N=82). In the first study we use a similar picture-viewing task to the discovery sample  to replicate an effect of the variant on emotion processing. In the second study we use a probabilistic reversal learning procedure to identify reward processing during decision-making under uncertainly (reward>punishment). In a region of interest (ROI) analysis of the bilateral VS, we show that the rs322931 genotype was associated with BOLD in the left VS during the positive>neutral contrast (PROI-CORRECTED=0.045) and during the reward>punishment contrast (PROI-CORRECTED=0.018), although the effect of passive picture viewing was in the opposite direction from that reported in the discovery sample. These findings suggest that the recently identified GWAS hit may influence positive emotion via individual differences in activity in the key hubs of the brain's reward system. Furthermore, these effects may not be limited to the passive viewing of positive emotional scenes, but may also be observed during dynamic decision-making. This study suggests that future studies of this GWAS locus may yield further insight into the biological mechanisms of psychopathologies characterised by deficits in reward processing and positive emotion.A recent genome-wide association study (GWAS) identified a significant single-nucleotide polymorphism (SNP) for trait-positive emotion at rs322931 on chromosome 1, which was also associated with brain activation in the reward system of healthy individuals when observing positive stimuli in a functional magnetic resonance imaging (fMRI) study. In the current study, we aimed to further validate the role of variation at rs322931 in reward processing. Using a similar fMRI approach, we use two paradigms that elicit a strong ventral striatum (VS) blood oxygen-level dependency (BOLD) response in a sample of young, healthy individuals ( A recent GWAS studied individual differences in positive emotional experiences (PE),4 identifying a GWAS significant single-nucleotide polymorphism  associated with PE.Genome-wide association studies (GWAS) are helping to uncover the specific biological factors that support personality4 The authors hypothesise that the loci may increase an individual's propensity to display positive affect through alteration in reward circuitry that supports the positive valuation of stimuli. The GWAS hit situated at rs322931 was further associated with reduced negative affect and increased resilience, suggesting a potential protective effect against environmental stressors and genetic factors that confer risk to psychopathology.Systems biology approaches such as functional magnetic resonance imaging (fMRI) can offer mechanistic explanations, by which genetic loci can influence heritable traits identified via GWAS. The genetic variant was also associated with expression of blood and brain miR-181a, miR181-b and blood oxygen-level dependency (BOLD) in the ventral striatum (VS) and the amygdala while viewing positive scenes compared with neutral scenes.5 to identify potential neural effects of the rs322931 SNP on VS BOLD. Second, we use a probabilistic reversal learning procedure (as previously described6), a reliable paradigm to assess reward-related brain activity,7 to identify potential neural effects of rs322931 associated with VS BOLD during receipt of reward, compared with punishment.In the current study we aim to further quantify the neural effects of the PE loci using two cognitive paradigms designed to elicit BOLD in the key hubs in the reward system such as the VS. We expand upon the initial association between rs322931 and VS BOLD during the passive viewing of positive stimuli and aim to identify further mechanistic roles of the variant in the brain's reward circuitry and valuation system. The observations from the study may help us to understand the role of the genetic variant in the brain's valuation system in health and clinical traits characterised by a reduction in positive emotion. Using a similar approach as described in the discovery study, we first use a passive viewing paradigm of positive and neutral picture stimuliWe hypothesise that the minor allele of rs322391, which has been associated with PE, will be associated with an increased VS BOLD response to (a) positive stimuli (as previously observed) and during (b) the receipt of monetary reward compared with punishment. The findings from the study will elucidate whether the variant has pleiotropic effects on the brains reward system, or whether the effects of the SNP are specific to the viewing of positive stimuli.8 or use of psychotropic medication. Informed consent was obtained for all individuals before the study, which was approved by the ethics committee of the School of Psychology, Cardiff University. A sample of N=81 (picture viewing) and N=82  participants was included in the final sample after removing individuals with failed quality control of genetic data (n=10) or incomplete imaging data .One hundred right-handed Caucasian (of western European descent) volunteers aged 19–47 were recruited from Cardiff University (staff and/or students) for a study involving several MRI, magnetoencephalography and behavioural paradigms. No participants reported any psychiatric illness9 to ensure genotypes did not display ambiguous sex, cryptic relatedness up to third-degree relatives by identity of descent or genotyping completeness <97%. We also removed non-European ethnicity admixture detected as outliers in iterative EIGENSTRAT analyses of an LD-pruned data set.10 SNPs were excluded where the minor allele frequency was <1%, if the call rate <98% or if the χ2-test for Hardy–Weinberg equilibrium had a P-value <1e−04. After quality control, rs322931 genotype information was available for all ninety successfully genotyped individuals.Genomic DNA was obtained from saliva using Oragene OG-500  saliva kits. Genotyping was performed using custom genotyping arrays (Illumina HumanCoreExome-24 BeadChip), which contain 570 038 genetic variants . Quality control was implemented in PLINK5 or Internet resources. We included 18 neutral IAPS pictures having a mean normative valence rating of 4.87  and 9 positive IAPS pictures having a mean normative valence rating of 6.99. Images taken from other resources had been used and validated in a previous study.11 Picture categories were comparable with regard to semantic homogeneity and perceptual complexity: neutral pictures showed household objects and positive images depicted appetitive food. Each block lasted 8 s, in which an array consisting of either four random positive or four random neutral images were presented at a rate of 2 s per image. This process was repeated 10 times for each participant. To keep individuals engaged in the task, we included a 1-back monitoring task in which participants had to confirm with a button press each time an image was presented twice in a row within a trial block. For each participant, we embedded four picture repetitions at random positions within the entire sequence of picture viewing blocks. The number of picture repetitions was balanced across picture categories. There were four picture repetitions for each participant, with an equal number of repetitions occurring for each picture category. Participants viewed a total of 40 stimuli per condition. Intertrial intervals were randomly jittered (6–10 s) in order to sample the haemodynamic response at different time points. To keep individuals engaged in the task, participants had to confirm with a button press each time an image was presented twice in a row within a trial block.Eighty-one participants randomly viewed affectively positive and neutral stimuli taken from the International Affective Picture System (IAPS)vice versa. Participants were instructed to maximise their earnings during the learning session, which consisted of 12 reversal episodes in total . Within each reversal episode we included either 1 or 2 PE (probabilistic error) trials, in which ‘wrong' feedback was given for correct choices, even though the reward contingencies had not changed. At the start of each choice trial, participants were presented with a response cue consisting of two white frames surrounding the colours and prompting the participants to press the left or right button on a response box to choose one colour. Response feedback (choice outcome) was given subsequently using a centrally presented white ‘smiley' (correct choice) or red ‘frowny' (incorrect choice) face and an earnings' counter changing incrementally by ±1p. In trials following reversal or PE events, that is, in those trials used for fMRI analysis (see below), response cues and feedback stimuli were presented with a jittered duration  intertrial interval). To reduce scanning time, in all other (standard) trials we used fixed and shorter stimulus durations . ITIs showed the two colours without response cue or feedback and were 0.5 s long after standard trials and between 4 and 8 s (mean 5.5 s) after PEs and reversals. BOLD response analysis focused on brain activation differences as a function choice outcomes (reward>punishment) in post PE and post-reversal trials. We selected those trials for analysis as they yielded a comparatively balanced number of rewards or/and punishments (correct versus incorrect choices) compared with standard trials . We have previously published fMRI data from this experiment in relation to polygenic risk for schizophrenia.6Eighty-two participants learned to choose one of two simultaneously presented colours (‘blue' and ‘green‘) by receiving monetary reward for correct choices and monetary punishment for wrong choices  for ‘blue' and −1p for ‘green‘). After 7–11 trials, reward/punishment contingencies were reversed so that the previously rewarded colour was now punished and Overall learning performance was assessed as the accumulated earnings across all 108 choice trials. We also calculated trial-based average accuracies  for the trials directly following PE and reversal events  for each participant. Post PE and post-reversal accuracy scores allowed us to measure impulsive choice behaviour (high switch rates/low accuracies after PEs) and perseverative tendencies .12Gradient echoplanar imaging data were acquired for each subject using a 3 T GT HDx system with an eight-channel receiver at Cardiff University Brain Research Imaging Centre, School of Psychology, Cardiff University ; 220 mm, repetition time (TR) 2000 ms, echo time (TE) 35 ms, flip angle 90°, acceleration (ASSET) factor; 2). High-resolution three-dimensional T1-weighted images were also acquired using a three-dimensional fast spoiled gradient echo sequence with 172 contiguous sagittal slices of 1 mm thickness  450 ms, flip angle 20°, FOV 256 × 256 × 176 mm, matrix size 256 × 256 × 192 to yield 1 mm isotropic voxel resolution images). All functional images were first motion-scrubbed, where TRs with a framewise displacement >0.9 were removed, as previously recommended.www.fmrib.ox.ac.uk/fsl). The following pre-statistics processing was applied; motion correction using MCFLIRT;13 slice-timing correction using Fourier-space time series phase-shifting; non-brain removal using BET (Brain Extraction Tool);14 spatial smoothing using a Gaussian kernel of FWHM 5 mm; grand-mean intensity normalisation of the entire 4D data set by a single multiplicative factor; and high-pass temporal filtering . Registration to high-resolution structural ) and standard space (group-level GLM) images was carried out using FLIRT.13 Time series analysis was carried out using FMRIB's Improved Linear Model with local autocorrelation correction.15 To further correct for any potential movement confounds, motion regressors estimated via MCFLIRT and scrubbed TRs were added as covariates of no interest to the design matrix. Group-level analysis was carried out using FLAME .16Image processing and statistical analyses were conducted using statistical parametric mapping methods as implemented in the FMRI Expert Analysis Tool  convolved with a canonical haemodynamic response function. Predictor time courses were locked to the onset of feedback stimuli, with a fixed duration of 3750 ms, which corresponded to the earliest possible start of the next choice trial, and convolved with a canonical haemodynamic response function. For each subject, statistical contrast images reward>punishment were obtained.4) possessed by each participant ; therefore, a positive association would represent a T allele dose-dependent increase in BOLD. We explored the (a) group-level contrasts (one sample t-tests) and (b) rs322931 genotype effects (multiple regressions) in the VS region of interest (ROI), defined as the bilateral accumbens in the Harvard–Oxford Subcortical Structural Atlas. The familywise error rate was controlled in all cases with nonparametric permutation testing (5000 permutations) and threshold-free cluster enhancement, which effectively controls for multiple comparisons, compared with cluster extent thresholding.18We ran multiple regression using the combined first-level contrasts  for each subject covarying for the rs322931 genotype and potential confounds (age and sex). The rs322931 genotype status was entered into the multiple regression as the number of T alleles .The rs322931 was not over-represented in either gender or was associated with age in either fMRI experiment. There were no associations between rs322931 genotype and performance during the reversal learning procedure  positive>neutral stimuli and (b) reward>punishment. After correcting for the familywise error rate using threshold-free cluster enhancement across the VS (PROI-CORRCETED<0.05), we found widespread activation in the VS for both contrasts. Specifically, in the positive>emotion contrast, we observed two clusters: k=247 , k=206 , PROI-CORRECTED=<0.001 in both cases. In the reward>punishment contrast, we observed k=241, k=219 , PROI-CORRECTED<0.001 in both cases . There were no rs322931 genotype related differences that survived correction for multiple comparisons across the whole brain (PCORRECTED >0.26). There were no positive associations rs322931 and BOLD in the VS or across the whole brain . For the Reward > Punishment contrast, we observed a positive association between rs322931 genotype and BOLD within a cluster in the left VS, k=113 , PROI-CORRECTED=0.018 .Within the Positive > Neutral Stimuli contrast, we observed a negative association between rs322931 genotype and BOLD within a cluster in the left VS, k=9 , PP>0.05, in all cases). Modelling behavioural performance during the reversal learning paradigm did not affect the association between VS BOLD and rs322931 genotype (PROI-CORRECTED=0.025).There were no significant correlations between the parameters estimates from the rs322931 linked clusters (that survived correction for family wise error across the VS) and behavioural performance during reversal learning  than the fMRI sample in the discovery sample (mean age: 40±12.8). Consistent with our broader hypothesis, we also show that increased VS BOLD was associated with the minor allele of rs322931 during the receipt of rewarding (compared to punishing) stimuli. The observation suggests that rs322931 may have pleotropic effects on the reward system that underpin its association with increased positive emotion, increased resilience and reduced negative affect.We found rs322931 genotype-related differences in VS BOLD during the processing of rewarding stimuli in a sample of healthy individuals. However, unlike the discovery study, we found that the minor allele (T) at rs322931 was associated with reduced VS BOLD when viewing positively valence stimuli , as opposed to the positive association between rs322931 T allele and VS BOLD described in the discovery sample. This discrepancy may be attributed to differences in the positively affective stimuli between the discovery sample and our study, as we showed positive affective pictures of appetitive food rather than complex visual scenery involving facial information. Furthermore, in the present study, task compliance/attentiveness was monitored with a 1-back design, whereas Wingo  and in individuals with increased genetic risk to psychopathology,26 suggesting that altered VS BOLD may be a neural antecedent that confers risk to psychopathology. Striatal BOLD for rewarding stimuli during reversal learning has also been associated with individual differences in extrinsic and intrinsic motivation,27 suggesting that BOLD during this condition may reflect individual differences in orientation towards incentivised stimuli. Across a wider range of paradigms that elicit VS BOLD, individual differences in this key hub within reward circuitry may also relate to individual differences in traits such as reward sensitivity29 and clinical symptoms such as anhedonia, avolition and apathy.35 Together, these studies suggest that risk and resilience to psychopathology may be (in part) related to VS BOLD and related behavioural manifestations.Several studies suggest that VS BOLD is altered in psychiatric disorders25 This response may also be influenced by factors such as maternal smoking behaviour,36 environmental stressors and early-life trauma.39 We did not assay these environmental stressors and acknowledge that our findings should be considered with these confounds in mind.We suggest that our findings are regarded with the following considerations. First, the results are based on a limited sample size (we had ~37–53% power in the current study) and will need to be replicated in larger, independent cohorts. Second, we did not have a behavioural measure of PE. These would have been useful in validating behavioural differences between rs322931 genotypes in the sample and exploring putative mediation models between genotype, VS BOLD and behaviour. Last, the direction of association between BOLD for positive stimuli and rs322931 genotype was in the opposing direction (attenuated BOLD was associated with positive emotion VS BOLD in our study) as anticipated and previously observed. Furthermore, it must be appreciated that the VS BOLD response to reward is not only shaped by genetic effects.40 and may shape an individual's risk or resilience to environmental stressors and related psychopathology.37 Elucidating the genetic architecture that underpins traits such as PE may help us to understand mechanisms of risk and resilience for psychopathology across nonclinical populations. Translating the findings of GWAS loci using techniques such as fMRI are essential in establishing links between genetics and transdiagnostic phenotypes such as those indexed by positive valence systems by the Research Domain Criteria.32In conclusion, we provide independent evidence that the GWAS significant locus at rs322931 may influence positive emotion via individual differences in VS BOLD response to rewarding stimuli. We suggest that this effect is not limited to the passive viewing of positive emotional scenes but can also be observed during reward feedback when individuals make decisions during periods of uncertainty. These findings add to a growing body of work, suggesting that individual differences in brain reward systems are heritable


Sirtuin3 (SIRT3) is an important protein deacetylase which predominantly presents in mitochondria and exhibits broad bioactivities including regulating energy metabolism and counteracting inflammatory effect. Since inflammatory cascade was proved to be critical for pathological damage following subarachnoid hemorrhage (SAH), we investigated the overall expression and cell-specific distribution of SIRT3 in the cerebral cortex of Sprague-Dawley rats with experimental SAH induced by internal carotid perforation. Results suggested that SIRT3 was expressed abundantly in neurons and endothelia but rarely in gliocytes in normal cerebral cortex. After experimental SAH, mRNA and protein expressions of SIRT3 decreased significantly as early as 8 hours and dropped to the minimum value at 24 h after SAH. By contrast, SOD2 expression increased slowly as early as 12 hours after experimental SAH, rose up sharply at the following 12 hours, and then was maintained at a higher level. In conclusion, attenuated SIRT3 expression in cortical neurons was associated closely with enhanced reactive oxygen species generation and cellular apoptosis, implying that SIRT3 might play an important neuroprotective role during early brain injury following SAH. Subarachnoid hemorrhage (SAH), especially following rupture of an aneurysm, is a devastating neurological disease associated with high morbidity and mortality . VictimsRecently, a large body of aneurysmal SAH literature has indicated strongly that early brain injury (EBI) might play a more pivotal role in neurological impairment and poor prognosis after SAH , 7. EBI +-dependent enzymes, have been shown to participate in transcriptional silencing and regulation of mitochondrial functions [The sirtuins, as a family of highly conservative NADunctions . As one unctions –15. It iunctions .Although hypoxia, ischemic injury, and other types of oxidative stress are closely involved in SAH, especially at the stage of EBI , there an = 6 for each subgroup). Rats were sacrificed at indicated time points during the following experiments.All experimental procedures were approved by the Animal Care and Use Committee of Second Military Medical University and complied with the Guide for the Care and Use of Laboratory Animals by National Institutes of Health. Male Sprague-Dawley (SD) rats (280 to 330 g) were raised ordinarily and randomly divided into sham group and SAH groups . Then the brain tissues were immediately transferred to an oven, dried at 110°C for 72 h, and weighed again (dry weight). Brain water content was determined according to the equation below: brain water content (%) = (wet weight − dry weight)/wet weight × 100%.The permeability of BBB was assessed 24 h after experimental SAH and quantified according to the method of Evans blue (EB) extravasations. Briefly, EB dye was injected through tail vein 1 h before and rinsed off just before sacrifice. All brain tissue specimens were refrigerated and centrifuged, and the absorbance of EB supernatant was detected at 610 nm by using a spectrophotometer.Rats were anesthetized and underwent transcardial perfusion with 0.9% ice-cold heparinized saline via the left cardiac ventricle for brain tissue preparation. For real-time PCR analysis and western blot, cortex of temporal lobe near the hematoma was harvested and preserved in liquid nitrogen. For immunohistochemistry, immunofluorescence, and TUNEL experiments, rats were perfused additionally with 4% buffered paraformaldehyde for 8 h, and the cortex of temporal lobe was embedded in paraffin and sectioned for further experiments.RNAs were extracted from temporal cortex ordinarily. The following primers were synthesized for real-time PCR: GAPDH ; SIRT3 ; SOD2 .μg) of protein lysates were separated in each cell line. The following primary antibodies were used: anti-SIRT3 antibody (diluted 1 : 200), anti-SOD2 antibody (diluted 1 : 200), and β-actin (diluted 1 : 5000). The following secondary antibodies were used: HRP-conjugated anti-goat IgG (diluted 1 : 2000), HRP-conjugated anti-rabbit IgG (diluted 1 : 2000), and HRP- conjugated anti-mouse IgG (diluted 1 : 2000). All the antibodies were purchased from Santa Cruz Biotechnology.Western blot analysis was performed as reported previously . EquivalImmunocytochemistry experiments were performed and immunoreactivity score (IRS) was interpreted by two pathologists blinded to the experiment as described previously . The priμm thick sections. The sections were first cocultured with 5% normal fetal bovine serum in PBS including 0.1% Triton X-100 for 2 h and then with anti-neuron-specific nuclear protein (NeuN) antibody  and anti-SIRT3 antibody (1 : 100) or anti-glial fibrillary acidic protein (GFAP) antibody  and anti-SIRT3 antibody  overnight at 4°C. Secondary antibodies included goat anti-rabbit IgG (1 : 200) and goat anti-guinea pig IgG . The sections were incubated with secondary antibodies and counterstained with DAPI counterstain. The fluorescence images were acquired using Nikon TE200  with a Spot RT digital camera.Immunofluorescence was carried out based on standard protocol. Tissue blocks containing the temporal cortex were ordinarily fixed, dehydrated, and cut into 6 In order to evaluate cellular apoptosis in the temporal cortex, the TUNEL staining was adopted to detect the fragmentation of DNA in cell nuclei using an In Situ Cell Death Detection Kit . Cellular apoptosis was interpreted by two pathologists blinded to the experiment. Briefly, for each slide, five high-power (400x) fields were selected randomly for quantification. Total numbers of 500 cortical cells were counted, and percentage of positive cell was evaluated for every 100 cells. Apoptosis index was confirmed by mean value of 5 percentages mentioned above. t-test and one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc test. Statistical significance was defined at P < 0.05.The data were presented as means ± SEM. SPSS18.0 and GraphPad Prism 5.0 were used for statistical analysis using independent samples During the whole experimental period, all the animals showed stable weight, body temperature, mean arterial pressure, and arterial blood gas parameters (data not shown). After euthanasia, it could be detected that the blood clots were distributed near the circle of Willis, the ventral brainstem, and basilar arteries in the SAH group, but not in the sham group.P < 0.01), while brain water content in the SAH group was higher than that in the sham group , as well as the BBB permeability .To identify cerebral damage following experimental SAH, key events such as neurologic deficits, cerebral edema, and BBB disruption were assessed 24 h after experimental SAH, by comparing with conditions before SAH. As shown in We tested the homeostatic regulation of mitochondrial reactive oxygen species (mtROS), which was associated with oxidative stress-induced injury, during the experimental aneurismal subarachnoid hemorrhage (SAH). Results suggested that both mRNA and protein expressions of SOD2 were associated inversely with the downregulation of SIRT3, which increased slowly as early as 12 hours after experimental SAH, rose up sharply at the following 12 hours, and then was maintained at a higher level   showed a significant difference between the sham group and the 24 h post-SAH group  .In rats of the sham group, ubiquitous expression of SIRT3 was detected in NeuN-positive neurons rather than GFAP-positive astrocytes. In rats of the 24 h post-SAH group, expression of SIRT3 decreased in the cytoplasm of NeuN-positive cells but was not changed in GFAP-positive astrocytes .In rats of the sham group, few TUNEL-positive apoptotic cells were observed in the temporal cortex, while a number of apoptotic cells were observed in the SAH group .This pilot study was designed to explore the expression and cellular distribution of SIRT3 in the cortex in an experimental SAH rat model. Results suggested that both mRNA and protein expressions of SIRT3 were downregulated since the early hours and decreased to the minimum 24 h after SAH in a time-dependent manner. IHC and immunofluorescence staining showed that SIRT3 protein was predominantly expressed in neurons, rather than in astrocytes in the lesion foci. These results suggest that SIRT3 might serve as an endogenous neuroprotective factor during the secondary early brain injury that may result in the unfavorable prognosis in SAH. in vitro experiment demonstrated that the upregulated protein expression of SIRT3 in PC12 cells secondary to oxygen-glucose deprivation (OGD) was an endogenous feedback response for neuroprotection [For decades, delayed vasospasm has been assumed to be the pivotal pathological mechanism for delayed neurologic deficits and poor prognosis, while the significance of EBI had not been noticed until therapies of vasospasm reversal failed to show distinct protective effects in improving long-term outcomes in clinical trials . The casotection . ConsistMammalian sirtuin family were initially recognized as histone deacetylases (HDAC) which are involved in a variety of physiological functions, such as stress reaction, metabolic regulation, gene silencing, and aging . Previou in vitro experiment showed that the overexpression of SIRT3 diminishes the sensitivity of PC12 neurons to both oxidative and apoptotic insults. Meanwhile, another study reported that SIRT3 protects PC12 cells against hypoxia damage through PGC-1a and MnSOD pathways by reducing ROS and maintaining ATP production [To date, considerable evidence indicates that oxidative stress is the key pathophysiological process in EBI secondary to aneurysm rupture; therefore blocking oxidative damage as early as possible could significantly improve the outcome of the patients . Due to oduction .In summary, this preliminary study for the first time characterized the expression pattern of SIRT3 and its cellular distribution in cerebral cortex after experimental SAH. Significant downregulation of SIRT3 was observed in the cortex during EBI. Further taking into account the previous findings from the abovementioned studies, we hypothesize that SIRT3 could provide neuroprotection effects in EBI secondary to SAH. It is necessary to comprehensively elucidate the full mechanism involved in this neuroprotection by further studies.


Acrophobia, an abnormal fear of heights, is a specific phobia characterized as apprehension cued by the occurrence or anticipation of elevated spaces. It is considered a complex trait with onset influenced by both genetic and environmental factors. Identification of genetic risk variants would provide novel insight into the genetic basis of the fear of heights phenotype and contribute to the molecular-level understanding of its aetiology. Genetic isolates may facilitate identification of susceptibility alleles due to reduced genetic heterogeneity. We took advantage of an internal genetic isolate in Finland in which a distinct acrophobia phenotype appears to be segregating in pedigrees originally ascertained for schizophrenia. We conducted parametric, nonparametric, joint linkage and linkage disequilibrium analyses using a microsatellite marker panel, genotyped in families to search for chromosomal regions correlated with acrophobia. Our results implicated a few regions with suggestive evidence for linkage on chromosomes 4q28 (LOD = 2.17), 8q24 (LOD = 2.09) and 13q21-q22 (LOD = 2.22). We observed no risk haplotypes shared between different families. These results suggest that genetic predisposition to acrophobia in this genetic isolate is unlikely to be mediated by a small number of shared high-risk alleles, but rather has a complex genetic architecture. Acrophobia is a pervasive mental disorder, also known as an irrational fear of heights, affecting approximately five percent of the world’s population24The symptoms of individuals suffering from acrophobia involve changes in behavioural, cognitive and physiological functioning, such as confusion and dizziness, when exposed to heightsAlthough the biological mechanisms of acrophobia have been thoroughly investigated12To reduce genetic heterogeneity we have used an isolated homogenous population from north-eastern Finland with small number of founders. This population, described elsewhere1517Our sample was composed mostly of large multigenerational pedigrees with multiple individuals affected with acrophobia and at least one parent born in the isolate . It incl17We first carried out parametric two-point linkage analysis to identify genetic regions linked with acrophobia. The strongest evidence for linkage in the acrophobia sample (including cases with comorbid schizophrenia) was obtained with marker D5S2115 (LOD = 2.16) using a dominant model. In the pure acrophobia sample, the same marker yielded a LOD score of 0.58 (dominant model), suggesting that this finding may be mainly driven by the schizophrenia phenotype. This was further confirmed by analysing all individuals with schizophrenia without comorbid acrophobia . For the pure acrophobia sample, we obtained the highest LOD score with marker D8S373 (LOD = 2.09) adopting a recessive inheritance model. Since in the acrophobia with comorbid schizophrenia sample the same marker yielded a LOD score of 0.51 (recessive model) and in the pure schizophrenia sample a LOD score of 0.00 (recessive model), this signal may be produced mainly by the acrophobia phenotype. We carried out parametric multipoint analysis for chromosomes with markers and models which yielded LOD score of >2.0 in at least one parametric two-point analysis . In the acrophobia sample (including cases with comorbid schizophrenia), marker D5S2115 yielded a maximum LOD score of 0.054 . In the pure acrophobia sample the highest LOD score on chromosome 8 was with marker D8S373 .We next carried out multipoint nonparametric genome-wide linkage analysis with empirical NPL_ALL model , measuriWe hypothesized that reduced genetic heterogeneity in the genetic isolate would lead to the majority of the cases carrying the same predisposing variant, detectable as linkage disequilibrium (LD). Therefore, we performed PSEUDOMARKER analysis of LD conditional on linkage . The strTo test the statistical power of the analysed sample, we performed simulation with SLINKWe aimed to find genetic loci predisposing to acrophobia in a genetically isolated population with the hypothesis that due to reduced genetic heterogeneity, a few risk alleles predisposing to the phenotype might be identified. While several loci attained LOD score of >2.0, we observed no genome-wide significant evidence for linkage at any of the studied markers.PCDH20) gene in this region, has been previously connected to positive symptom dimension in a genome-wide association study (GWAS) of schizophrenia (P = 3 × 10−6)We detected the strongest evidence of linkage to acrophobia on chromosomal region 13q21-q22 with a peak on marker D13S162 . To our knowledge, this region has not been previously associated with phobias or other anxiety disorders. SNP rs2323266, located 14.01 Mb from marker D13S162 and close to the protocadherin 20 . Again, in our study, this signal appeared to be mainly coming from acrophobia and not schizophrenia phenotype . This region has not been previously associated with anxiety disorders or schizophrenia.KCNQ3)24ADCY8)LYNX1)313233ARC)Chromosomal region 8q24.2-q24.3 with marker D8S373 (LOD = 2.09) was the third region most strongly linked to acrophobia. It has previously been associated with bipolar disorder2425Several of the chromosomal regions we identified seemed mainly specific for schizophrenia. The most significant of those regions were 5q31 and 1q32, discussed above for markers D5S2115 and D1S2817. Both of them have been previously associated with schizophrenia in numerous studies353637Furthermore, several markers on the long arm of chromosome 13, localized between 13q31 and 13q33, showed evidence of linkage (LOD > 2.0) to both pure acrophobia and acrophobia with comorbid schizophrenia, and also weak linkage to pure schizophrenia (maximum LOD = 1.26). Therefore, this region may harbour variants influencing susceptibility to both phenotypes. As specific phobia subtypes have high comorbidity with other anxiety disordersWe recognise the hypothesis-generating character of our study. It included a large number of genotyped markers (570) and analysed models (6). Although this serves to strengthen the study, it inevitably lead to multiple statistical testing. However, as the tests carried out are not completely independent, the adjustment for multiple testing is not straightforwardIn recent years, single nucleotide polymorphisms (SNPs) have replaced microsatellite markers due to their lower genotyping costs. This technical advance has enabled genome-wide association studies (GWASs) in which thousands of individuals are genotyped. Consequently, the usage of microsatellite markers, a class of short tandem repeats (STRs), has severely decreased. However, due to their highly polymorphic nature microsatellites are still considered more informative than the diallelic SNPs1117In conclusion, our findings suggest that the genetic basis of acrophobia is highly complex, even in this genetic isolate, as we were not able to identify high-risk variants shared by several families. However, we identified several chromosomal regions with suggestive evidence for linkage which could be investigated further in other acrophobia samples and meta-analyses of such datasets having an increased statistical power.The sample was composed mostly of large multigenerational pedigrees with multiple affected individuals and at least one parent born in the isolate . It comp171846th edition (DSM-IV) criteriaData from psychiatric case notes and treatment facilities concerning affected individuals and blood samples were collected between 1991 and 2002We analysed 575 autosomal microsatellite markers across the genome that had been genotyped as a part of earlier linkage-based gene mapping studies17unknown, due to the fact that unaffected family members were not systematically assessedWe performed statistical analyses separately for acrophobia with comorbid schizophrenia and pure acrophobia sample to discriminate between the possible shared and separate genetic signals associated with the phenotypes. We further followed with statistical analysis of the pure schizophrenia sample for the most interesting results. We first checked all genotypes for Mendelian inconsistencies with PedCheck softwareWe performed two-point parametric linkage analysis with statistical software package FASTLINK 4.1 P under a recessive and dominant mode of inheritance with, respectively, penetrance of 0.001% and 90%, disease allele frequency of 0.00001 and 0.01, and phenocopy rates of 0 and 0.01. FASTLINK 4.1 P program was implemented in a helper program AUTOGSCANWe carried out multipoint parametric and nonparametric linkage analysis with SimWalk2 version 2.965455We performed the linkage disequilibrium conditional on linkage analysis with PSEUDOMARKER software package under default dominant and recessive models58The power of the analysed pedigrees to detect linkage was estimated with SLINK simulation program and the replicates were analysed with ISIM analysis program implemented in SLINK packageHow to cite this article: Misiewicz, Z. et al. A genome-wide screen for acrophobia susceptibility loci in a Finnish isolate. Sci. Rep.6, 39345; doi: 10.1038/srep39345 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Genome sequencing and subsequent gene annotation of genomes has led to the elucidation of many genes, but in vertebrates the actual number of protein coding genes are very consistent across species . Seven years after sequencing the cattle genome, there are still genes that have limited annotation and the function of many genes are still not understood, or partly understood at best. Based on the assumption that genes with similar patterns of expression across a vast array of tissues and experimental conditions are likely to encode proteins with related functions or participate within a given pathway, we constructed a genome-wide Cattle Gene Co-expression Network (CGCN) using 72 microarray datasets that contained a total of 1470 Affymetrix Genechip Bovine Genome Arrays that were retrieved from either NCBI GEO or EBI ArrayExpress.LOC509513), RNA processing (LOC100848208), nucleic acid metabolic process (LOC100850151) and organic-acid metabolic process (MGC137211). Such highly connected genes should be investigated more closely as they likely to have key regulatory roles.The total of 16,607 probe sets, which represented 11,397 genes, with unique Entrez ID were consolidated into 32 co-expression modules that contained between 29 and 2569 probe sets. All of the identified modules showed strong functional enrichment for gene ontology (GO) terms and Reactome pathways. For example, modules with important biological functions such as response to virus, response to bacteria, energy metabolism, cell signaling and cell cycle have been identified. Moreover, gene co-expression networks using “guilt-by-association” principle have been used to predict the potential function of 132 genes with no functional annotation. Four unknown Hub genes were identified in modules highly enriched for GO terms related to leukocyte activation  contains supplementary material, which is available to authorized users. The completion of a draft genome assembly simply marks the “end of the beginning” of genome exploration in that species . After aThe large amount of biological data deposited in public databases provides an opportunity to computationally annotate functional and regulatory connections among genes. A challenge in this post-genomic era is to properly integrate available information so as to reconstruct, as accurately as possible, valuable information from large volumes of data . It is whttp://www.animalgenome.org/cgi-bin/host/reecylab/d.In this study, a condition independent gene co-expression network was generated to provide additional functional annotation information for genes in the cattle genome. We have annotated genes with possible putative functions and possible regulatory mechanisms. This effort will accelerate discovery of genes and lead to elucidation of the biological features responsible for economic traits. Network information is publically available at R2) shows approximate scale free topology in CGCN . Interrogation of the cattle protein interaction network database  revealedThe Molecular Function (MF) GO terms of the genes included in this module were related to regulation of gene expression such as: single-stranded RNA binding, double-stranded RNA binding, NAD+ ADP-ribosyltransferase activity and exonuclease activity, active with either ribo- or deoxyribonucleic acids and producing 5′-phosphomonoesters  had high interaction scores   > 0.01) with known genes (Table LOC509513), RNA processing (LOC100848208), nucleic acid metabolic process (LOC100850151) and organic-acid metabolic process (MGC137211). Such highly connected genes should be investigated more closely as they likely to have key regulatory roles in the cattle.The potential function of 133 genes with no previous functional annotation, e.g. no associated/assigned GO terms, was predicted based on functional uniformity among the associated genes  encodes for an interferon induced ubiquitin like (UbL) protein, which plays a key role in the innate immune response to viral infection either via its conjugation to both host and viral proteins (ISGylation) or via its action as a free or unconjugated protein [UBA7 and UBE2L6, encode for ubiquitin/ISG15 activating and conjugating enzymes, respectively [HERC6, has ligase activity and is involved in the UbL conjugation pathway [HERC6 has been reported to be important in the antiviral response [ISG15 conjugation in mouse cells [Tau (IFNT) has been shown in cattle endometrium [Tau shows antiproliferative effects and antiviral activities that have less toxicity than the other type-I IFNs [USP18) had the highest intra-modularconnectivity and is the hub node for the white module  with 40 % of the proteins encoded in the white module (25 out of 63 proteins)  [The white module had several close interconnected over-represented GO terms and Reactome pathways related to immune response to virus Fig. . This mo protein . The ISG protein . The othectively . Another pathway . HERC6 hresponse , where ise cells . The expometrium . Interfee-I IFNs . Ubiquitproteins  as evideproteins . USP18 pproteins . In addis (IFNs) , which ps (IFNs) .IRF7 and IRF9 has been reported to be associated with both Bovine Herpesvirus1 [Regulation of gene expression is determined in large part by the activity of transcriptional activator proteins. Also, transcriptional regulation enables cells to respond to environmental cues such as viral infection . Two memesvirus1  and Footesvirus1 , 47 infeIL10, IL1B, IL1RN and PF4. The antibacterial activity of these well-known genes with the highest quality annotation scoring in the UniProt database [S100A9 plays a prominent role in the regulation of inflammatory processes and immune response [2+, which is essential for bacterial growth [S100A9 acts as an alarmin or a danger associated molecular pattern (DAMP) and can stimulate innate immune cells via binding to pattern recognition receptors such as Toll-Like Receptor 4 (TLR4) [The saddle brown module was highly enriched for several BP GO terms related to response to bacterium  .TP53 Regulates Metabolic Genes pathway, in the light green module, the TP53 gene was not included in the module. This gene was included in the yellow module that showed high enrichment for GO terms related to gene expression and RNA processing  revealed tight connectivity (TOM > 0.01) between the probe sets that mapped to these genes and TP53. This result indicates that TP53 might be an intermediate node between the yellow and light green modules. COX5A gene had the highest connectivity in the module and considered as an intra-modular hub node. This gene encodes for mitochondrial Cytochrome c oxidase subunit 5A, which is the heme A-containing chain of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport and has a key role in cell energy production [The light yellow module had several over-represented GO terms and Reactome pathways related to different aspects of energy metabolism , G2/S (CDKN1A), G2/M (CCNA2 and CDKN1A) and cell division  [CDCA8 gene had the highest connectivity in the red module and considered as the intra-modular hub node  . CDCA8 gThe fact that functionally related genes are connected together in co-expression networks provides evidence for prediction of the cellular roles for hypothetical genes based on a GBA principle . NeighboGenes with little to no associated functional information generally have no gene symbol and so are automatically assigned an identifier such as LOC533597. Gene nomenclature, i.e. the scientific naming of genes, tries to standardized representation of genes within an organism, but not necessarily between organisms, based on the biological process or pathway in which they are involved. Although the results of the current study cannot be used directly for nomenclature purposes as they have no supporting biological information, they provide a rich resource for experimental biologists to begin to define the real biological function and thereby helping to assign gene symbols to such genes.In summary, these analyses indicate that the cattle gene co-expression network and corresponding regulons provides rich information for experimental biologists to design experiments, interpret experimental results, and develop novel hypotheses on gene function in cattle. Combinatorial approaches that integrate multiple omics findings will provide an important resource that should lead to the elucidation of molecular mechanisms underlying traits of interest in cattle.Affymetrix Genechip Bovine Genome Array (Bos taurus) were downloaded from either NCBI GEO [Affymetrix Genechip Bovine Genome Array were removed from the sample data. Probe-set IDs that did not map to an Entrez gene ID or Probe-set IDs that mapped to multiple Entrez gene IDs were discarded. The parametric Bayes Combat algorithm [CEL files for 1470 publicly available NCBI GEO  or EBI ANCBI GEO  , which was the square of the correlation between log (p (k)) and log (k). A β coefficient of seven with R2 of 0.9 was used to develop a weighted adjacency matrix. The weighted adjacency matrix was used to then develop the topological overlap matrix (TOM) as described by Langfelder et al. [The WGCNA R package  was usedfficient  between fficient , the adjr et al. . The TOMr et al. . Dynamicr et al. , which ur et al. , was usePotential interaction between genes included in each module were evaluated with the protein interaction network (v10) from String database . String p-value ≤ 0.05) were included in the analysis. Kappa statistics were used to link and grouping of the enriched terms and functional grouping of them as described in [To decipher the potential mechanism of action of detected modules, ClueGO , a widelribed in . The min


Purpose. To report unusual presentation of type 1A idiopathic macular telangiectasia (IMT). Methods. Two middle-aged women with bilateral IMT were examined. Results. Both patients presented with a gradual vision loss in both eyes. Fundus examination was unremarkable in one case and showed small macular telangiectasia in both eyes in the other case. Fluorescein angiography (FA) revealed early bilateral macular punctuated hyperfluorescence corresponding to the dilated capillaries in both cases. FA and fundus examination confirmed also the absence of vascular abnormalities in the middle or anterior fundus periphery in one case. Spectral-domain optical coherence tomography (SD-OCT) showed cystoid macular edema in both cases. No signs of retinal vein occlusions were detected in both cases and other differential diagnoses were excluded. Based on these findings, the patients were diagnosed with bilateral type 1A IMT according to Gass and Blodi classification and were treated with intravitreal antivascular endothelial growth factor (anti-VEGF) injections and focal laser photocoagulation. Twelve months later, SD-OCT revealed partial regression of the exudative signs and significant VA improvement. Conclusion. We described two patients with an unusual presentation of type 1A IMT with bilateral presentation, affecting two middle-aged women, with occult and without peripheral involvement in one case. The description of more cases of bilateral type 1 IMT should be helpful to more precisely define the pathophysiologic mechanism that could be different from a localized Coats' disease of the macula area. Idiopathic macular telangiectasia (IMT) was first described by Gass and Oyakawa in 1982 as juxtafoveal capillary abnormalities and dilatations . Both clμm RE and to 345 μm LE  was 42 letters in the right eye (RE) and 61 letters in the left eye (LE). The anterior segment examination was unremarkable. Fundus examination revealed small pigment epithelium alterations in the foveal area without capillary abnormalities or dilatations on both eyes Figures . Early-p Figures . Autoflu Figures .Since there was no medical history of diabetes, arterial hypertension, or X-ray radiation and considering the absence of evidence of Irvine-Gass syndrome or retinal vein occlusion on FA examination we conclude the diagnosis of bilateral type 1 IMT. Moreover the absence of typical intraretinal pseudocysts on OCT rendered the diagnosis of type 2 IMT not compatible.μm to the fovea in the temporal area of her RE. This treatment allowed almost complete macular edema resorption with functional stability. Laser focal photocoagulation allowed increasing of the interval of injections during followup and the patient required less injections in the RE thereafter.The patient was treated with anti-VEGF intravitreal injections in both eyes according to a PRN regimen associated with focal laser photocoagulation on telangiectasia located more than 1000 A 60-year-old phakic woman presented at our clinic complaining of gradual vision loss in her both eyes since one year. She previously received 6 anti-VEGF and one dexamethasone intravitreal injections in her RE and 8 anti-VEGF and one dexamethasone intravitreal injections in her LE. She was also treated with focal laser photocoagulation in the macular temporal area on her LE during last one year. Her past medical history included also well-balanced arterial hypertension and no medical history of diabetes was found. Her best-corrected visual acuity (BCVA) was 74 letters in the right eye (RE) and 45 letters in the left eye (LE). The anterior segment examination was unremarkable. Fundus examination revealed perifoveal exudates with small perifoveal capillary abnormalities on both eyes Figures . Left eyμm on RE and to 334 μm LE  showed cystoid macular edema on both eyes with subretinal fluid in the RE and central macular thickness was measured to 538  Figures . Several Figures .μm to the fovea. One month after 3 anti-VEGF injections, visual acuity improved to 86 letters RE and 64 letters LE and OCT showed complete subretinal fluid resorption RE and partial macular edema resorption on both eyes. Central macular thickness reduced to 447 μm RE and 295 μm LE.As in the case 1, we conclude the diagnosis of bilateral type 1 IMT since there was no medical history of diabetes or X-ray radiation and considering the absence of evidence of retinal vein occlusion on FA examination. Moreover the absence of typical intraretinal pseudocysts on OCT excluded the diagnosis of type 2 IMT. Despite a medical history of arterial hypertension, we did not consider the diagnosis of hypertensive maculopathy, since the absence of other retinal typical abnormalities and since the disease was under control. The patient was treated with anti-VEGF intravitreal injections in both eyes according to a PRN regimen without new focal laser photocoagulation since telangiectasia was located less than 1000 Here we reported two cases with unusual presentation of bilateral type 1A IMT. Previous publications reported rare presentation of unilateral type 1 IMT associated with choroidal neovascularization , 5. To oBefore considering the diagnosis of type 1A IMT, we made sure to exclude other diagnoses. Indeed type 1 IMT must be differentiated from secondary telangiectasia caused by other retinal vascular diseases especially retinal venous occlusions, diabetic retinopathy, radiation retinopathy, Irvine-Gass syndrome, or hypertensive retinopathy. No medical history of diabetes or X-ray treatment was noted in our patients. In both cases FA showed no evidence of capillary dilatations related to a previous branch retinal vein occlusion and confirmed also the absence of late staining of the optic disc in case 1 excluding the diagnosis of Irvine-Gass syndrome. Moreover in case 2 we made sure that arterial hypertension was well-balanced.According to the classification by Gass and Blodi , diagnosOur cases may also suggest that middle-aged women can be affected with idiopathic type 1 Mac Tel, but less frequently. Indeed according to Gass and Blodi, type 1A IMT typically affects young males in 90% of cases with an average age of 39 years . MoreoveRegarding the aspect of telangiectasia, it is not visible in fundus examination in case 1. However telangiectasia is typically visible in type 1 IMT and appears like aneurysmal lesions and occult presentation is usually observed in type 2 IMT . These oWe described two middle-aged women presenting with bilateral type 1A IMT and occult and without any vascular abnormalities in the middle or anterior fundus periphery in one case. Our cases emphasize that these unusual presentations do not exclude the diagnosis of type 1A IMT. Case 1 may probably be related to another pathophysiological mechanism than a localized Coats' disease.The description of more cases of bilateral type 1A IMT would be useful in order to more precisely define their clinical presentation and their pathophysiological occurrence.


CSA is an established cause of post-transplantation central nervous system (CNS) toxicity, typically characterised by posterior reversible encephalopathy syndrome (PRES).4 Even though neurological complications of CSA seldom result in mortality, the necessitated withdrawal of this potent anti-GvHD agent can have major implications on clinical outcome particularly in the face of on-going GvHD. The spectrum of CSA-related neurological complications has been well characterised in several case series of paediatric HSCT recipients,7 however, long-term follow-up data of those children who experience neurotoxicity are lacking. Here, we present the outcome of 26 children who developed CSA-neurotoxicity following allogeneic HSCT.With increasing numbers of children and adults undergoing allogeneic haematopoietic stem cell transplantation (HSCT), neurotoxicity is emerging as an important cause of transplant-related morbidity.7 were included. Those with obvious alternate causes for neurotoxicity were excluded. All children who suffered CNS complications were clinically evaluated by a neurologist. Supplementary investigations such as computed tomography (CT) or magnetic resonance imaging (MRI) scan of head, cerebrospinal fluid analysis and electroencephalogram were used as appropriate to identify the cause. PRES was diagnosed in accordance with its well described MRI appearance.8 Hypodense lesions in the parieto-occipital areas were considered to be CT findings consistent with PRES. TMA was diagnosed based on clinical and laboratory features of microangiopathic haemolytic anaemia and thrombocytopenia. GvHD was diagnosed clinically and supported with histological confirmation where possible.9 IV CSA administration at a dose of 5 mg/kg/day in two divided doses was commenced either on day −1 (2003–2008) or day −3 (2009 onwards). Levels were monitored twice weekly. Target trough serum levels were 100–150 ng/mL for HLA-identical sibling transplants and 150–200 ng/mL for unrelated donor transplants.From a cohort of 569 consecutive paediatric allogeneic HSCT recipients at our institution between 1 January 2003 and 31 December 2013, we retrospectively identified those children who developed neurological complications at any time after the start of conditioning whilst receiving CSA using a computerised search of our patient database. Children with clinical symptoms of PRES or neurological manifestations of thrombotic microangiopathy (TMA)n=19), followed by CSA+MTX (n=4) and CSA alone (n=3). In one patient (subject #19), CSA was changed to Tacrolimus because of renal toxicity before the development of neurotoxicity that is, in this patient neurotoxicity occurred while being treated with Tacrolimus. Twenty-one children experienced CI-related neurotoxicity during their primary transplant procedure and 5 children following their second HSCT, having tolerated CSA without adverse events during their first HSCT.During the 11-year study period, 569 paediatric allogeneic HSCTs were performed at this centre. Twenty-six children developed neurotoxicity associated with CSA (4.6%). Their demographics, disease characteristics and transplant-related parameters are summarised in Median time from HSCT to neurotoxicity was 47.5 days (range day –1 to +545) . ClinicaManagement of CI-induced neurotoxicity in addition to supportive care consisted of discontinuation of CSA/Tacrolimus in 25/26 patients. Neurological symptoms fully resolved in all patients except in patient #9 who developed TMA and died shortly thereafter and patient #3 who continues to require anticonvulsants despite normalisation MRI appearance. One patient (#12) who presented with seizures on day +24 and had non-specific features on MRI with a normal EEG was continued on IV CSA along with anticonvulsants and had no further neurological events.After a median of 11 days post onset of CSA neurotoxicity (range 0–597 days) a CI was reintroduced in 18 patients: in 10 patients CSA was used and in 12 patients Tacrolimus, 3 of whom had failed re-challenge with CSA . One patDespite these measures, 7/9 patients (78%) who had no prior GvHD, developed acute/late acute GvHD after onset of CSA neurotoxicity whereas 13/17 had recurrence, persistence or progression of acute/late acute GvHD. Twenty-three patients were evaluable for chronic GvHD as 2 died before day +100 and one patient had second HSCT before day +100. Eighteen out of 23 (78%) had chronic GvHD and this was extensive in 14 patients: 13 patients developed chronic GvHD following CSA-neurotoxicity whereas pre-existing chronic GvHD persisted or progressed in 5 patients .At a median of 176 days (range 7–1889 days) following development of CSA-induced neurotoxicity 15/26 (58%) patients died, most due to progressive GvHD or its complications . There w11 In our study, outcome was particularly poor in the 10 patients who had severe GvHD (Grade III/IV) before the development of CSA-neurotoxicity: 8/10 died and 2 are alive with extensive vitiligo. In the same time period at our institution, other patients with severe GvHD, but without the added complication of CSA neurotoxicity had a far superior survival rate of 70%.12 Hence it would seem that the development of CSA neurotoxicity and consequent inability to tolerate CI, adversely affected their prognosis. In future studies with larger patient numbers, it would be useful to substantiate our findings with multivariate analyses.This case series of paediatric HSCT recipients who developed CSA-related neurotoxicity illustrates that outcome following this complication is notably poor: high non-relapse mortality of 58% and significant morbidity with 36% of survivors living with late effects of extensive chronic GvHD. These findings are similar to those in large case series of adult allogeneic HSCT recipients who developed CSA-related neurotoxicity reporting 43–52% mortality due to progressive GvHD/infection.14 as seen in 5 patients in our case series. Nevertheless, re-challenge with a CI was tolerated in 56% of patients in this series, a similar proportion as that reported by others.10 In those patients where re-challenge resulted in recurrence of symptoms and use of CI was precluded permanently, outcome appears particularly dismal with overall survival of 13% (1/8) in this case series.Development of CSA-related neurotoxicity poses a complex clinical management situation as one of the most effective drugs in the treatment and prevention of GvHD needs to be discontinued promptly; sometimes in patients with on-going GvHD. As symptoms of CSA-neurotoxicity usually resolve over several days, re-challenge could be a viable option. Tacrolimus has been used as alternative agent in the event of CSA-related neurotoxicity but as it is also a CI, its use is similarly associated with significant neurotoxicity 13 and on its own it does not provide satisfactory GvHD prophylaxis or treatment. Sirolimus, which has a completely different mechanism of action to the CI provides another option. Although Sirolimus can also cause neurotoxicity, this has mostly been reported when it has been used in combination with CSA.15 In this series, Sirolimus was only used in five patients but this agent may potentially be increasingly used in this clinical situation in future.Evidence for the optimal approach of prophylaxis/management of GvHD when use of CI is contra-indicated is not available. In this case series, all but one patient received corticosteroids. In addition, as the combination of CSA and MMF is the most common GvHD prophylaxis regimen in our centre, the majority of patients were already receiving MMF at the time of development of CSA neurotoxicity. The efficacy of MMF as sole anti-GvHD agent is limited,In conclusion, this case series illustrates the dismal prognosis in patients following the development of CI-related neurotoxicity and the complexities of managing GvHD in this situation. There is a need for further studies to determine the optimal treatment approach to improve outcome following this rare but serious complication.


Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities  governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. Gene regulation by miRNAs is triggered by target mRNA degradation, translational repression or destabilization of target mRNAs through de-capping and poly-A removal12345789Progress in next-generation sequencing introduced numerous datasets for transcript analyses, which are used for diagnosis and prognostics in various diseases, in particular in several cancer types1213Recently, computational approaches on high throughput data investigated miRNA expression levels relative to their respective target mRNA levels. The results revealed only a weak correlation and suggested that expression levels do not concisely explain the regulatory effects1515171517Furthermore, miRNAs with similar cellular concentration, as detected in RNAseq read counts or alternative quantification methods, exhibited a wide range of functional outputs. High-throughput assays for miRNA activity20The increasing complexity and detailed expression analyses did so far not result in improved functional reporters. Commonly luciferase reporter assays get involved in exhaustive biochemical characterizations2223To generate an easy, reliable and accessible assay for single cell level analysis of miRNA activity, we implemented a single plasmid based dual reporter gene system. Within the construct one fluorescence-encoding gene contains a complementary miRNA-target region within its 3′UTR, while the second fluorescence gene served as a reference. The outputs of various reporter-constructs were integrated with a previously reported mathematical model for miRNA-repressionUntranslated Trans Assay (UTA), by using a synthetic non-human targeting siRNA. This allowed us to incorporate the experimental validation with the model derived predictions to describe gene regulation on the single cell level. Three repression groups were revealed and supported both by experimental data and modeling, i.e. low, mid and high functional miRNAs, based on the interaction between relative expression and binding capabilities, defined in the model by two parameters θ (concentration) and λ(binding), respectively.We experimentally examined the feasibility of integrating the mathematical model for our single plasmid reporter-system, which is referred to as To validate the system for endogenous miRNA analysis, we generated small-RNA profiles from HEK293 cells in triplicate and built a read-count based list of 29 miRNAs with high, medium or low expression signatures. Specific complementary targets for the individual candidates were integrated into one of the two different fluorescent proteins on the UTA constructs and used in the subsequent single cell analysis. Our measurements revealed that miRNA-activities match the predicted functional groups, but also indicated that repression activity does not directly correlate with the expression levels of the 29 endogenous miRNAs that have been evaluated.Furthermore, we showed that UTA system can be applied also in HeLa cells, where the ordinal categorical functional groups  were also observed. Furthermore, some individual miRNAs displayed their functionality according to their subcellular location, low functional and high expressed miRNA were at least partially located in the nucleus, while some mid expressed but high functional miRNAs were seen mainly cytoplasmic.λ-θ relations for synthetic siRNAs and corresponding target-reporters with stronger or weaker siRNA-binding sites, ii) estimate this relation from experimental data for endogenous miRNAs and evaluate their overall functionality and iii) show that miRNA expression should be closely evaluated in context with functionality and iv) show that some miRNA’s functionality is independent of their overall expression, but their specific cellular location.With the UTA system we were able to i) validate in vitro and in silico analysis, Utilizing this system we could demonstrate that expression level and functionality do not act proportionally for numerous endogenous human miRNAs. This system could help to improve the understanding of miRNA levels and effects in complex situations, including cancer development and progression or cell differentiation.In summary, we introduced a system that enables a detailed evaluation of individual miRNAs via a single sensor construct and combined it with downstream 2.Human HEK293 cells were maintained in DMEM (Dulbecco’s modified Eagle’s Medium) supplemented with 15% FCS and 1% antibiotics solution (Pen/Strep), HeLa cells were maintained in RPMI-1640 medium supplemented with 10% FCS and 1% antibiotics solution (Pen/Strep) cells were maintained at 37 °C and 5% COwww.mirbase.org) and oligonucleotides  were designed with NotI and XhoI overhangs annealed and ligated into fluorescence plasmid and PsiCheck2.Plasmids were constructed using key features of the PsiCheck2  dual luciferase reporter plasmid: promoters, XhoI and NotI sites were kept and fluorescent  proteins were inserted . Small RFor a complete list of ligated target site oligonucleotides, see the 2. Fluorescence microscopy pictures were taken using an Axio Observer microscope . Exposure time was 90 ms for GFP (Filter BP 525/50) and 900 ms for RFP (Filter BP 605/70).HEK293 cells and HeLa (50000 cells/well) were transfected with Lipofectamine 2000  or calcium phosphate precipitation method, cells were incubated for 72 h at 37 °C and 5% CO2 and subsequently prepared for flow cytometry.Plasmid and siRNA-GL2 or Lamin A/C  were transfected into HEK293 cells (50000 cells/well in 24 well plate) with Lipofectamine 2000 and incubated for 72 h at 37 °C and 5% COFlow cytometry was performed using a BD LSR II instrument . The filters employed were 550LP- BP575/26 and BP450/50 for YFP and CFP respectively. YFP positive cells were selected using the FACSdiva™ software and FCS files were exported for analysis in R .2. Luciferase activities were determined with the Dual-Glo® Luciferase Assay . psiCHECK-2 transfected cells were treated in accordance with the manufacturer’s protocols and luminescence was assayed on a Synergy 2 microplate reader . Luminescence intensities for the Renilla luciferase (Rluc) were normalized against the simultaneously examined firefly luciferase (Fluc). Normalized Rluc/Fluc values were calculated according to manufacturer’s guidelines.HEK293 cells (50000 cells/well) were transfected with Lipofectamine 2000  and incubated for 72 h at 37 °C and 5% COTotal RNA was isolated from HEK293 and HeLa cells via Phenol/Chloroform extraction according to manufacturer’s protocol . The BioAnalyzer System was used to measure the quality and quantity of total RNA. . 2 μg of total RNA was used for RNA purification with magnetic bead cleanup module . Purified RNA was ligated with sequencing adapters, reverse transcribed and purified . Finally, barcodes were added and library size and amount was detected via BioAnalyzer HS Chip .Diluted library (18pM) was clonally amplified by emulsion PCR in the IonTorrent OneTouch System according to manufacturer´s protocol . Amplified library purification was performed by OneTouch ES System. Templated ISPs were loaded onto IonTorrent 316 Chip and sequenced via IonTorrent Personal Genome Machine. Raw reads were trimmed and high quality reads were analyzed via custom Perl scripts as describedTo analyze sRNA contents the Ago2 Co-Immunoprecipitation from HEK293 data was retrieved from the public database GEO: GSE58127Suite Software  and high quality reads were then aligned to the miRbase21 and snoRNA base v3 using bowtie2HeLa cells small RNA data was retrieved from GEO GSE50057R), its decay rate (γr), the free mRNA interaction with miRNAs, which concentration is assumed to be constant, forming mRNA-miRNA complex (r*) at on rate (kon). This complex can dissociate either to its individual components (koff) or degrade the targeted mRNA (γr). These processes are described in the following We adapted a previously published simple model that explains threshold on miRNA repression activity in terms of miRNAs binding capabilities and miRNA concentration*):In this context, total miRNA concentration is considered as the free miRNAs plus the miRNAs included in the complex  suitable for microscopy  and ii) For using the system in fluorescence microscopy settings, a green fluorescent protein (GFP) was used as gene-expression reference while a red fluorescent protein (RFP) contained a specific complementary target sequence  as reporter for miRNA activity . By compTo quantify the miRNA inhibitory activity on a larger number of cells we developed in parallel constructs that were suited for flow cytometry, including the set of fluorescent proteins that fit the most commonly used laser/filter sets . For floThe intensity of CFP for miR-27a-3p is not proportional for low YFP intensities until it reaches high YFP intensities, suggesting a deviation from linearity. In contrast, for miR-23a-3p targeted constructs the CFP/YFP ratio decreased but it keeps a linear behavior .For a direct comparison with luciferase based miRNA-activity analysis, YFP positive cells were gated and the ratio between log (CFP) and log (YFP) was calculated . The outThe output of UTA-experiments after flow cytometry from controls and the two individual miRNA-candidates displayed different results: the empty vector and several non-cognate inserts showed a linear shape relation between reporters , miR-23-38The advances on miRNA regulation analysis through mathematical models introduced a multilevel understanding of miRNA on an intricate set of nodes, which included targeted expression, feedback positive or negative loops for transcription factors , unbinding constant (koff) and decaying rate (γr*)  as function of transcription in absence of miRNA regulation (r0), which here is measured by YFP intensity. The miRNA targeted mRNA transcript (CFP) includes a rate constant  and (λ). Theta (θ) is proportional to the total miRNA concentration and lambda (λ) to the dissociation constant of the miRNA-mRNA complex (see methods). Using values within the YFP intensities as r0, we simulated steady state solutions for several θ and λ, and function shapes suggest that the threshold depends highly on binding capability (1/ λ) while the expression levels (θ) governs the shift of function (The steady state solutions for r (CFP) generate two parameters that control the shape of the function and are proportional to binding capabilities and miRNA levels,  we introduced either one or three target site-copies, or a bulged target site, to shift the threshold (changing θ) we transfected increasing concentration of siRNA (GL-2) and simplified the resulting FACS outputs through transfer functions  changes the threshold shape, while increasing concentration by siRNA transfection shift the transfer function away from the non-transfected control .θ and λ ranges, the simulation graphs described 3 different functional groups  according to the shape of the curve  had no observable threshold but a parallel function shift from the non-transfected control. One and three copies of perfect complementary target sites exhibited threshold differences, for high functional concentrations the curve is non linear, while for mid functional the curve displayed the expected intermediate shape .In model terms, the low functional miRNAs have a low mRNA repression within several λ at low expression levels θ, were no threshold is observed and just a shift on the intercept of the linear behavior is observed. For high and mid functional groups, the function shape depends on 1/λ and θ coupled interaction, because high values for 1/λ could potentially pull medium-expressed miRNAs towards the high functional group (blue), while highly expressed and low 1/λ would rather bring the function close to the mid functional (red) miRNA group .To test the relation between expressed miRNAs and the UTA measured repression, three independent HEK293 small-RNA libraries were generated and sequenced using the Ion Torrent platform. The microRNAs contained in the miR-Base 21 were selected as a reference dataset and the individual read-counts were normalized using the DEseq package on Bioconductor. The three independent libraries have highly similar read-count distributions and in particular the miRNAs share similar expression profiles . We usedThe endogenous miRNA transfer functions displayed the same threshold behavior that was observed on raw flow cytometry data . The miRComparing the shape of the curves from endogenous miRNAs with the simulations and the GL-2 experiments, we observed the defined groups : First aSecondly, high functional miRNAs have an observed transfer function with a notorious threshold (low CFP intensities from a considerable range of YFP intensities), before it reaches a linear behavior  and the Also, when we compared the mean of transformed read counts among the described functional groups we observed no differences between low-mid and high-mid functional groups but a clear and significant difference between the low and high functional. This finding indicates that differences at inhibitory potential might appear below 1000 reads, reflecting previously discussed expression thresholds seen in bulk luciferase and fluorescence assays171920To examine whether the described groups may rather be reflected by RISC associated miRNAs than by the total miRNA content, we analyzed public available Ago2 co-IP data sets on HEK293 cellsSince the GL-2 siRNA experiment proved that UTA out puts are remarkably valid for the model predictions. We decided to define its reliability for endogenous miRNAs we switch the threshold sharpness of miR-27a-3p introducing different copies for perfect match and bulged target sites . We obseθ were calculated. Remarkably θ kept a constant value for perfect and bulged copies  were co-transfected with UTA reporters for low, mid and high functional endogenous miRNAs. As expected the transfer function shifted upon siRNA co-transfection but no change on the sharpness was observed .θ), we derived these parameters for all tested miRNA-candidates and their transfer functions. We observed significant differences between the calculated 1/λ for low functional and the other tested groups, while there was no significant differences among mid and high functional groups  and miRNA quantity , while one high functional miRNA with expression lower than the median of mid functional miRNAs expression was selected (let-7c-5p).The UTA transfer functions for candidates were plotted . InteresIn summary, our findings and results underline that the UTA construct system is applicable to other cells types. The functional data is useful to define alternative hypothesis based on its outputs, and that miRNAs subcellular location also defines its functionality, displaying that expression profiles without a systematic and detailed functional outputs could be misleading.Recent studies have deep sequenced miRNAs present in the total small RNA population in various cell types and tissues. The underlying assumption among these studies is that miRNA quantification accurately reflect miRISC activity, since aligned reads from sRNA libraries display Ago2 guide strand stabilization at miRNA genomic loci4649Nevertheless, the complex and dynamic cellular function of miRNAs introduces uncertainty to the expression profiling conclusions. For instance: Ago proteins (Ago 1–4) quantities are lower than those of miRNA molecules, these Ago sub-stoichiometric amounts deal with the miRNA excess by multiple rounds of recruitments on miRNA-mRNA free complex, showing that Ago2 can interact with pre-made double stranded RNAFurthermore, studies of miRISCs sub-cellular locations suggested that only ER associated fractions perform gene silencing at the mRNA level1819At functional level, lentiviral based high-throughput methods, which include antibiotic-selection and cell sorting, confirmed the breach silencing-expression1917We reason that a high-resolution functional miRNA evaluation must be included and might bring clarifying evidence, since a great number of studies test the miRNA-functionality through artificial expression or luciferase based assays. These assays could misjudge the miRNA’s physiological activity and provide only whole population measurements within a rather narrow detection limit1718To set up a single cell resolution data analysis, we adapted and tested the reliability of a molecular titration modelWe propose that UTA drastically simplifies the experimental workflow and can expand the model to other cell types. It was created with new and established features suitable for all transfectable cell types. Our dual fluorescence system uses common constitutive promoters .Additionally, we also consider that our tool, which integrates the titration modelOutside of classic miRNAs mechanisms, the increased number of studies showing small RNAs association with Ago2 or miRNA like size101758591017616262636465666722Finally, functional studies between different conditions and further mechanistic definitions would be necessary to understand the three levels of miRNA-functionality. For example, a highly expressed miRNA included in Ago2 but bearing a characterized as low functional, might have high target-to-miRNA ratio, a different cellular location that inhibits its activity or even non-canonical binding. Importantly, these observations can only be reached through functional assay with high resolution as we introduced here, while simple profiling can lead to misinterpretation.How to cite this article: Lemus-Diaz, N. et al. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system. Sci. Rep.7, 45197; doi: 10.1038/srep45197 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.