Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retards cell division
© Shoubridge et al; licensee BioMed Central Ltd. 2010
Received: 17 October 2009
Accepted: 5 January 2010
Published: 5 January 2010
Aristaless related homeobox (ARX) is a paired-type homeobox gene. ARX function is frequently affected by naturally occurring mutations. Nonsense mutations, polyalanine tract expansions and missense mutations in ARX cause a range of intellectual disability and epilepsy phenotypes with or without additional features including hand dystonia, lissencephaly, autism or dysarthria. Severe malformation phenotypes, such as X-linked lissencephaly with ambiguous genitalia (XLAG), are frequently observed in individuals with protein truncating or missense mutations clustered in the highly conserved paired-type homeodomain.
We have identified two novel point mutations in the R379 residue of the ARX homeodomain; c.1135C>A, p.R379S in a patient with infantile spasms and intellectual disability and c.1136G>T, p.R379L in a patient with XLAG. We investigated these and other missense mutations (R332P, R332H, R332C, T333N: associated with XLAG and Proud syndrome) predicted to affect the nuclear localisation sequences (NLS) flanking either end of the ARX homeodomain. The NLS regions are required for correct nuclear import facilitated by Importin 13 (IPO13). We demonstrate that missense mutations in either the N- or C-terminal NLS regions of the homeodomain cause significant disruption to nuclear localisation of the ARX protein in vitro. Surprisingly, none of these mutations abolished the binding of ARX to IPO13. This was confirmed by co-immunoprecipitation and immmuno fluorescence studies. Instead, tagged and endogenous IPO13 remained bound to the mutant ARX proteins, even in the RanGTP rich nuclear environment. We also identify the microtubule protein TUBA1A as a novel interacting protein for ARX and show cells expressing mutant ARX protein accumulate in mitosis, indicating normal cell division may be disrupted.
We show that the most likely, common pathogenic mechanism of the missense mutations in NLS regions of the ARX homeodomain is inadequate accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13.
The genes on the X-chromosome contribute significantly to genetic aetiology of intellectual disability . The Aristaless related homeobox gene (ARX) [GenBank: NM_139058.2] is one of the more frequent contributors . ARX belongs to a subset of Aristaless-related Paired-class (Prd-class) homeodomain proteins  and contains multiple domains, including the aristaless domain, homeodomain, the octapeptide and 4 polyalanine tracts . ARX mutations cause intellectual disability with or without additional features including epilepsy, infantile spasms, dystonia, lissencephaly, autism and dysarthia [3–5]. To date, over 90 families and individual cases with 40 different types of mutations have been reported. These include missense mutations, protein truncations but most frequently, polyalanine tract expansions [6–19].
When Arx was first ablated in mouse , the phenotype recapitulated many clinical aspects of X-linked lissencephaly with ambiguous genitalia [XLAG; MIM 300215] [20, 21]. Subsequently, this led to the identification of ARX mutations in patients with XLAG . Lissencephaly is one of a heterogenous group of disorders arising from aberrant neuronal migration. The characteristic 'smooth brain', due to a paucity of normal gyri and sulci, is due to either the arrest of neuronal migration (classical) or an over-migration of neurons (cobblestone). Mutations in the X-linked gene DCX (MIM 300121)  and four autosomal genes: LIS1 (MIM 601545) , RELN (MIM 600514) , TUBA1A (MIM 602529) [25, 26] and VLDLR (MIM 192977)  have been associated with distinct lissencephaly syndromes. However, mutations in ARX are the only identified genetic cause underlying the distinct syndrome of XLAG. This syndrome differs from the other forms of lissencephaly, displaying a thickened cortex with posterior to anterior gradient of gyral malformation, agenesis of the corpus callosum and ambiguous genitalia .
There are currently 31 families reported with XLAG phenotypes, with and without additional features, due to 27 different mutations in ARX. The majority of these families are predicted to arise from protein truncation and the loss-of-function of the mature ARX protein [5, 7, 9, 12, 28–31]. In the remaining families with XLAG, single nucleotide substitutions clustered in the homeodomain or, in one case, just prior to the aristaless domain, are predicted to give rise to amino acid substitutions in the mature protein. Several of these single nucleotide substitutions occur in residues of nuclear localization sequences (NLS) that flank both ends of the ARX homeodomain. In the N-terminal NLS (NLS2) there are four naturally occurring point mutations - R332P, R332H and R332C which cause XLAG and a residue adjacent to this arginine, T333N, which causes Proud syndrome (agenesis of the corpus callosum with ambiguous genitalia) [ACC/AG; MIM 30004]. We have recently identified novel point mutations in a single residue of the NLS3 region flanking the C-terminal portion of the homeodomain, R379. In one case, a substitution of R379 with lysine (L) has been identified in a patient presenting a phenotype of XLAG (D Coman, unpublished data). In contrast, a second change in the same residue resulting in the substitution of R379 with serine (S) was identified in a proband and his female relatives . Interestingly, the phenotype includes infantile spasms and severe mental retardation without obvious brain malformation.
The basic residues of ARX NLS2 and NLS3 are important for the correct localization of ARX in the nucleus through interaction with Importin 13 (IPO13) . IPO13 is a member of the importin-β superfamily involved in nuclear import and export of a variety of proteins [34, 35]. In addition to IPO13, a recent study suggests that multiple importins are utlized to import murine Arx into the nucleus, including importin β1 . Regardless of the importin utilized, the driving force for nuclear protein import is provided by RanGTP and its interaction with the importin-cargo complex . We show that missense mutations of NLS2 and NLS3 cause significant disruption to the nuclear localization of ARX in vitro. However, these mutations do not abolish the binding of ARX to IPO13. Interestingly, the binding of mutant ARX protein to endogenous IPO13 is indistinguishable from the binding to N-terminally truncated IPO13 lacking the RanGTPase binding domain. Hence, the ability of the mutant ARX-IPO13 complex to uncouple in the RanGTP rich nuclear environment appears to be compromised. As part of our investigation into the pathogenic mechanism underlying these mutations we identify TUBA1A, a component of the cytoskeleton important in mitosis, as a novel protein partner for ARX and examine the impact of sequestration of mutant ARX cargo with IPO13 on mitosis and cell division.
Materials and methods
Subjects and families
The relevant research ethics committees and institutional review boards of collaborating institutions approved this study. Clinical information and DNA samples were collected with informed consent.
Cloning of mutant full-length ARXconstructs
The cloning of the full-length human ARX cDNA construct in pCMV-Myc vectors (ARX-Wt; 1-562 aa) and the V5-IPO13 (aa 217-963; minus the RanGTPase binding site) have been described previously . Single nucleotide substitutions were introduced into the pCMV-Myc-ARX-Wt full-length construct via site directed mutagenesis (Stratagene) following the manufacturer's instructions. The mutations included c.995G>C leading to p.R332P (R332P) in the NLS2 region, c.998C>A leading to p.T333N (T333N) close to NLS2, c.1136G>T leading to p.R3479L (R379L) and c.1135C>A leading to p.R379S (R379S) in the NLS3 region. A double mutant construct was engineered with a mutation in each the NLS2 (c.995G>C) and NLS3 (c.1136G>T) regions in the same cDNA construct (R332P-R379L). In addition, a construct containing a c.1058C>T change leading to p.P353L (P353L) was engineered to provide an ARX mutation located within the homeodomain but outside of the NLS regions. All clone preparations were verified by sequencing of the entire coding region.
Cell culture and transient transfection
HEK293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. All cells were cultured in the presence of 100 U/ml sodium penicillin and 100 μg/ml of streptomycin sulphate in 5% CO2 at 37°C. Cells plated at 4 × 105 per well in a six- well plate the day before transfection in media lacking antibiotics. Routinely, cells were transfected with a total of 1 μg of plasmid DNA using Lipofectamine 2000 (Invitrogen, CA, USA) following the manufacturer's instructions.
The following antibodies were used for immunofluorescence and/or western immunoblot analysis: mouse anti-ARX antibody (2 μg/ml final); goat anti-IPO13 (1:500) (Imgenex, CA, USA); rabbit-anti-alpha-tubulin (1:1500) (Rockland, PA, USA); rabbit anti-V5 antibody (1:5000) (Bethyl Laboratories, Tx, USA). The secondary antibodies were either fluorescent labelled; goat anti-mouse-IgG conjugated to CY3 (1:1000) (Jackson Laboratories, Maine, USA); goat anti-mouse-IgG conjugated to FITC (1:1000) (Dako, Glostrup, Sweden); goat anti-rabbit-IgG Alexa Fluor 488 (1:800) (Invitrogen); donkey anti-goat-IgG Alexa Fluor 555 (1:1000) (Invitrogen) or horseradish-peroxidase (HRP) conjugated; mouse anti-Myc HRP conjugated antibody (1:5000) (Invitrogen); mouse anti-V5 HRP conjugated antibody (1:5000) (Invitrogen); goat anti-mouse-HRP antibody (1:1000) (Dako); goat anti-rabbit HRP antibody (1:1000) (Dako); rabbit anti-goat HRP (1:2000).
Immunofluorescence and microscopy
Transfected cells were harvested at 24 and 48 h post-transfection by fixation in 3.7% formaldehyde-phosphate buffered saline (PBS) (v/v) and permeabilized in 0.2% (v/v) triton PBS for 5 min. Blocking of non-specific binding of the secondary antibody was achieved routinely by addition of 5% skim-milk powder (w/v) in Tris buffered saline and 0.5% Tween (v/v) (TBS-T) before incubation of the primary and secondary antibodies diluted in 1% milk (w/v) in TBS-T. Removal of excess block and antibody was achieved with multiple washes of TBS-T. Nuclei were counterstained with DAPI (Molecular probes, Invitrogen).
For subcellular localization studies: Between 1000-4000 transfected cells were counted for each construct from at least three different transfection reactions using standard fluorescence microscopy. The percent of transfected cells with abnormal localization or aggregates was determined as the number of cells containing abnormal localization or aggregates divided by the total number of ARX positive cells. Sub-cellular localization images were captured by Leica TCS SP5 spectral confocal microscope using a 100× Plan apochromat objective. Z-stacks were taken at 0.25 μm intervals and maximal projections were made for all cell images.
For mitosis studies: Between 1200 and 4000 cells were analysed at 24, 48 and 72 h post-transfection for: (i) percentage of cells transfected; (ii) percentage of mitotic cells in both transfected and un-transfected cells; and (iii) and the phase of mitosis for each cell undergoing division.
Yeast-2 hybrid screening
Plasmids encoding the ARX homeodomain (ARX-HD) were fused to the GAL4 DNA binding domain and the library protein were fused to GAL4-activation domain. The MaV203 yeast strain was co-transformed with the ARX-HD construct and human fetal brain cDNA library (ProQuest, Invitrogen). Colonies were selected on media lacking leucine, tryptophan and histidine and positive clones were analysed for expression of three reporter genes (HIS3, URA3 and LacZ) by measuring β-galactosidase activity. Library inserts of positive clones that activated more than one reporter gene were amplified by polymerase chain reaction and sequenced to determine the identity of the library clone.
Cells transfected with Myc-ARX, both with and without V5-IPO13 were harvested at 24 hrs post-transfection and cell lysates prepared using lysis buffer (120 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% NP-40 (v/v), 1× protease inhibitor cocktail (Sigma, AZ, USA), 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF). Lysates were clarified by centrifugation (15 min, 13,000 g at 4°C). Aliquots of extracts were immunoprecipitated (IP) overnight at 4°C. Protein-A sepharose was pre-treated with un-transfected HEK293T cell lysate to ameliorate non-specific binding of cell proteins. The IP reactions were incubated with the pre-treated protein-A sepharose for 1 h at 4°C before removal of non-specifically bound proteins with four changes of high stringency wash buffer (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40 (v/v)) to ensure adequate removal of non-specific binding of alanine tract containing ARX protein. Proteins bound to the protein-A sepharose were eluted in SDS loading buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS (v/v), 10% glycerol (v/v), 5% β-Mercaptoethanol (v/v), 0.001% bromophenol blue (w/v), heated to 65°C before addition and incubated for 3 min). IP proteins were subjected to SDS-PAGE and transferred to nitrocellulose membrane. Lysates from HEK293T cells producing either Myc-ARX alone or V5-IPO13 alone were used as controls.
In co-transfected cells; Myc-ARX protein was IP with 0.5 μg of anti-Myc antibody (Santa Cruz Biotech, CA, USA) or the reciprocal co-immunoprecipitation (Co-IP) with 0.5 μg of rabbit anti-V5 antibody immunoprecipitating V5-IPO13 protein. IP proteins were analysed for the presence of Myc-ARX and V5-IPO13 by western immunblotting. In cells transfected with ARX alone, endogenous IPO13 was IP with 1 μg of goat anti-IPO13 and Co-IP of Myc-ARX protein was identified using mouse anti-Myc HRP conjugated antibody.
To verify the interaction between Myc-ARX and TUBA1A, HEK293T cells transfected with Myc-ARX constructs were lysed and immunoprecipitated with mouse anti-Myc antibody. IP proteins were analysed for the presence of Myc-ARX and endogenous alpha-tubulin by western immunoblotting.
All data are reported as mean ± standard error of mean, determined from a minimum of three separate transfection reactions, with the number of cells counted indicated in each figure legend. Differences in the percentage of transfected cells with abnormal subcellular localization were analysed by Kruskal-Wallis Test and when significance was reached a one-tailed pair-wise comparison was achieved using Wilcoxon-Mann-Whitney U test. In the case of the mitosis data, differences in the proportions of cells in various phases of mitosis at increasing times post-transfection were compared by two-way ANOVA, with mutations and time as factors. P < 0.05 was considered significant.
Novel mutations in ARX homeodomain
Missense mutations in NLS2 and NLS3 disrupt nuclear localization of ARX
Missense mutations in NLS2 and NLS3 do not abolish ARX interaction with N-terminally truncated IPO13
Endogenous IPO13 binds to ARX protein containing missense mutations in NLS2 and NLS3
The binding of N-terminally truncated V5-IPO13 to ARX-Wt increases the proportion of cells with abnormal ARX protein localization
Potential pathogenic mechanism of NLS mutations in ARX
Alpha Tubulin is a novel interacting protein partner of ARX
Sequestration of mutant ARX and IPO13 may compromise mitosis
The effects of the sequestration of mutant ARX with IPO13 on mitosis and cell division were investigated at 24, 48 and 72 h post-transfection and compared with ARX-Wt. Co-staining of ARX (transfected cells expressing ARX protein), alpha-tublin (mitotic microtubules) and nuclear material (DAPI) was conducted to assist characterization of mitotic cells. At each time point cells were analysed for: (i) percentage of cells transfected; (ii) percentage of mitotic cells in both transfected and untransfected cells; and (iii) and the phase of mitosis for each cell undergoing division.
When mitotic cells expressing mutant ARX proteins were analysed we saw an accumulation of cells undergoing mitosis compared to the cells expressing ARX-Wt (Figure 9B). In particular, there was a significant increase in the proportion of cells in the early phase of mitosis at 24, 48 and 72 h post-transfection in cells expressing the ARX NLS mutant proteins compared to cells expressing ARX-Wt (with the exception of T333N at 72 h). In cells expressing the ARX NLS mutations there was also a significant increase in the proportion of cells in the middle phase of mitosis at both 24 and 48 h post-transfection compared to the ARX-Wt protein. Interestingly, the percentages of cells in the late phase of mitosis expressing NLS mutations were also significantly higher than ARX-Wt at 48 h post-transfection (Figure 9B) but not at 72 h (data not shown). This indicates that cells expressing the ARX NLS mutation proteins are progressing through cell division at a slower rate compared to the ARX-Wt protein.
We have established that naturally occurring patient mutations leading to non-synonymous changes of single, specific residues of the NLS regions cause significant disruption to nuclear localization of the mutant ARX protein in vitro. These findings were consistent in all NLS2 and NLS3 mutations tested, indicating the mechanism involved may be similar for missense mutations in both NLS2 and NLS3 regions of the ARX homeodomain. Consistent with the severity of clinical outcomes, mutations leading to severe malformation phenotypes had the highest percentages of cells with abnormal mutant protein localization while the mutation causing ISSX but not brain malformation gave the smallest, but still significant, increase in cells with abnormal protein localization compared to the ARX-Wt protein. In contrast to the NLS mutations, another ARX mutation in the middle of the homeodomain, but outside of the NLS regions, did not disrupt the nuclear localization of the mutant protein with the subcellular localization of the mutant protein the same as the ARX-Wt protein.
The NLS regions flanking the ARX homeodomain are predicted to be important in the bi-partite binding to IPO13 [33, 34]. We wanted to investigate if changing single, specific residues of the NLS regions would diminish or potentially abolish binding of the mutant NLS with IPO13. The interaction of importins and cargo proteins are often difficult to visualize or measure, as these interactions are highly transient with efficient recycling of the importin proteins back to the cytoplasm after delivery of the cargo to the nucleus. In order to capture this interaction, N-terminal truncated V5-IPO13 was engineered to enable binding to cargo and transport across the nucleus coupled with an inability to interact with RanGTP and dissociate upon reaching the nucleus . Both the Co-IP and co-localization results indicate single amino acid substitutions in either the NLS2 or NLS3 regions of the ARX homeodomain did not abolish binding of the mutant ARX protein to N-terminally truncated V5-IPO13. Moreover, protein containing both R332P and R379L mutations was still able to bind to V5-IPO13.
Interestingly, binding of mutant ARX protein to endogenous IPO13 was indistinguishable from the binding to the N-terminally truncated IPO13 lacking the RanGTPase binding domain. The interaction of endogenous IPO13 with mutant ARX protein implies that the binding of these two proteins not only occurs but that this complex is either unable to correctly transport across the nuclear pore or unable to discharge the ARX cargo once inside the nucleus, or both. We contend that the inability of mutant ARX to uncouple from endogenous IPO13 contributes to the overall increase in cells with abnormal localisation of ARX protein. In support of this suggestion, co-transfection of ARX-Wt protein with the N-terminally truncated IPO13 led to a marked increase in the proportion of transfected cells with abnormal localization compared to cells transfected with ARX-Wt alone. Hence, disruption of normal nuclear delivery and distribution of mutant ARX protein may be due to a compromised ability of the mutant ARX-IPO13 complex to uncouple in the presence of RanGTP. Inadequate nuclear accumulation of a transcription factor due to impaired interaction with importin-β has been suggested as the pathogenic mechanism behind mutations in the C-terminal NLS of the SRY gene leading to XY genotype developing as females . A similar scenario of inadequate accumulation and distribution of the mutant ARX protein within the nucleus might ultimately mimic complete absence of ARX and contribute to the catastrophic phenotypic consequences routinely observed in patients with these mutations. This prediction fits with the emerging genotype-phenotype pattern for mutations in ARX. In particular, naturally occurring mutations such as insertions [5, 7, 9, 12], deletions [5, 28–31], nonsense changes  and splice mutations  in ARX that result in protein truncation and loss-of function of the mature ARX protein invariably lead to severe phenotypes, including XLAG.
Both arginine residues examined in this study, R332 in NLS2 and R379 in NLS3 of ARX, are invariant in all 26 paired-type homeodomain proteins. Within this family of proteins these two basic residues are frequent sites of missense and nonsense mutations associated with a range of diseases. A mutation in the homologous NLS2 residue has been reported for ALX4 (MIM 605420)  and mutations in the homologous NLS3 residue have been identified in CRX (MIM 602225) , OTX2 (MIM 600037)  and SHOX (MIM 312865) [42, 43]. Mutations in both residues corresponding to the R332 and R379 of ARX have been identified in PAX3 (MIM 606597) [44, 45], PAX6 (MIM 607108) [46, 47], PITX2 (MIM 601542) [48–50] and PROP1 (MIM 601538) [51–54]. A mutation in the conserved residue at position 50 of the paired-type homeodomain SHOX results in the abolition of DNA binding . This residue corresponds to R379 of ARX. However, this residue is not part of the NLS region of the SHOX protein. When a residue within the NLS is mutated (R173C of SHOX) this change does not affect the DNA binding but instead disrupts the nuclear transport of SHOX. This may also be the case with the mutations tested in our study. There is no doubt that some of the missense mutations in the ARX homeodomain are likely to be important in either specificity of binding or in the actual binding to DNA. For example, the P353L mutation did not disrupt nuclear location of the resulting protein. Perhaps this mutation leads to the severe XMESID phenotype due to changes in binding to DNA targets. The recent identification of a specific transcription factor-binding site for Arx in a study ablating Arx in the sub-palluim of the mouse brain  will provide an important tool to investigate the potential mechanisms underlying the pathogenesis of mutations in the ARX homeodomain in the future.
A recent examination of heterozygous females from families with known mutations in ARX has highlighted the fact that in some cases mutations that disrupt ARX in females may have pathogenic consequences . In particular, a number of females with the T333N and R379S mutations display mental retardation or learning disabilities, although other female carriers of the same mutation are phenotypically normal [30, 32]. Our data indicates that in addition to a predicted loss of normal transcription factor activity of ARX, sequestration of mutant ARX with IPO13 may lead to a disruption of interactions with other protein partners, contributing to the disease phenotype. An emerging feature of many forms of lissencephaly and pachygyria is the potential disruption of key elements of microtubule behaviour. For example, one of the key genes mutated in lissencephaly is doublecortin (DCX). Mutations in DCX are clustered in two tubulin binding domains and impair the polymerization of microtubules and as such correct neuronal migration . Interestingly, DCX does not bind to the tubulin heterodimer itself but acts to nucleate the microtubule growth and to stabilize microtubules. More recently, mutations in TUBA1A have been reported to cause lissencephaly with a distinct clinical presentation, ranging from perisylvian pachygyria in the less severe form, to posteriorly predominant pachygyria in the most severe form, in association with dysgenesis of the anterior limb of the internal capsule and mild to severe cerebellar hypoplasia . A recurrent mutation in TUBA1A compromises the efficiency of de novo alpha/beta-tubulin heterodimer formation . We have shown that ARX interacts with TUBA1A by Co-IP, but the two proteins do not appear to co-localize during normal cell growth. However, in cells expressing mutant ARX we observed a disruption of the mitotic spindle structures and an accumulation of cells in the early stages of mitosis. Hence, neurons, which normally express ARX during the development of the sequestration of mutant ARX, might compromise the interaction with TUBA1A, disrupt normal microtubule formation and subsequently retard efficient cell division.
We cannot rule out the possibility that sequestration of IPO13 in these cells may also contribute to pathogenesis. The dynamic cycling of Ran between the GTP and GDP bound forms is exquisitely controlled by specific regulators differentially localized within the cellular environment . The RanGAP, which accelerates GTP hydrolysis, is cytoplasmic. The Ran guanine nucleotide exchange factor, also known as RCC1 (regulator of chromatin condensation 1), is located in the nucleus bound to chromatin. Hence, the levels of RanGTP increase in proximity to chromatin. Importin proteins bind and inhibit spindle accessory factors (SAF) everywhere in the mitotic cytosol, except in the vicinity of the chromosome. The high levels of RanGTP close to the chromatin relieve the inhibition of SAFs by importins and subsequently allow local spindle assembly. RanGTP binding to importin protein generates conformation change in the Importin molecule that in turn alters the binding site of the importin for the cargo proteins . Therefore, IPO13 trapped in complex with mutant ARX may not be able to adequately bind SAFs and as such compromise spindle assembly. Samples of the cortex taken at autopsy from the patient with the R379L mutation were stained with neurofilament immunoperoxidase and, interestingly, a paucity of neurons was noted (D Coman, unpublished data). The authors could not rule out the contribution of perinatal ischemia to the histological findings. However, in light of our findings, it is interesting to speculate that disruption of the mitotic spindle due to sequestration of mutant ARX with IPO13 retards cell division, potentially contributing to a disruption in neuronal migration and correct lamination patterns of the brain leading to lissencephaly and associated clinical outcomes.
Aristaless related homeobox
magnetic resonance imaging
Nuclear localization sequence
phosphate buffered saline
spindle accessory factor
tris buffered saline
X-linked lissencephaly with ambiguous genitalia
X-linked intellectual disability
X-linked myoclonic epilepsy with spasticity and intellectual disability.
The authors would like to thank the patients and their family members and Nancy Briggs (University of Adelaide, Australia) for assistance with statistical analysis and Dr Helger Yntema (Radboud University Nijmegen Medical Centre, The Netherlands) for performing ARX analysis in family 2. This work was supported by an Australian NHMRC programme grant (JG), a NHMRC Training Fellowship (CS) and a NHMRC Senior Research Fellowship (JG).
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