Human Molecular Genetics, 2002, Vol. 11, No. 7 853-860
© 2002 Oxford University Press
Hyperekplexia associated with compound heterozygote mutations in the ß-subunit of the human inhibitory glycine receptor (GLRB)
1Department of Psychological Medicine and 2Department of Medical Genetics, University of Wales College of Medicine, Cardiff CF14 4XN, UK, 3The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia, 4Department of Medical Genetics and 5Department of Paediatrics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium and 6Department of Molecular Medicine, University of Auckland Medical School, Private Bag 92019, Auckland, New Zealand
Received January 23, 2002; Revised and Accepted January 30, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hyperekplexia (MIM: 149400) is a neurological disorder characterized by an excessive startle response which can be caused by mutations in the
1-subunit (GLRA1) of the heteropentameric human inhibitory glycine receptor (hGlyR). These receptors facilitate fast-response, inhibitory glycinergic neurotransmission in the brainstem and spinal cord leading to a rapid modification and reduction of the excitatory startle response. Mutations in the ß-subunit of GlyR (glrb) occur in a murine model of hyperekplexia (spastic), but have not been detected in human hyperekplexia. Following mutation analysis of the human ß-subunit of hGlyR (GLRB) in a cohort of 22 hyperekplexia patients, we provide evidence to confirm that GLRB mutations can cause human hyperekplexia. A missense (G920A resulting in G229D) and a splice site mutation (IVS5+5G
A) occurred together in a compound heterozygote with a transient hyperekplexia phenotype. Exon trap analysis revealed that IVS5+5GA results in the exclusion of exon 5 from GLRB transcripts. Electrophysiological studies showed reduced sensitivity to agonist mediated activation of the 1ß (G229D) GlyR suggesting that GlyR ß-subunits are not restricted to conferring modulatory influences and maintaining structural integrity, but may also play a functional role in hGlyR ligand binding. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The human inhibitory glycine receptor (hGlyR) is a heteropentameric, ligand-gated ion channel (LGIC) composed of three ligand-binding
1-subunits (GLRA1) plus two ß-subunits (GLRB) (1–3). These receptors facilitate fast-response, inhibitory glycinergic neurotransmission in the brainstem and spinal cord and are clustered on the post-synaptic membrane by association of the ß-subunit with the organizational protein, gephyrin (4–11). A similar pentameric arrangement of subunits is observed in other LGICs such as nicotinic acetylcholine receptors, GABAA+C (
-aminobutyric acid) receptors and serotonin 5HT3 receptors (12–14). Each subunit contains four membrane spanning regions (M1–M4), a large extracellular N-terminal domain, an extended intracellular loop between M3 and M4, and a M2 domain forming an internal channel pore (15–17).
A degree of receptor heterogeneity is generated by the expression and alternative splicing of a further three agonist binding -subunits (2–4) in the central nervous system (CNS), although the 1-subunit predominates in neonatal and adult CNS whereas the 2-subunits are expressed as homo-oligomers in embryonic CNS (18–21). The ß-subunit is widely expressed in the developing and adult CNS, but absent in embryonic and fetal tissues (22). As well as conferring a structural role, GLRB contributes to chloride channel functioning as demonstrated by changes in single channel conductance and picrotoxin (PTX) sensitivity of heteromeric 1ß receptors in comparison to the homomeric 1 receptor (23–25).
Dominant (26–32) and recessive (33–35) missense and nonsense mutations in GLRA1 occur in a proportion of familial and sporadic hyperekplexia, a neurological disorder characterized by an abnormal startle response, neonatal hypertonia, nocturnal myoclonus and a chronic accumulation of injuries caused by startle-induced falls (36–37). The deleterious effects of the GLRA1 mutations have been demonstrated by electrophysiological studies of recombinantly expressed receptors, confirming the altered receptor functioning (38–41). A homozygous deletion of GLRA1 exons 1–6 confirmed that a null GLRA1 genotype is not lethal in humans, in contrast to the glra1 murine model, oscillator (42–44). Consequently, human neurotransmission must have developed alternative, compensatory inhibitory mechanism(s) that buffer the quantitative/qualitative loss of hGlyR receptor function but not with sufficient effect to avoid the onset of an excessive startle response. Two further murine models of hyperekplexia have been extensively characterized, namely spasmodic (glra1 point mutation) and spastic, the latter exhibiting a complex neuromotor phenotype caused by a recessive intronic LINE-1 insertion into glrb (45–47). This insertion acts to drastically reduce expression of murine glrb leading to a low synaptic density of functional GlyRs on the postsynaptic membrane (48). To date, no mutations in GLRB have been found in human hyperekplexia and spastic remains a solitary but indicative example of GLRB candidacy.
Despite the apparent absence of GLRB mutations in human hyperekplexia (49), we hypothesized that a proportion of sporadic hyperekplexia would be caused by GLRB mutations and detectable by using a combination of mutation detection techniques. For one patient, we present evidence of compound heterozygote mutations (missense and nonsense) associated with a transient hyperekplexia phenotype. The maternal nonsense mutation at an exon 5 splice junction generates GLRB transcripts lacking exon 5, whereas electrophysiological characterization of the paternal missense mutation indicates that ß-subunits are not restricted to conferring receptor modulation or maintaining structural integrity, but may also play a functional role in GlyR ligand binding.
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RESULTS |
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Clinical presentation
A total of 22 patients with sporadic hyperekplexia were obtained from several neurological centres in the UK and northern Europe, with permission from patients or parents of affected patients. Our diagnostic criteria for hyperekplexia included a history of (i) neonatal hypertonia; (ii) the nose tap reflex; and (iii) a fall down startle response to sudden and unexpected stimulus.
The patient subsequently identified as a compound heterozygote for GLRB mutations was born at term (Apgar, 6/8; weight, 3750 g; length, 53 cm; OFC, 34 cm) and was the first child of healthy, non-consanguineous parents. He was born with vacuum extraction and was slightly hypotonic after birth. He was admitted at the Neonatal Intensive Care Unit 3 h after birth because of generalized hypertonia and hyper-reactivity. Physical examination revealed exaggerated startle responses, normal spontaneous movements, hypertonia, and myoclonus elicited by testing the tendon reflexes or tapping on the back and absent Moro reflex. Treatment with clonazepam (0.05 mg/kg/day) resulted in reduced hypertonia but had no effect on the startle responses. Greatly exaggerated startle responses and brisk tendon reflexes persisted and were noted at 2, 4, 8 and 10 months. Mental development seemed normal, though motor development was slightly delayed. He sat unsupported at 8–9 months of age, walked with support at 13 months and walked unsupported at 18 months. By 16 months hypertonia and hyper-reflexia were less evident on physical examination but a hiatus hernia with gastro-esophageal reflux was noted. At 20 months tone was normal, though the tendon reflexes were still brisk. However, by 3 years of age the phenotype had improved such that an abnormal startle response could only be invoked by tactile stimulus of the perioral region.
GLRB mutations
Having excluded mutations in GLRA1 for the majority of the patient cohort (35), we screened the exons and flanking intronic regions of GLRB in the 22 unrelated patients with sporadic hyperekplexia using a combination of single strand conformation polymorphism (SSCP) and bi-directional di-deoxy fingerprinting (ddF) mutation detection (see Materials and Methods). Variant profiles were detected in exons 3, 5, 7 and 8, with exon 5 exhibiting two distinct patterns of variation. Sequencing revealed several polymorphisms present in both patients and controls (Fig. 1A and Table 1). However, two sequence changes were found in a single child with hyperekplexia (Fig. 1B) but not in 96 unrelated controls or the remaining cases. These mutations were in intron 5 (IVS5+5GA) and in exon 7 (G920A). The nucleotide change in exon 7 was predicted to result in an aspartic acid (GGC) for glycine (GAC) substitution in position 229 (G229D), 13 residues preceding the first transmembrane domain (TM1) of the GLRB polypeptide.
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Sequencing DNA from the unaffected parents indicated that the child is a compound heterozygote, with the IVS5+5G
A mutation inherited from the mother and the G920A mutation from the father (Fig. 1B and C). A search of the Cardiff Human Gene Mutation Database (http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html) revealed that substitution of conserved G at IVS+5 has been documented on 90 occasions in 64 distinct genetic diseases, causing radically altered splicing patterns (50–55). Despite successful 3'-rapid amplification of cDNA ends (RACE) of GLRB cDNA from a human brain RNA control (data not shown), the GLRB message was not detected in either patient or control lymphocytes. Consequently we adopted an in vitro exon trap assay to assess the functional effect of GLRB IVS5+5GA.
Splice site assay
Exon trap analysis (56) of a patient-specific genomic fragment encompassing exons 4 and 5 was used to demonstrate that the IVS5 mutation decreased the efficiency of splicing in of exon 5 in the GLRB gene. RT–PCR of exon trap products yielded three PCR products which correspond to the splicing in, or deletion, of exons 4 and 5 (Fig. 2A). These were confirmed by direct sequencing of each PCR product. In triplicate experiments, the presence of the IVS5 mutation resulted in more of the products corresponding to the deletion of exon 5 (+exon4, 
exon5) relative to products containing exon 5 (+exon4, +exon5) compared with wild-type sequence. The levels of exon5 PCR products for the IVS5 mutation were determined semi-quantitatively and demonstrated a significant increase of 38% (P < 0.0005, Student’s t-test) compared with wild-type sequence (Fig. 2B).
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Mutagenesis and electrophysiology
The
1 and ß-subunit cDNA constructs were co-transfected into 293 cells at a ratio of 1:10 to assist in expression of heteromeric receptors. As heteromeric 1ß GlyRs are less sensitive to inhibition by PTX in comparison to homomeric 1 GlyRs, the IC50 for PTX was used here as an assessment of the incorporation of the ß-subunit. A PTX inhibition curve was obtained for each cell to confirm heteromeric receptor expression before continuing the experiment. For homomeric 1 GlyRs the PTX IC50 was 15.39 ± 1.11 µM (n = 5), whereas wild-type 1ß GlyRs displayed an IC50 of 346.1 ± 24.5 µM (n = 4) and 1ß(G229D) receptors were found to have an IC50 of 371.7 ± 29.4 µM (n = 6) (Fig. 3A).
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Concentration-response curves were constructed from the peak whole-cell current recorded in response to a range of concentrations of glycine applied to the cells expressing wild-type
1ß or 1ß(G229D) receptors and the data were fitted with the Hill equation (Fig. 3B). The 1ß(G229D) GlyRs displayed a significant 3.4-fold increase in EC50 for glycine (60.9 ± 6.2 µM, n = 6) when compared to wild-type 1ß GlyRs (17.9 ± 2.7 µM, n = 4; P = 0.010, randomization test), with little effect upon the Hill coefficient (2.2 ± 0.3 versus 2.9 ± 0.3, respectively). There was also no discernible difference in the maximum currents for 1ß (4.5 ± 4.0 nA) and 1ß(G229D) GlyRs (4.7 ± 2.9 nA).
Single channel recordings were made from outside-out patches at approximately equipotent concentrations of glycine for 1ß and 1ß(G229D) GlyRs. Examples of channel openings are shown in Figure 4A. The single channel chord conductance (assuming a reversal potential of 0 mV) was not different between the 1ß (46.9 ± 0.7 pS; n = 4) and 1ß(G229D) GlyRs (46.3 ± 1.4 pS; n = 3). Open period distributions were fitted with a mixture of three exponential densities in each case (Fig. 4B). The mean time constants of each exponential density did not show any significant difference between the wild-type and mutant (Table 2). The overall mean open time was 1.31 ± 0.28 ms (n = 4) for 1ß and 1.18 ± 0.27 ms (n = 3) for 1ß(G229D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Compound heterozygote mutations were identified in a young boy with a transient hyperekplexia phenotype. The fact that neither parent was, or had been, affected suggests that each mutation alone is not sufficient to cause hyperekplexia and the maternal splice-site mutation demonstrates that hyperekplexia is not susceptible to haploinsufficiency of the GlyR ß-subunit. The IVS5 mutation leads to a significant increase in the levels of GLRB mRNA that lack exon 5. This will lead to the production of truncated polypeptides which would not assemble with GlyR
-subunits to form functional GlyRs in synapses resulting in a marked reduction in the number of functional inhibitory GlyRs. Since the majority of maternal GLRB transcripts are compromised, the patient is transcriptionally recessive for the paternal G229D GLRB message which evidently fails to exert a dominant effect. Indeed, the G920A mutation is outside the M1–M2 loop and the M2–M3 region where dominant hyperekplexia mutations have been described in GLRA1. Additionally, there are no descriptions of dominant hyperekplexia pedigrees mapping to linkage intervals containing GLRB (4q31.3).
The small but significant shift in the EC50 observed for the 1ß (G229D) GlyR is consistent with the recessive nature of the mutation. The reduced sensitivity to agonist-mediated activation of the 1ß(G229D) GlyR could be due to either a decrease in the affinity for the agonist or a decrease in the ability of the receptor to undergo the conformational change that opens the channel pore. Single channel analysis showed no change in the time constants from the fitted open period distributions and this suggests that there is little or no effect upon the ability of the receptor channel to open. The alternative hypothesis is that the ß-subunits contribute in some way to the binding of agonist to the receptor, either directly or via an allosteric interaction, and that this is impaired by the mutation. This would seem to be corroborated by the location of the mutation within the ß-subunit protein.
The location of Gly229 between Cys221 and Cys233 in GLRB is similar to the Cys-loop between Cys198 and Cys209 in the GLRA1 (57). Within this loop of the GLRA1, a number of residues are involved in ligand binding, forming a predicted ß-sheet structure followed by a ß-turn (58). Alignment of human and phylogenetic GlyR subunits demonstrates conservation of the glycine residue at this position (Fig. 5) and puts the Gly229 residue of the GLRB subunit in line with the residue Gly205 of the GLRA1 subunit that is thought to form the ß-turn. If this sequence forms a similar ß-sheet and ß-turn in the GLRB subunit, then substitution of Gly229 for a charged aspartic acid residue may well disrupt this structure.
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The largely transient nature of the phenotype in our case is in contrast to a more severe phenotype observed in patients with GLRA1 mutations, but is consistent with the transient neuromotor phenotype described in a transgene rescue strain of spastic (59). These mice show a gradual improvement in startle and tremor symptoms that correlates with a low but compensatory expression of glrb, thereby rescuing the strain from the more severe features of the spastic phenotype. The transient improvement of our patient’s phenotype may be due to compensation by GABAA neurotransmission, although other mechanisms such as a progressive increase in the number of gephyrin-mediated GlyR clusters at the synapse or compensation by homomeric
1-subunit GlyRs cannot be excluded.
Our results and those of another study (49) suggest that GLRB mutations are a rare cause of hyperekplexia and are unlikely to account for the majority of those cases that do not have mutations in GLRA1. A possible candidate gene for these cases is gephyrin, the protein which secures and clusters 1ß GlyR to the post-synaptic membrane (5–6,8–9,60). Indeed, heterozygote knock-out mice for gephyrin have features reminiscent of startle disease (61).
In conclusion, we confirm genetic heterogeneity in hyperekplexia by describing the first human example of disease-associated mutations in GLRB. Our functional data also suggests that GlyR ß-subunits may contribute to ligand binding action of heteropentameric receptors, a function hitherto attributed exclusively to the -subunits. Further mutagenesis experiments of critical regions within GLRB will be needed to evaluate the effects of this subunit on ligand binding and receptor function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Polymerase chain reaction
Oligonucleotide sequences for GLRB were taken from reference sources (49,62) and encompassed all GLRB exons and their immediate flanking intronic regions. Each reaction consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles of 30 s at the primer annealing temperature, 30 s for primer extension at 72°C and 30 s denaturation at 94°C. Each 25 µl reaction contained 60 ng of genomic DNA, 10 pmol of each primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 200 µM of dNTPs (Pharmacia) and 1 U Taq polymerase (Qiagen or Bohringer-Mannheim).
SSCP
Samples were prepared by denaturing 5 µl of PCR product with 7 µl of formamide dye at 94°C for 10 min. Samples were cooled and applied to 10% non-denaturing gels (Sigma 49:1) and run at 70 V for 12–16 h at room temperature or 4°C. All SSCP gels were silver-stained as described previously (63) and assessed for heterogeneous profiles.
Bi-directional ddF
Bi-directional ddF analysis was carried out as detailed by Sarkar et al. (64) and Liu et al. (65) with slight variations. The di-deoxy reactions were set up using 2.5 µl of sample-specific post-purified PCR product and 5 µl of reaction mixture (7.5 µl total volume reaction) composed of 2 µl of dNTPs (7 µM GATC), 0.25 µl of a [-33P]ddNTP (Amersham), 0.25 µl of each primer used in the initial PCR amplification, 0.75 µl of thermosequenase buffer and 1 U thermosequenase enzyme (Amersham). Following addition of the thermosequenase, a three-step sequencing protocol (Hybaid) was initiated using 35 cycles of primer annealing temperatures at 50°C for 30 s, primer extension at 72°C for 1 min and denaturation at 94°C for 30 s. Denatured samples were applied to a 10% non-denaturing MDE gel (BMA Bioproducts) and run at 15W for 4–5 h at 4°C. Gels were viewed by autoradiography with X-ograph film (Kodak) and carefully assessed for variant profiles.
Sequencing of GLRB exons
The reaction mixture was comprised of 2.0 µl of thermosequenase buffer, 1 µl of the sequencing primer at 10 pmol/µl, 1 µl of thermosequenase (4 U/µl), the 30 ng of gel-purified PCR product and H2O to make a 20 µl final volume. For each sample, four tubes were labelled G, A, T and C; to each tube, 2.5 µl of a 1:4 mixture consisting of the corresponding labelled [-33P] ddNTP and 7 µM [GATC] dNTP mix was added. To each of these tubes, 5 µl of the reaction mixture was added and the constituents mixed. Sequencing was initiated using a denaturation stage at 94°C for 5 min followed by 45 cycles of primer annealing temperatures at 55°C for 30 s, primer extension at 72°C for 1min and denaturation at 94°C for 30 s. Denatured samples were applied to a 6% denaturing gels (National Diagnostics) and run at 85 W for 1–2 h at room temperature.
Exon trap analysis
Amplification of a patient-specific genomic fragment encompassing exons 4 and 5 of GLRB was cloned into a pGEM vector (Stratagene). Clones which contained either the wild-type or IVS5 mutation in exon 5 of GLRB gene, along with 132 bp and 226 bp of 5' and 3' flanking intronic sequence, were subcloned into the exon trap vector pSPL3 (Gibco BRL). HEK293 cells were plated into six-well plates and allowed to recover for 24 h prior to transfection with the exon trap constructs using the Fugene 6 reagent according to the manufacturer’s instructions (Roche). Cells were collected 48 h post-transfection and total RNA was isolated for exon trap analysis as described previously (56).
Mutagenesis and expression of heteromeric human GlyRs
The cDNA encoding the ß-subunit of the hGlyR (53) was subcloned into the pIRES-EGFP vector (Clontech). Mutations were introduced using an oligonucleotide-directed polymerase chain reaction method (66) and were confirmed by complete sequencing of the cDNA clones. Plasmid DNA encoding wild-type or mutated ß-subunits of the hGlyR was transiently transfected into exponentially growing 293 cells (adenovirus transformed human embryonic kidney cells; ATCC CRL 1573) along with cDNA for the 1 hGlyR subunit (22) in the pCIS expression vector (67) and cDNA for the CD4 protein (ratio of 10:1:5, respectively). Transfections were performed using a modified calcium phosphate precipitation method as described previously (62). Transfected cells were identified by labelling with CD4 Dyanbeads (Dynal).
Electrophysiology
Whole-cell current recordings were made at room temperature (20–22°C) using an Axopatch 1D (Axon Instruments) amplifier, low-pass filtered at 1 kHz (4-pole Bessel, –3 dB) and digitized at 4 kHz. Single channel recordings were made at a bandwidth of 10 kHz onto digital audio tape and upon playback were low-passed filtered at 2.5 kHz (8-pole Bessel, –3 dB) and digitized at 25 kHz. All data collection was done using pCLAMP v.6 software and a DigiData 1200B interface (both Axon Instruments). Cells were continually superfused with a bathing solution of composition: 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose pH 7.4 adjusted with NaOH. Patch pipettes were pulled from thick-walled borosilicate glass (GC150F; Clark Electromedical Instruments) on a horizontal puller (P-87, Sutter Instrument Co.), coated with Sylgard® (Dow Corning 184) and fire polished before use to a final resistance of 3–6 M
. Whole-cell currents were recorded at a holding potential of –50 mV and single channel currents at –100 mV, with a pipette solution of composition: 120 mM CsCl, 20 mM TEA Cl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 11 mM EGTA pH 7.2 adjusted with CsOH.
Glycine was applied to whole-cell patches with a modified U-tube (68), with a gravity feed and PTX was co-applied with glycine in the same way. Concentration-response curves were constructed from the peak current response to the application of glycine. The responses, y, to agonists concentration [A], were fitted empirically with the Hill equation:
where ymax is the maximum response, EC50 is the agonist concentration for 50% maximum response and nH is the Hill coefficient. PTX inhibition curves were determined in the presence of 20 µM glycine and the IC50 estimated from fitting the curves with the Hill equation, with responses normalized to that obtained from 20 µM glycine in the absence of PTX.
Single channel recordings were analysed by the method of time course fitting using SCAN software (D.Colquhoun, http://www.ucl.ac.uk/Pharmacology/dc.html). A fixed time resolution of 40 µs (which gave a false-event rate of less than 10–8 s–1) was imposed on the open and shut times before analysis of the distributions. Open periods are defined in the same manner as described by Lewis et al. (40). Open period distributions were constructed with logarithmic bin widths and displayed using the square root of the event frequency (69). Distributions were fitted with a mixture of exponential densities by the method of maximum likelihood (EKDIST program; D.Colquhoun, http://www.ucl.ac.uk/Pharmacology/dc.html).
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Medical Research Council (MRC programme grant G9309834), the National Health and Medical Research Council of Australia (Block grant 993050) and the Health Research Council for New Zealand.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Neuropsychiatric Genetics Unit, Tenovus Building, University of Wales College of Medicine, Cardiff, CF14 4XN, UK. Tel: +44 02920 743058; Fax: +44 02920 746554; Email : owenmj@cardiff.ac.uk
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