Chaperone-mediated coupling of subunit availability to activation of flagellar Type III secretion.

Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilize the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, by using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.

Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein.

BACKGROUND: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. RESULTS: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. CONCLUSIONS: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

A chain mechanism for flagellum growth.

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip.

Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin.

While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as µNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

Large recurrent microdeletions associated with schizophrenia.

Reduced fecundity, associated with severe mental disorders, places negative selection pressure on risk alleles and may explain, in part, why common variants have not been found that confer risk of disorders such as autism, schizophrenia and mental retardation. Thus, rare variants may account for a larger fraction of the overall genetic risk than previously assumed. In contrast to rare single nucleotide mutations, rare copy number variations (CNVs) can be detected using genome-wide single nucleotide polymorphism arrays. This has led to the identification of CNVs associated with mental retardation and autism. In a genome-wide search for CNVs associating with schizophrenia, we used a population-based sample to identify de novo CNVs by analysing 9,878 transmissions from parents to offspring. The 66 de novo CNVs identified were tested for association in a sample of 1,433 schizophrenia cases and 33,250 controls. Three deletions at 1q21.1, 15q11.2 and 15q13.3 showing nominal association with schizophrenia in the first sample (phase I) were followed up in a second sample of 3,285 cases and 7,951 controls (phase II). All three deletions significantly associate with schizophrenia and related psychoses in the combined sample. The identification of these rare, recurrent risk variants, having occurred independently in multiple founders and being subject to negative selection, is important in itself. CNV analysis may also point the way to the identification of additional and more prevalent risk variants in genes and pathways involved in schizophrenia.

Genomic analysis of DNA binding and gene regulation by homologous nucleoid-associated proteins IHF and HU in Escherichia coli K12.

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ∼30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Common variants at VRK2 and TCF4 conferring risk of schizophrenia.

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.09, P = 1.9 × 10(-9)] and rs4309482[A], between coiled-coiled domain containing 68 (CCDC68) and TCF4, about 400 kb from the previously described risk allele, but not accounted for by its association (OR = 1.09, P = 7.8 × 10(-9)).

Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli.

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these--H-NS and Fis--bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.

Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity.

Cyclic-di-GMP is a bacterial second messenger that controls the switch between motile and sessile states. It is synthesized by proteins containing the enzymatic GGDEF domain and degraded by the EAL domain. Many bacterial genomes encode several copies of proteins containing these domains, raising questions on how the activities of parallel c-di-GMP signalling systems are segregated to avoid potentially deleterious cross-talk. Moreover, many 'hybrid' proteins contain both GGDEF and EAL domains; the relationship between the two apparently opposing enzymatic activities has been termed a 'biochemical conundrum'. Here, we present a computational analysis of 11 248 GGDEF- and EAL-containing proteins in 867 prokaryotic genomes to address these two outstanding questions. Over half of these proteins contain a signal for cell-surface localization, and a majority accommodate a signal-sensing partner domain; these indicate widespread prevalence of post-translational regulation that may segregate the activities of proteins that are co-expressed. By examining the conservation of amino acid residues in the GGDEF and EAL catalytic sites, we show that there are predominantly two types of hybrid proteins. In the first, both sites are intact; an additional regulatory partner domain, present in most of these proteins, might determine the balance between the two enzymatic activities. In the second type, only the EAL catalytic site is intact; these--unlike EAL-only proteins--generally contain a signal-sensing partner domain, suggesting distinct modes of regulation for EAL activity under different sequence contexts. Finally, we discuss the role of proteins that have lost GGDEF and EAL catalytic sites as potential c-di-GMP-binding effectors. Our findings will serve as a genomic framework for interpreting ongoing molecular investigations of these proteins.

A genome-wide investigation of SNPs and CNVs in schizophrenia.

We report a genome-wide assessment of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in schizophrenia. We investigated SNPs using 871 patients and 863 controls, following up the top hits in four independent cohorts comprising 1,460 patients and 12,995 controls, all of European origin. We found no genome-wide significant associations, nor could we provide support for any previously reported candidate gene or genome-wide associations. We went on to examine CNVs using a subset of 1,013 cases and 1,084 controls of European ancestry, and a further set of 60 cases and 64 controls of African ancestry. We found that eight cases and zero controls carried deletions greater than 2 Mb, of which two, at 8p22 and 16p13.11-p12.4, are newly reported here. A further evaluation of 1,378 controls identified no deletions greater than 2 Mb, suggesting a high prior probability of disease involvement when such deletions are observed in cases. We also provide further evidence for some smaller, previously reported, schizophrenia-associated CNVs, such as those in NRXN1 and APBA2. We could not provide strong support for the hypothesis that schizophrenia patients have a significantly greater "load" of large (>100 kb), rare CNVs, nor could we find common CNVs that associate with schizophrenia. Finally, we did not provide support for the suggestion that schizophrenia-associated CNVs may preferentially disrupt genes in neurodevelopmental pathways. Collectively, these analyses provide the first integrated study of SNPs and CNVs in schizophrenia and support the emerging view that rare deleterious variants may be more important in schizophrenia predisposition than common polymorphisms. While our analyses do not suggest that implicated CNVs impinge on particular key pathways, we do support the contribution of specific genomic regions in schizophrenia, presumably due to recurrent mutation. On balance, these data suggest that very few schizophrenia patients share identical genomic causation, potentially complicating efforts to personalize treatment regimens.

Regulation of bacterial Type III Secretion System export gate opening by substrates and the FliJ stalk of the flagellar ATPase.

Type III Secretion Systems (T3SS) transport proteins from the bacterial cytosol for assembly into cell surface nanomachines or direct delivery into target eukaryotic cells. At the core of the flagellar T3SS, the FlhAB-FliPQR export gate regulates protein entry into the export channel whilst maintaining the integrity of the cell membrane. Here, we identify critical residues in the export gate FliR plug that stabilise the closed conformation, preserving the membrane permeability barrier, and we show that the gate opens and closes in response to export substrate availability. Our data indicate that FlhAB-FliPQR gate opening, which is triggered by substrate export signals, is energised by FlhA in a proton motive force-dependent manner. We present evidence that the export substrate and the FliJ stalk of the flagellar ATPase provide mechanistically distinct, non-redundant gate-activating signals that are critical for efficient export.

Recognition of discrete export signals in early flagellar subunits during bacterial type III secretion.

Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, 'stalled' at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.

Disruption of the neurexin 1 gene is associated with schizophrenia.

Deletions within the neurexin 1 gene (NRXN1; 2p16.3) are associated with autism and have also been reported in two families with schizophrenia. We examined NRXN1, and the closely related NRXN2 and NRXN3 genes, for copy number variants (CNVs) in 2977 schizophrenia patients and 33 746 controls from seven European populations (Iceland, Finland, Norway, Germany, The Netherlands, Italy and UK) using microarray data. We found 66 deletions and 5 duplications in NRXN1, including a de novo deletion: 12 deletions and 2 duplications occurred in schizophrenia cases (0.47%) compared to 49 and 3 (0.15%) in controls. There was no common breakpoint and the CNVs varied from 18 to 420 kb. No CNVs were found in NRXN2 or NRXN3. We performed a Cochran-Mantel-Haenszel exact test to estimate association between all CNVs and schizophrenia (P = 0.13; OR = 1.73; 95% CI 0.81-3.50). Because the penetrance of NRXN1 CNVs may vary according to the level of functional impact on the gene, we next restricted the association analysis to CNVs that disrupt exons (0.24% of cases and 0.015% of controls). These were significantly associated with a high odds ratio (P = 0.0027; OR 8.97, 95% CI 1.8-51.9). We conclude that NRXN1 deletions affecting exons confer risk of schizophrenia.

Behavioural risk of bipolar disorder in an analogue population: the role of cognitive, developmental and interpersonal factors.

Research to date has identified the contribution of a number of cognitive, developmental and interpersonal risk factors in the development of bipolar affective disorder. However, further work is needed to understand the mechanisms and interactions between these risk factors in relation to bipolar mood instability. The aim of this study is to explore the possibility of identifying high risk of bipolar disorder through cognitive and interpersonal factors and to further expand our knowledge regarding the relationship between such factors. The findings from this work demonstrate that when both cognitive and interpersonal variables are entered into one model to predict bipolar high risk, direct effects are observed for the interpersonal factors, which then have a fully mediational effect on the cognitive factors. This work proposes that interpersonal factors develop and maintain cognitive risk factors and that future formulations and treatment of bipolar disorder need to focus on addressing such interpersonal issues.

Development of an apparatus to produce fractures from short-duration high-impulse loading with an application in the lower leg.

Axial loading of the lower leg during impact events can cause significant fractures of the tibia. The magnitude of lower leg axial loading that occurs during short-duration high-impulse events, such as antivehicular landmine blasts, can lead to life-altering injuries. These events achieve higher forces over shorter durations than car crashes, the current standard used for protective measures. In order to determine appropriate injury limits for the lower limb, a testing apparatus has been designed that can simulate these types of events for testing of anthropomorphic test device (ATD) lower legs as well as cadaveric specimens. Moreover, the design allows for the velocity at which the specimen is struck to be varied independently of the force applied, thus allowing independent investigation into the effect of momentum or energy on fracture strength. Test specimens are supported on a low-friction bearing system, and receive the controlled impulse from a projectile of variable mass that is accelerated using pneumatics. The apparatus includes velocity sensors, a six-degree-of-freedom load cell, and an accelerometer to completely quantify the loading event. The apparatus' performance was validated against an ATD lower leg. It was able to create impulse events with forces from 0.5 kN to 17.0 kN, and projectile speeds of 2.3-13.9 m/s. Various momenta could be achieved at a constant force level by varying the mass of the projectile, with a maximum difference of 65%, whereas kinetic energy was inherently linked to the impact force. This apparatus will be useful in future studies for determining the appropriateness of currently used injury limits for the lower limb to high-impulse events, as well as for quantifying the relationship between cadaveric fracture response and ATD measurements. This device can also be readily applied to other bones of the body, to create realistic fracture patterns for known injury mechanisms.

The effect of multiplanar distal radius fractures on forearm rotation: in vitro biomechanical study.

PURPOSE: Many patients develop distal radioulnar joint (DRUJ) pain and loss of forearm rotation after distal radial fractures. Residual distal radial deformity is one potential cause of DRUJ dysfunction; however, the parameters of distal radial fracture alignment that lead to an acceptable functional outcome are poorly defined in the literature. METHODS: We used 8 fresh-frozen cadaveric specimens in this in vitro study to examine the effect of simulated distal radius fracture misalignment on forearm rotation. A distal radial osteotomy was performed just proximal to the DRUJ and a custom-made, 3-degrees-of-freedom modular implant designed to simulate distal radius fracture deformities was secured in place. This allowed for accurate simulation of dorsal angulation, dorsal translation, and radial shortening, both independently and in combination. We examined the effects of distal radius deformity in the setting of both an intact and sectioned triangular fibrocartilage complex. RESULTS: Pronation was not significantly affected until dorsal angulation reached 30 degrees . Dorsal translation of up to 10 mm or radial shortening up to 5 mm had no effect on forearm rotation. Combined deformities had a greater effect on forearm motion than isolated malpositions. Dorsal angulation of > or =20 degrees combined with 10 mm of dorsal translation or 20 degrees of angulation with 2.5 mm of radial shortening resulted in a significant decrease in forearm pronation. There was no effect of distal radial deformities, either isolated or combined, on the magnitude of forearm rotation after sectioning the triangular fibrocartilage complex. CONCLUSIONS: This study demonstrates that a broad range of distal radius fracture malpositions can be tolerated before a notable loss in forearm range of motion is evident. Combined deformities are more likely to result in a clinically important loss of forearm rotation, and this should be considered when choosing the optimal management of patients with displaced distal radial fractures. Disruption of the triangular fibrocartilage releases the tether on the DRUJ, allowing for preservation of forearm motion even in the setting of marked osseous deformities.

Prevention and management of urinary catheter blockages in community settings.

Self-management of long-term urinary catheters can be challenging for patients, and recurrent catheter blockages may cause concern among patients, carers and healthcare professionals. Catheter blockages are a significant challenge for nurses practising in community settings, because frequent and unplanned catheter changes can be costly to healthcare services in terms of time and resources. This article details evidence-based recommendations for the assessment and diagnosis of catheter blockages, as well as the identification of risk factors. It also explains the interventions that can be used to prevent and manage catheter blockages and describes the role of the nurse in supporting patients with a long-term catheter in situ in community settings.

Confirmation of the genetic association between the U2AF homology motif (UHM) kinase 1 (UHMK1) gene and schizophrenia on chromosome 1q23.3.

UHMK1 has previously been implicated as a susceptibility gene for schizophrenia in the 1q23.3 region by significant evidence of allelic and haplotypic association between schizophrenia and several genetic markers at UHMK1 in a London-based case-control sample. Further fine mapping of the UHMK1 gene locus in the University College London schizophrenia case-control sample was carried out with tagging SNPs. Two additional SNPs were found to be associated with schizophrenia (rs6604863 P = 0.02, rs10753578 P = 0.017). Tests of allelic and haplotypic association were then carried out in a second independent sample from Aberdeen consisting of 858 individuals with schizophrenia and 591 controls. Two of these SNPs also showed association in the Aberdeen sample (rs7513662 P = 0.0087, rs10753578 P = 0.022) and several haplotypes were associated (global permutation P = 0.0004). When the UCL and Aberdeen samples were combined three SNPs (rs7513662 P = 0.0007, rs6427680 P = 0.0252, rs6694863 P = 0.015) and several haplotypes showed association (eg HAP-A, HAP-B, HAP-C permutation P = 0.00005). The finding of allelic association with markers in the UHMK1 gene might help explain why it has not been possible, despite great effort, to satisfactorily confirm previously reported associations between schizophrenia and the genes RGS4 and NOS1AP/CAPON. These genes flank UHMK1 and all three loci are within a 700 kb region showing linkage to schizophrenia. The confirmation of association between UHMK1 and schizophrenia, rather than RGS4 and NOS1AP in the London sample, points to the possibility that previous efforts to accurately fine map a gene in the 1q23.3 region have lacked accuracy or may have suffered from methodological flaws.