCl2 PCR buffer and 0.1 U of Tag Polymerase. Each reaction was denatured for 5 min at 94°C, followed by 35 cycles of 30 sec at 94°C, 30 sec at 50- 65°C (depending on the primer) and 4 sec at 74 C C, as well as a final 5 min extension at 72°C.
- the repeat sequence is (CA)24 and the primers used to amplify this marker were: BP2F 5 ' -TTG CTT GAA GGG CAC CTG-3' and BP2R 5'- AAC ATC CTG GGT ACA TGC-3'.
- the GT strand oligonucleotide primer flanking the (GT)n sequence was end-labelled with polynucleotide kinase (USB) .
- PCR was performed using standard conditions in a 25 ⁇ l reaction volume in a mixture containing 100 ng of genomic DNA, 1 ⁇ M each oligonucleotide primer, 0.2 mM dNTPs, 1.5 mM MgCl2 PCR buffer and 0.1 U of Taq Polymerase.
- the amplification conditions consisted of an initial denaturation step of 5 min at 94 °C, followed by 30 cycles of 30 sec at 94°C, 30 sec at 58-65°C (depending on the primer) and 40 sec at 74 °C, as well as a final 5 min extension at 72°C.
- Reaction products (2 ⁇ l) were mixed with 2 X cf formamide stop solution and electrophoresed in a 6% pclyacrylamide DNA sequencing gel at 40 W for 3.5 to 4 hours. Gels were dried and autoradiographed for 4-12 hours by exposure to X-ray film (Curix, Agfa) at -80°C Markers D15S322 , D15S1040 and NTRK3-BP2 were amplified following the same above reaction conditions but reverse primers were not end labelled. Instead, 2 ⁇ l of reaction products were mixed with 2 ⁇ l formamide stop solution and electrophoresed in a 5% poiyacrylamide DNA sequencing gel at 40 W for 4-5 hours. Gels were silver stained and dried.
- the 15q24-25 duplication was entered in the analysis as at affection status or dichotomous locus type.
- the genotype inherited from the respective parent and present in the zygote as the true ger.ctype, which is DUP/DUP, DUP/+ or +/+; being DUP the Xeleterious duplicated allele" carrying the 15q24-25 mutation and + the non duplicated wild type allele.
- the c served phenotype originates from this zygote genotype, vr.icr. is observed by FISH and includes mosaicism.
- the penetrance values for this locus trait are the probabilities tc observe by FISH a final phenotype given the zygote original genotype. Additionally, we have also considered the possibility of cytogenetic diagnostic errors (c) due to probe efficiency or missinterpretation. In this way we considered 3 liability classes (LC) : LCI was built for the unaffected or non-duplicated cases, coded as "1 1" in the linkage pedigree standard format (for affection status and liability class, respectively) . The penetrances (1-P) for LX were 0.9 (+/+), 0.05 (+/DUP) and 0.01 (DUP/DUP).
- LC2 was built for "duplicated in heterozygosis", entered as “2 2" in the linkage pedigree format.
- the LC2 penetrances (P) were 0.05 (+/+), 0.90 (+/DUP) and 0.05 (DUP/DUP).
- LC3 was built for "duplicated in homozygosis” cases, entered as “2 3" and with the following penetrances (P) : 0.01 (+/+), 0.05 (+/DUP) and 0.90 (DUP/DUP).
- penetrances P
- FISH with cosmids t216-l (in green) and c251-3 (in red) shows the interstitial duplication of both probes in a quite more centromeric region of chromosome 15, while the normal chromosome 15 shows the normal pattern of hybridisation of both probes.
- Figure 2 Physical map of the 15q24-25 region, which is duplicated in patients with anxiety and joint hypermobility disorders. The order of markers is based on available information
- TEL ends.
- the markers are spaced at equal intervals.
- FISH analysis in the interphase nuclei of lymphocytes and sperm of patients with panic disorder and joint hypermobility syndrome a/ Two interphase nuclei (Gl state) of a patient with the 15q24-25 in heterozygosity hybridised with cosmid t216-l (in green) and a control cosmid of chromosomal region 21q22.1 (in red), b/ Two interphase nuclei of an heterozygous patient for dupl5q24-25 hybridised with cosmid t21 ⁇ -l indicating mosaicism (100 cells were examined) .
- Ratio values for heterozygous patients are higher than the ones for control samples .
- the homozygous patient has higher peak ratio values than heterozygous individuals (1.3 fold) and the control subject (about 1.7 fold).
- the genotypes are n/n for the normal subject, T/n for the heterozygous for the telomeric duplication, and T/C for a compound heterozygous for the telomeric and centromeric duplications.
- the upper band corresponds to the target cDNA (IL16) and the lower fragment to varying dilutions of the internal standard (I.St.), which are 32, 19.2, 9.6, 2.8, 1.6, 0.9 and 0.3 attomoles, for lanes 1 to 8, respectively.
- Lanes 9 to 11 correspond to the I.St., IL1 6 and blank controls, respectively.
- the equivalence point for a 1:1 ratio between IL16 and I.St, in the control individual corresponds to dilution 3 (9.6 attomoles) ; while in the homozygous patient the equivalence point lies between lane 1 (32 attomoles) and lane 2 (19.2 attomoles) .
- the genotypes n/n and T/C correspond to normal and compound heterozygous for the centromeric and telomeric 15q24-25 duplications.
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AU51600/99A AU5160099A (en) | 1998-07-23 | 1999-07-14 | Duplications of human chromosome 15q24-25 and anxiety disorders, diagnostic methods for their detection |
JP2000561353A JP2003525579A (en) | 1998-07-23 | 1999-07-14 | Association between duplication of human chromosome 15q24-25 and anxiety disease, and diagnostic method for detecting them |
EP99936540A EP1100967A2 (en) | 1998-07-23 | 1999-07-14 | DUPLICATIONS OF HUMAN CHROMOSOME 15q24-25 AND ANXIETY DISORDERS, DIAGNOSTIC METHODS FOR THEIR DETECTION |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001006848A1 (en) * | 1999-07-23 | 2001-02-01 | Xavier Estivill Palleja | Transgenic mice and overexpression model of the gene ntrk3 (trkc) based thereon for the study and monitoring of treatments of anxiety, depression and related psychiatric diseases |
WO2006048896A1 (en) * | 2004-09-20 | 2006-05-11 | Decode Genetics Ehf. | Association of gene expression and psychiatric disorders |
WO2009043584A2 (en) * | 2007-10-05 | 2009-04-09 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Use of polymorphisms in the tmem132d gene in the prediction and treatment of anxiety disorders |
JP2013101152A (en) * | 2013-02-25 | 2013-05-23 | Atsuo Sekiyama | Index agent for living body load and method for measuring living body load |
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WO2004074512A1 (en) * | 2003-02-19 | 2004-09-02 | UNIVERSITé LAVAL | Method for determining susceptibility to schizophrenia |
EP1680518A2 (en) * | 2003-09-19 | 2006-07-19 | Decode Genetics EHF. | Inversion on chromosome 8p23 is a risk factor for anxiety disorders, depression and bipolar |
US8309539B2 (en) * | 2005-09-09 | 2012-11-13 | Pherin Pharmaceuticals, Inc. | Acute treatment of social phobia |
US9389588B2 (en) | 2011-12-09 | 2016-07-12 | Cartier International Ag | Method for adjusting the chronometry of a timepiece movement intended to operate in a low-pressure atmosphere |
US20180330050A1 (en) * | 2015-11-16 | 2018-11-15 | Mayo Foundation For Medical Education And Research | Detecting copy number variations |
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EP0512181A1 (en) * | 1991-05-07 | 1992-11-11 | N.V. Innogenetics S.A. | Process for the in vitro diagnosis of chromosomal anomalies liable to be correlated with CMT1A disease |
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- 1999-07-14 EP EP99936540A patent/EP1100967A2/en not_active Withdrawn
- 1999-07-14 AU AU51600/99A patent/AU5160099A/en not_active Abandoned
- 1999-07-14 JP JP2000561353A patent/JP2003525579A/en active Pending
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001006848A1 (en) * | 1999-07-23 | 2001-02-01 | Xavier Estivill Palleja | Transgenic mice and overexpression model of the gene ntrk3 (trkc) based thereon for the study and monitoring of treatments of anxiety, depression and related psychiatric diseases |
WO2006048896A1 (en) * | 2004-09-20 | 2006-05-11 | Decode Genetics Ehf. | Association of gene expression and psychiatric disorders |
WO2009043584A2 (en) * | 2007-10-05 | 2009-04-09 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Use of polymorphisms in the tmem132d gene in the prediction and treatment of anxiety disorders |
WO2009043584A3 (en) * | 2007-10-05 | 2009-06-18 | Max Planck Gesellschaft | Use of polymorphisms in the tmem132d gene in the prediction and treatment of anxiety disorders |
JP2013101152A (en) * | 2013-02-25 | 2013-05-23 | Atsuo Sekiyama | Index agent for living body load and method for measuring living body load |
Also Published As
Publication number | Publication date |
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AU5160099A (en) | 2000-02-14 |
US6225057B1 (en) | 2001-05-01 |
EP1100967A2 (en) | 2001-05-23 |
JP2003525579A (en) | 2003-09-02 |
WO2000005407A3 (en) | 2000-04-27 |
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