Fine mapping of this locus identified a number of polymorphic candidate genes, however only one, beta haemoglobin, was differentially expressed in both the founder strain and F6 generation. These findings provide novel insights into the genetic substrates and putative neurobiological mechanisms of impulsivity with broader translational relevance for impulsivity-related disorders in humans.Impulsivity describes the tendency to act prematurely without appropriate foresight and is symptomatic of a number of neuropsychiatric disorders. Although a number of genes for impulsivity have been identified, no study to date has carried out an unbiased, genome-wide approach to identify genetic markers associated with impulsivity in experimental animals. Herein we report a linkage study of a six-generational pedigree of adult rats phenotyped for one dimension of impulsivity, namely premature responding on the five-choice serial reaction time task, combined with genome wide sequencing and transcriptome analysis to identify candidate genes associated with the expression of the impulsivity trait. Premature responding was found to be heritable (h Although acting impulsively can occasionally be advantageous, it more often results in negative outcomes, especially when co-expressed in disorders of personality2 and mood3, drug addiction4, and attention-deficit hyperactivity disorder (ADHD)5. As such, impulsivity has emerged as a key dimensional construct in psychiatry with utility as an endophenotype to inform gene discovery and etiological mechanisms for a variety of disorders7.Impulsivity describes an individual’s predisposition towards premature, excessively risky, poorly planned and inappropriate actions and decisions8. Several groupings have been proposed to capture the evident heterogeneity of impulsivity; for example, impulsive action that depends on self-restraint to avoid or stop a response, and impulsive choice involving preferences for small, immediate rewards over larger but delayed rewards11. Research into the neural mechanisms of impulsivity has been greatly accelerated by the development of objective, translatable measures of impulsivity in rodents, humans and other species7. Indeed, convergent studies in humans and experimental animals suggest that interactions of the ventral striatum, including the nucleus accumbens core and shell , with the prefrontal cortex (PFC) and hippocampus mediate different forms of impulsivity, with important contributions from the ascending dopaminergic, serotonergic and noradrenergic systems8.Impulsivity is a complex, multidimensional trait mediated by distinct psychological and neural mechanisms15. Findings in humans indicate that impulsivity may be a heritable risk factor for the development of harmful drug use4, consistent with a substantial genetic etiology of this disorder17 and the impulsivity trait18. To investigate further the genetic mechanisms of impulsivity we assessed the heritability of a single quantitative measure of impulsivity in male and female rats, premature responding on the 5-choice serial reaction time task (5CSRTT). Premature responding on this task reflects the failure of a subject to inhibit anticipatory responding to a reward-predictive cue – a form of waiting impulsivity8 – and has been shown to vary considerably in outbred strains of rats21. Persisently high levels of premature responding on the 5CSRTT predicts increased escalation of cocaine and nicotine self-administration22, an increased risk for relapse after punishment- induced drug abstinence23 and the subsequent emergence of compulsive cocaine self-administration that persists despite concurrent punishment24.The personality trait of impulsivity is prevalent in substance use disorders acting as both a determinant and consequence of drug abuse and addictionIn the present study we established a six-generational pedigree of animals selectively bred for high and low levels of impulsivity on the 5CSRTT. We specifically investigated the heritability of this ‘trait-like’ characteristic prior to carrying out a non-parametric linkage analysis with next generation sequencing to identify and fine map significant quantitative trait loci. Microarray- based gene expression profiling of brain regions previously implicated in impulsive behavior on the 5CSRTT  was subsequently carried out to reveal differentially-expressed genes with underlying sequence variation within the linkage region. = 11.087, p<0.01, main effect of phenotype F = 401.885, p < 0.00001, breeding x phenotype interaction F = 2.879, p < 0.05 MANOVA, . When iVA, Fig. . AnalysiVA, Fig.  and absoVA, Fig.  number o,69 = 11.Our study identified 1,536 single nucleotide polymorphisms (SNPs) in generations F1–5 of the established pedigree. Figure th generation. The single nucleotide variants and indel sequence variants detected are shown in Supplementary Tables We repeated the genome-wide linkage analysis of the first five generations using the average number of premature responses as the quantitative trait. This identified a second locus at chromosome 1q31–1q34, which resided at ~ 213 Mbp . Importantly, for each quantitative trait (percentage and absolute premature responding), linkage results were confirmed using different sets of families (obtained by PedCut and Jenti), suggesting the identified QTL was robust and not dependent on the family structure used in the linkage analysis. In order to detect sequence variants potentially associated with impulsivity, in addition to the already analysed SNPs, we carried out whole genome sequencing (WGS) in four extremely-discordant sib-pairs chosen from the 5Hbb and Nmb  Fig. . Of the Nmb Fig. .Figure 4Pde3b, Sv2b, and Hbb; Supplementary Table Sv2b, Rlbp1, Far1, Grm5, Pde3b, Usp47, Ppfibp2, Prc1; Supplementary Table Hbb was found to be consistently differentially-expressed in both the base and F6 generations so we sought to confirm its differential expression independently. Quantitative real time PCR from tissue from a separate cohort of phenotyped rats confirmed Hbb expression was significantly reduced in the IL cortex  and NAcS  but not in the NAcC of HI rats  nevertheless support the conclusion that the impulsivity phenotype on the 5CSRTT is heritable and enriched by selective inbreeding.The heritability estimates we report  for the impulsivity trait are comparable with estimates for other behavioral and physiological phenotypes in rodents36, novelty reactivity37, novelty preference38 and incentive salience attribution  demonstrate moderate rates of heritability in rats and mice. While there is evidence to suggest the heritability of these susceptibility traits are primarily driven by an inter-relationship with novelty reactivity (e.g.39), rats impulsive on the 5CSRTT fail to show enhanced measures of novelty reactivity40. Further, impulsivity and novelty reactivity have been shown to predict distinct aspects of addiction-like behavior24 suggesting the addiction vulnerability phenotype in rats may involve multiple genetic contributions. Given there was no difference in the proportion of HI vs LI rats across each generation it is unlikely the heritability of impulsivity was driven by a relationship with an indirect variable such as maternal behaviour41.The observed heritability of impulsivity is also consistent with other traits shown to confer susceptibility to addiction-like behaviors in rodents. Thus, anxiety42, which in turn may underlie sexual dimorphism observed in the expression of related neuropsychiatric disorders such as addiction43 and ADHD44. However, no significant difference was observed when incorporating the effect of breeding and sex into trait heritability estimates. Further, no evidence of linkage was observed on either sex chromosome, suggesting that alternative mechanisms may underlie the observed sex-dependent breeding effects; for example as a result of sex-specific regulatory elements on autosomal gene expression45 and/or the impact of circulating sex hormones on 5CSRTT performance46.The expression of the impulsivity phenotype, as measured by premature responding in the 5CSRTT, was moderated by sex, with HI male offspring, specifically bred for high impulsivity maintaining quantitative extremes of their phenotype across multiple generations, with females and males bred for low impulsivity showing lower levels of impulsivity. This observation aligns with clinical studies demonstrating the influence of gender on impulsivity, with greater heritability of this trait in males8. This network of corticostriatal structures encompasses parallel and interacting circuits involving the NAcbC and NAcbS with the infralimbic cortex47, cingulate cortex48, insula49 and ventral hippocampus50. Rats expressing the HI phenotype show a reduction in D2/3 receptor availability in the NAcb19, decreased grey matter density and γ-amino-butyric acid (GABA) levels and markers of GABA function and dendritic spine density in the NAcb52, and structural abnormalities in the insula49.Since more than 300 genes were present within the identified linkage region, including 126 polymorphic genes, we utilised a transcriptome analysis to investigate gene expression in key brain regions of the ‘waiting impulsivity’ networkGrm5 (metabotropic glutamate receptor 5) shown recently to modulate impulsivity53 and cocaine reinforcement55 and whose deletion is associated with ADHD56; Sv2b (Synaptic vesicle glycoprotein 2B) implicated in neurotransmitter release through regulation of the synaptic release vesicles and exocytosis58; Rlbp1  associated with alcohol preference in mice59.A number of transcripts located within the linkage region were differentially regulated between high and low impulsive rats, some of which were also sequence variant. Notably, some of these genes have previously described roles in impulsivity and associated disorders including: Hbb, demonstrated significantly altered gene expression in both the founder strain and generation six of the pedigree. This finding was additionally confirmed in an independent cohort of impulsive rats, which showed a significant reduction in the level of this transcript in the NAcS and IL cortex, potentially implicating a novel role for this gene in impulsivity. Hbb encodes for the beta chain subunit of the haemoglobin complex and is traditionally considered in the context of oxygen transport in vertebrate blood erythrocytes60. However, Hbb is also expressed in neurons of invertebrates, rats and humans64 where it is hypothesised to contribute to oxygen homeostasis and mitochondrial respiration61. It is therefore not clear whether the observed reduction in Hbb transcript in HI rats represents blood or brain hemoglobin. It is possible that differences in expression may reflect a regional perfusion deficit. However, transcript levels for other blood proteins present on the array  were unaltered between HI and LI rats, suggesting the reduction in Hbb was more likely a neuronal or glial-derived transcript.Of these identified polymorphic candidates, only one gene, 64 with reductions in neuronal haemoglobin immunoreactivity reported in Alzheimer’s and Parkinson’s disease65. Thus, low expression may be one variable contributing to oxidative stress and neuronal abnormalities in corticostriatal networks related to impulsivity. Since GABAergic medium spiny neurons (MSNs) are highly susceptible to oxidative stress after transient global cerebral ischemia66 with prenatal ischemia leading to reduced dendritic branching and spine density in MSNs67, we hypothesise that impaired neuronal Hbb function may increase the vulnerability of MSNs to oxidative stress with consequent effects on dendritic spine density and function in MSNs of the NAcb, potentially as a manifestation of antenatal and developmental hypoxia. This conclusion could be tested by tissue-specific interference of Hbb in relevant brain regions.Hbb expression is highest in midbrain dopamine neurons, striatal GABAergic neurons, cortical pyramidal neurons and glial cellsWe report a number of transcriptomic alterations associated with impulsive behavior in the 5CSRTT. However, genetic variation linked to impulsive behavior may also have other functional effects on the genome; for example, through epigenetic modification and variation in RNA splicing proteins. In addition, since our gene expression analysis was limited to adult tissue, we cannot discount the possibility that differential expression of these genes within the QTL affected key stages of neurodevelopment, which in turn affected the function of the mature ‘waiting impulsivity’ network in adults.8.The endpoint of this study was to demonstrate the heritability of the impulsivity trait in rats and to identify linkage of discrete chromosomal regions with impulsivity, as indexed by premature responding in the 5CSRTT. Our findings indicate that impulsive responding in the 5CSRTT is a heritable trait and therefore a valid endophenotype to investigate the brain mechanisms of premature responding and more broadly waiting impulsivity. Our integrative approach enabled us to identify a target locus harboring candidate genes relevant to the etiology of impulsivity, with translational potential to inform our understanding of analogous forms of impulsivity present in ADHD, drug addiction, depression, and other impulsivity-related disordersad libitum with sufficient food provided to maintain body weights at no less than 90% of free-feeding weights (20 g chow/day). Rats were housed four per cage, under temperature- and humidity-controlled conditions and a reversed 12-hour light/dark cycle (white lights off/red light on at 07:00 h). All regulated procedures conformed to the Animals (Scientific Procedures) Act of 1986 and approved by the animal welfare ethical review board at the University of Cambridge.Founding male and female adult Lister-hooded rats were purchased from Charles River , aged 2–3 months. Behavioral training commenced 1 week after acclimatisation to the animal house (for the founding generation) or at 3 months of age for derived animals. Water was available 68. A PC using WhiskerServer software and FiveChoice client69 controlled the behavioral chambers . Rats were trained to nose-poke into one of five apertures, for a food-predictive, light stimulus. Correct responses were rewarded, while incorrect, premature and omitted responses were punished with a 5 s time-out. Daily training sessions consisted of 100 discrete trials or 30 minutes, which ever was shorter. Each trial was initiated by the entry of the animal into the food magazine. Following an inter-trial interval (ITI) of 5 s, a brief light stimulus (0.7 s in duration) was presented randomly in one of the five apertures. A nose poke into the corresponding aperture was rewarded with delivery of one food pellet . Failure of the animal to respond within 5 s (‘omission’) or a nose poke into the incorrect aperture (‘incorrect’ response) resulted in a 5 s time-out, during which time no new trials could be initiated and the house light was extinguished. A nose poke response prior to the onset of the light stimulus (‘premature’ response) also resulted in a 5 s time-out. Daily training sessions continued for 8–12 weeks (5 daily sessions/week) until performance on the task was stable .The founding cohort of 48 male adult rats was trained daily on the 5CSRTT, as described previouslyScreening for impulsivity involved challenging the animals with a series of long inter-trial interval (LITI) sessions where the waiting interval leading up to the presentation of the light stimulus increased from 5 s to 7 s. A single LITI challenge session was employed once a week, on three occasions, with two baseline sessions (i.e. ITI 5 s) on the days leading up to the LITI session and two baseline sessions after the LITI challenge. LITI sessions consisted of 100 trials and a session duration of 45 min. The LITI challenge ended when rats had completed 100 trials or when 45 min had elapsed. Rats were ranked for their level of impulsivity based on the number of premature responses across the three LITI challenge session. Animals making on average ≥ 49 premature responses across the three challenge sessions were deemed high impulsive (HI), while rats making on average ≤ 48 premature responses were deemed low impulsive (LI). Impulsivity phenotypes  were quantified using measures of both percentage premature  and absolute number of premature responses averaged across the three challenge sessions. Screening for impulsivity required approximately 5 months from the start of training on the 5CSRTT.2-induced asphyxiation and their brains and spleens excised and frozen over liquid nitrogen for storage at −80 °C for later analysis.A six-generation experimental cross was generated (n = 629), derived from one male HI proband from the founder cohort and nine non-phenotyped females, followed by a selective breeding strategy from generation 1 to 6, according to impulsivity phenotype. The F2 and F3 generations were derived from pairs of rats of the same impulsivity phenotype but from different litters, to enrich for alelles involved in the expression of this trait, before selective breeding, where the most extreme male and female individuals from each litter were mated to establish HI and LI lines respectively and to reduce genetic variability, limiting epistatic effects and the impact of alelles unrelated to the expression of the impulsivity trait on transcriptome analysis. Individual pairings are shown in Fig. 70, in generations F2-F6, and confirmed using linear variance component models using MCMCglmm package71 in R to account for the in-breeding within our pedigree, with sex and breeding as random effects. All variance components models were run with a minimum of 1 × 106 iterations and burn-in periods of 1 × 104. Convergence of each model was tested using the Heidelberg stationary test. Posterior mean of heritability estimates are reported.Genomic DNA was purified from spleen samples  and diluted to a final concentration ≥40 ng/µl. To attribute the contribution of variability in impulsivity to genetic factors we estimated trait heritability using the correlation between the quantitative phenotype of parents and offspring using R/QTLTo evaluate the heterogeneity of sequence variation in the Lister-hooded strain genome and to select polymorphic markers to be genotyped in the linkage study, we carried out pilot genome-wide genotyping  on six rats  randomly chosen from different families of the colony, from generations two, three and four of the experimental cross. We selected ~118,000 markers according to their Polymorphism Information Content (0.37 < PIC < 0.77) and NO CALL ratio (≤0.3) and submitted them to Illumina  for assay development evaluation. We then used the final Illumina genotyping score ≥0.5, position and distance (at least 1.5 Mbp distance between consecutive markers), to define a final data set of 1,536 SNPs that were used to genotype generations 1–5 of the experimental cross using the custom Illumina GoldenGate Bead Array.72, an algorithm designed for SNP calling in highly homozygous populations. It produces posterior probabilities for AA, AB and BB genotypes: P(AA); P(AB); and P(BB), even if the assay-failure probability P(NC) is high. In that case the correct P is the second highest posterior probability. To call reliable genotypes we applied a quality call score threshold of 0.8 and defined P <0.8 as a “no call”.Genotype calling was performed using Alchemy73 using variance component (VC) and non-parametric linkage (NPL) so to avoid confounds associated with the inbred nature of our pedigree (e.g.74). Further to this, and given MERLIN is not suitable for very large and complex pedigree structures, the original pedigree was divided into smaller families (max-bit-size = 24), using two different algorithms, PedCut75 and Jenti76, so to replicate our linkage findings. Mendelian inconsistencies were checked with Pedcheck77 and zeroed (5%). Hardy Weinberg Equilibrium (HWE) were tested with Pedstats78 and markers with HWE p < 0.0001 were removed (n = 56). A total of 894 autosomal polymorphic SNPs were selected for the final linkage analysis  from family data from generation 1 to 5, we generated an additional cross (F6) and carried out an additional linkage analysis adding data from this generation to the families (n = 76), to confirm segregation of the locus with impulsivity. An additional four heterozygous markers 80 with default parameters. Genomic variants, which included single nucleotide (SNV) and short insertion/deletions (indels) (1–15 bp), were detected using the Genome Analysis Toolkit (GATK version 1.0.6001)82 with default parameters. Before calling variants, data were pre-processed by masking of clonal reads using Picard tools (https://sourceforge.net/projects/picard/), realignment of reads around potential indels, and recalibration of base quality scores using GATK81. To exclude potential false positives, variant quality was recalibrated for both SNVs and indels. Polymorphic SNVs and indels where alleles in at least one rat differed from the reference BN rat genome were then extracted from the variant data.To fine map the QTL region of interest and analyze a denser polymorphic-loci map between HI and LI rats, we sequenced the genome of four extremely-discordant sib-pairs from generation 5, with high throughput sequencing. Illumina paired-end reads were mapped to the reference Brown Norway (BN) genome RGSC-3.483, these regions were bilaterally microdissected using biopsy needles  for RNA extraction and subsequent transcriptome analysis.Brains from HI and LI rats derived from sixth generation animals and an additional cohort of the original outbred base population  were sectioned into 120 µm coronal slices using a Leica CM 3000 Cryostat  at −20 °C. Based upon previous studies implicating the infralimbic cortex (IL), nucleus accumbens core (NAcbC) and shell (NAcbS) in the expression of impulsivity on the 5CSRTTOne-ml of TRIzol reagent  was added to each of the punched tissue samples. The resulting suspensions were homogenized by 20 passages through a 22 gauge needle. Chloroform was added and the RNA isolated by phase separation after centrifugation. The RNA-containing upper phases were carefully removed and purified with an RNeasy MinElute Cleanup Kit  and Genomic DNA eliminated with a DNase step (Qiagen). RNA yield and purity were assessed with a NanoDrop 1000 spectrophotometer  and samples having optical density 260/280 measurements in the range of 1.8 to 2.2 were kept for further analysis. RNA integrity was determined using an Agilent 2100 Bioanalyzer ; only samples with RNA integrity values above 8 were used.www.arrayanalysis.org) in R 14.2 software environment. Probe sets were summarized according to updated Entrez Gene definitions, version 14, and data subsequently quantile-normalised using the robust multi-array average function of the Affymetrix package from the Bioconductor project84. Genes with expression values below 80 were excluded to avoid confounding array background noise.RNA samples were subjected to microarray gene expression profiling. One-hundred ng of total RNA was amplified and labelled using the GeneChip 3’IVT Express Kit  according to the manufacturer’s protocol. Labelled RNA was hybridized on GeneChip Rat Genome 230 2.0 Arrays according to protocol and scanned using an Affymetrix GeneChip Scanner 3000 run with Affymetrix GeneChip Command Console Software (AGCC) at the microarray core facility at the University Hospital Mannheim. The obtained cell intensity (CEL) files were quality assessed using the array analysis Affymetrix quality control pipeline  from a separate cohort of outbred Lister Hooded rats . Total RNA was prepared from tissue biopsies specifically from the NAcS, NAcC and IL cortex as described previously by use of an RNAeasy Total RNA kit  and reverse transcribed using the RT2 first strand kit (Qiagen) according to the manufacturer’s protocol. RT-PCR amplification was performed using a QFX96 PCR detection system  with the use of RT2 SYBER Green Master Mix (Qiagen) with RT2 qPCR primer assay for each gene of interest (Qiagen). ACTB, HPRT, and TBP were used as housekeeping genes and samples run in triplicate. Relative gene expression was calculated using the delta Ct method.We identified one gene within the linkage region that was polymorphic and showed consistent changes in gene expression in both the founder and F6 strains. To confirm the findings of the array, we assessed transcript levels for this gene, Supplementary Information.


Bivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to monovalency upon differntiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely.We develop an approach for capturing genes undergoing transcriptional switching by detecting ‘bimodal’ gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state, and identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudotime approximation and use a hidden Markov model to infer gene activity switching pseudotimes, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets.Genes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state. Since chromatin bivalency is a hallmark of gene promoters poised for activity, this approach provides an alternative that complements conventional scRNA-seq analysis while focusing on genes near the ON/OFF boundary of activity. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data. All levels of chromatin organization in eukaryotes are associated with covalent histone modifications that expose or constrain the genome for regulation , 2. Becabivalent, marked by both the activating H3K4me3 mark and the repressive H3K27me3 mark [While nucleosomes at most promoters in most cells carry either activating or repressive histone modifications, a large number of promoters in embryonic stem cells are me3 mark . Increasme3 mark , 6.Our understanding of transcription is being revolutionized by single-cell RNA sequencing (scRNA-seq) , which pOne challenge is that scRNA-seq data commonly yields many genes with measured expression of exactly zero in many cells. Zeros may appear because of limited read depth in single cells, or from technical sources of zero inflation. In either case, the frequent zeros present challenges for both normalization and for detection of differential expression . In mostCell state-dependent genes in scRNA-seq data are often chosen first on the basis of high cell-to-cell variability. These Highly Variable Genes (HVG) may include genes differentially expressed in bulk transcriptomic studies, and may include genes indicative of underlying cell variability. HVG detection methods include exploiting the coefficient of variation  or squarMotivated by our interest in chromatin bivalency, our working model is that important stochastic expression changes occur right at the transcriptional boundary between active and inactive states . Genes tTo identify transcriptional switches at bivalent (including poised) promoters, we focus on genes that are sometimes silent, and other times expressed. We show that a modified Gaussian mixture model can identify genes undergoing transcriptional switches, even in the presence of the abundance of zeros common to scRNA-seq data. We refer to these as “bimodal” genes. We associate these bimodal genes with chromatin state from independent ChIP-seq studies and find that important developmental processes are enriched in bimodal genes carrying bivalent histone marks in pluripotent cells, and that bimodal bivalent genes can be used to characterize cell types.We employ binarized gene expression for feature selection, and identify known and novel genes activated or silenced upon differentiation of embryonic stem cells to distinct cell types. We show how fractional state proportion of binarized gene expression data is a useful complement to conventional differential expression measures for many genes, and allows us to identify genes that switch activity with cell state change.We apply a similar approach to time course differentiation data and develop a pseudotime ordering method exploiting probabilistic suffix trees of binarized data. The pseudotime ordering allows us to identify switching events using a hidden Markov model in pseudotime, when genes switch between mostly OFF and mostly ON states. We use the orchestration of these events in pseudotime to infer a regulatory network of gene switching.Bimodality-based variable gene detection identifies genes in ON/OFF transcriptional statesWe define expression variability as the toggle between active and inactive transcriptional states. Variable genes, therefore, are those that are active in some cells and inactive in others in a population. Across the population, we expect the variable genes to show clear separation of expression values, resulting in a bimodal distribution.p<0.01 and a smallest mean below 1 were selected as bimodal genes.For identification of bimodal genes, we define the underlying model of gene expression as a mixed lognormal distribution with possible zero inflation Eq. ” sectionWe applied this approach on two scRNA-seq datasets . The firThe two datasets presented two different experimental groupings. In H1 data, cells were grouped based on cell types, while in H9 data, cells were grouped based on experimental time points. We applied our bimodal gene identification method to H1 data to explore differentiation signature genes and genes with poised promoters, and later to H9 data to construct and analyze a pseudotime trajectory based on bimodality.−5  marks important developmental and differentiation regulatory genes . BivalenWe integrated the 2610 bimodal genes from scRNA-seq analysis of H1 data with independent ChIP-seq data of the same cell types to focus on those bimodal genes that are bivalently marked in H1 cells. This selection is meant to include those genes poised for transcription while in the undifferentiated state. This integrative analysis resulted in 644 bivalent, bimodal genes from the H1 data.When cells were clustered based on the expression profiles of bivalent bimodal genes, all of the cells clustered perfectly accordingly to the experimentally determined cell type Fig. a. This aIn contrast to bivalent bimodal genes, when we used monovalent H3K4me3-marked bimodal genes in H1 cells for clustering, the cell type-specific clustering was much weaker , and cells with expression value more than that were considered active (ON).We used the clustering obtained above to assign fractional gene activity between 0 and 1 by assigning cells to random windows within each cluster, converting the original 750 cells with binarized expression to 30 windows with fractional activity. Using these fractional expression profiles, we clustered bivalent bimodal genes into 8 groups  . In thisWe used the 4943 bimodal genes and asked whether we could use binarized bimodal genes to infer a pseudotime trajectory of cells. Unlike H1 data where a clear distinction between cell types was important to reveal marker genes, an overly clear distinction between time-dependent cell clusters in H9 data would potentially compromise pseudotime trajectory reconstruction. Therefore, we included H3K27me3 monovalent bimodal genes in addition to the H3K4me3/H3K27me3 bivalent bimodal genes. We also kept H3K27me3 monovalent-marked genes to identify genes that showed de-repression upon differentiation. A total of 1371 genes were selected.The selected genes characterized cells at each timepoint well, with the exception of cells from 72 and 96 hours of treatment  of binarized gene expression. To order the cells within each cluster, we used the adjacent time points (clusters) as anchors (sequences), we built a probabilistic suffix tree (PST) for each cluster, followed by sequence generation. If no unique sequence could be obtained, we selected the ordering with the highest probability based on the PST. We observed that all clusters generated unique sequences except for cluster 1. We believe this is because cluster 1 was formed from untreated control cells, so strictly speaking they were not on the same pseudotime trajectory as the other treated cells. This was also reflected in the t-SNE map . The switching points for each gene were determined based on the transition between active and inactive state paths in pseudotime-ordered cells. We classified genes into 17 switch groups, based on the initial state, final state, and the number of switches  gene set from MSigDB  using a Gene regulation by transcription factors often shows a correlation between a TF and its targets. Some of the transcription factor hits were bimodal genes themselves, so we examined whether the switching pattern of a TF was recapitulated in the switching pattern of predicted targets. We compared the expression of four bimodal TFs  to the aggregated number of transcriptionally active TF targets that are also transcriptionally switching Fig. . In eachA network of switching genes based on mutual informationconnector genes that are closely related to the pathway genes based on variation of information. We assumed that the connector genes are the mediating regulators that help to establish a more interconnected map. With 68 switching genes that were involved in the pathways identified from the earlier pathway search, we selected the top 20 most related connector genes.Based on the pathway genes identified from the earlier analysis, we wanted to draw a map of key regulatory genes involved in endoderm differentiation. In addition to the pathway genes, we identified Co-expression networks are mapped using a distance metric obtained from expression data , to estaThe ARACNE network is undirected, but we assigned direction to some edges based on the switching times of the connected nodes (genes). If gene A had undergone transcriptional switching earlier in pseudotime than gene B, the edge connecting A and B was directed from A to B. This was based on the assumption that a prior event would influence later events. The network may not represent the actual gene regulatory network, but displays the co-switching relationship between key pathway genes during endoderm differentiation.The network structure shows several interesting properties related to the earlier pathway and transcription factor analyses. Frizzled genes  and bone morphogenetic protein (BMP) genes  appeared at different parts of the network, indicating their involvement in different signaling events. In the motif analysis we had found that switching genes were enriched in LEF1 and NFAT targets. From the network map, the edge connecting FZD5 and TCF7 further supports the presence of the canonical Wnt/beta-catenin signaling pathway. On the opposite side of the network map, we saw a hub of genes around HAPLN1. The presence of FZD2 (Frizzled 2), CAMK2D , and DAAM1 (Dishevelled Associated Activator Of Morphogenesis 1) suggests that there is also non-canonical Wnt/Ca2+ signaling pathway present. The time-dependent differential expression of various Wnt and frizzled genes further supports the involvement of dynamic Wnt signaling events  then selected based on a mean-variance threshold . VariancIn this analysis, we instead model differential expression of signature genes happening across the transcriptional ON/OFF boundary, regardless of the actual magnitude of expression values. We choose this approach because the most functionally striking difference in expression is a switch from complete repression to activation, or vice versa. This is related to, but distinct from, the assumption behind variance-based methods.Crossing the transcriptional on/off boundary, or switching between gene activation and repression, does not ensure bimodally distributed expression values. However, in this study we used data having distinct experimental groupings: either grouped according to cell types (in H1 data) or according to experimental time points (in H9 data). We assumed that signature genes distinguishing the cell groups must have formed stable transcriptional states within each cell group distinct from the originating cells. Such shifts in transcriptional states would result in bimodally distributed expression values across the cell groups, leaving a clear separation between the expression values from the on/off transcriptional states.We also focused on the toggle between on/off transcriptional states because histone mark-driven transcriptional control mostly involves switching between repression and activation, especially in the poised promoter model of bivalency. This model is relevant for both the H1 and H9 embryonic stem cell differentiation data sets we used.In a typical population-based transcriptomic analysis, differential expression descriptors (such as “increased/decreased expression”) describe gene expression as a process on a continuous scale of change. Differential expression analysis then takes the quantified or normalized read counts and performs statistical tests to discover quantitative changes in expression levels between experimental groups . Genes iBimodality-based identification of variable genes, therefore, presents a complementary method of detecting gene expression changes in scRNA-seq by focusing on the on/off transcriptional states. We are not advocating any superiority of one method over the other, but rather presenting a different method of identifying variable genes. If a choice were to be made between variance-based and bimodality-based methods, it must depend on the purpose of the experiment.Advantages of binarizing expression dataSince bimodal genes present clearly distinguishable on/off transcriptional states, their expression values could be appropriately binarized, which presented several advantages for later analysis. In addition to using Fisher’s exact test for differential gene expression testing, binarized expression makes the data less susceptible to normalization accuracy and less biased towards genes with high expression counts. Binarized counts also enabled the use of mutual information-based analysis to detect patterns of co-regulatory genes. This is in contrast to many of the existing co-expression network methods based on expression correlation , and preIntegrating chromatin state information into transcriptomic analysisWe have seen that the presence of H3K4me3/H3K27me3 bivalency successfully identified cell type-specific bimodality in the differentiation datasets. We also saw that H3K4me3 monovalent bimodal genes were enriched in cell state-specific pathways such as cell cycle regulation. Our results suggest that chromatin states indeed offer valuable insight to further interrogate the transcriptomic data.Our approach may have work especially well in differentiation studies because of the poised promoters of bivalent genes. Several studies have shown that bivalently marked genes are differentiation signature genes , 22. SinFurther studies linking between chromatin state and gene expression are currently limited by the availability of experimental methods. Various challenges include simultaneously measuring both quantities and achieving single-cell level for chromatin state data.On the use of binarized data for regulatory network inferencelevels may be useful to infer regulatory networks and reveal hidden components of pathway activation.Some of the earliest gene regulatory network reconstruction methods based on microarray expression data used Boolean networks. Technologies to infer networks have advanced tremendously since then, as have technologies to measure gene expression. However, single-cell RNA-seq data offers the opportunity to observe – zero inflation notwithstanding – if a gene is transcriptionally on or off in a given cell, and the binary state of its activity is informative. As we show here, genes that might otherwise be missed because they are not expressed at high enough Histone mark modifications are strongly associated with transcriptional regulation, but direct analysis in single cells is limited by current technology. By adopting a strategy to integrate population-level measurements of histone modifications and single-cell transcriptomics, we identified transcriptional switching patterns of bivalent genes in pluripotent cells associated with cell type upon differentiation. The technical strategy, using a mixture model-based method to identify genes with multimodal on/off expression patterns across cell types, is amenable to both pairwise comparisons and time course scRNA-seq experiments.Binarizing transcript expression levels facilitates analysis of transcriptional state, and supports novel analysis analogous to conventional differential expression as well as novel pseudotime inference, estimate of switching points in pseudotime, and gene regulatory network reconstruction.ggplot2 [All analysis was performed in R  and mosggplot2 .DataChIP-seq files for H1 cells and H1 cells differentiated by treatment with BMP4 – into mesendoderm, trophoblast and neural progenitor cells were downloaded from the UCSD Human Reference Epigenome Mapping Project scRNA-seq files for baseline and differentiated H1 and H9 cells were downloaded from GEO Series GSE75748 .NormalizationFor normalization, we used Transcripts per kilobase million (TPM) to scale both datasets. Transcript lengths were based on the GRCh37 genome build from the Ensembl database through biomaRt . We compp<0.01.The two methods of normalization were compared based on the number of bimodal genes detected in H9 data, with the threshold of dip test Model description Let Yg denote the vector of observed gene expression values for a gene in a population of cells. Given a population of cells, the gene may be in active or inactive states in different cells. We define inactive (OFF) and active (ON) transcriptional states, XOFF and XON, to denote the gene expression model of one gene based on the bimodal nature of transcription. We assume that the log of normalized gene expression values in state i follows a Gaussian distribution with a mean of μi and standard deviation of σi.We take the threshold of 1 for mean gene expression values to distinguish the two states.Due to the nature of scRNA-seq, we add in in zero inflation component to the model, yielding a sum of two lognormal distributions for the data plus the zero inflation.We add a small amount of normally distributed noise after log transformation and model the result as a Gaussian mixture.ε=1×10−5 was optimized over a wide range of values choosing the value that resulted in clear single maximum number of bimodal genes for the given data set  , 32 implSignature analysishttp://software.broadinstitute.org/gsea/msigdb/annotate.jsp) at the Broad Institute, with default parameters. Genes were compared to C2 (curated gene sets) category, C3 (motif gene sets), and C5 (Gene Ontology gene sets) gene sets [Web-based signature analysis was performed using the MSigDB “Investigate Gene Sets” tool (, availabene sets .Pseudotime trajectory estimationinfotheo [PST package was used to build probabilistic suffix trees [Matrix of Mutual Information and variation of information between cells were calculated using the R package infotheo . The PSTix trees .Network analysisminet [Co-switching gene networks were generated using the R package minet  followedAdditional file 1 Additional File 1: Supplementary Figures S1–S9 and Supplementary Tables S1–S5.


To the Editor,5-Aminolevulinic acid (ALA) is utilized for photodynamic diagnosis to detect superficial bladder cancer and its adverse effect is hypotension –5. Compan = 66) or not used . The ALA group received 5-ALA (20 mg/kg) orally 3 h before the start of surgery. The change in noninvasive blood pressure (BP) on the day of surgery and the dosage of vasopressors used during spinal anesthesia were analyzed.The medical records of 129 patients who underwent TUR-BT under spinal anesthesia using 0.5% hyperbaric bupivacaine in Kyoto City Hospital between January 2018 and March 2019 were divided into two groups, based on whether 5-ALA was used (ALA group, No significant difference existed between the two groups in the patients’ preoperative characteristics. Intravenous fluid volume during anesthesia was significantly higher in the ALA group than in the control group Table . SystoliAdditive hypotensive effect induced by spinal anesthesia and 5-ALA was prominent after spinal anesthesia induction not only during surgery but also for 2 h after the ward admission, as shown by the BP changes in our ALA group. Since the duration of the hypotensive effect by 5-ALA is longer than that of hyperbaric bupivacaine of spinal anesthesia, the systolic BP in the ALA group did not return to the baseline level even 2 h after the ward admission, unlike in the control group. We cannot easily state that spinal anesthesia using hyperbaric bupivacaine is a safe technique from our result, when considering the postoperative period. Our reflection point is that hypotension may be encouraged by hypovolemia in the ALA group, because all patients did not have intravenous fluids after 5-ALA administration before spinal anesthesia. Other risk factors that increase the incidence of 5-ALA-induced hypotension have been reported to be the regular use of antihypertensive drugs, particularly renin-angiotensin system inhibitor drugs  and a hiIn conclusion, orally administered 5-ALA before TUR-BT under spinal anesthesia induced significant hypotension after spinal anesthesia induction until 2 h after surgery and an increased need for vasopressors and intravenous volume. Anesthesiologists should have a strategy for safe anesthetic management against long-lasting hypotension by 5-ALA.


Microcirculatory insufficiency has been hypothesized in glaucoma pathogenesis. There is a scarcity of data to comprehensively examine the changes in retinal microvasculature and its role in normal tension glaucoma (NTG). We conducted a cross-sectional case–control study and included 168 eyes from 100 NTG patients and 68 healthy subjects. Quantitative retinal arteriolar and venular metrics were measured from retinal photographs using a computer-assisted program. Radial peripapillary capillary network was imaged with OCT-A and quantitative capillary metrics (circumpapillary vessel density (cpVD) and circumpapillary fractal dimension (cpFD)) were measured with a customized MATLAB program. We found that NTG was associated with decreased arteriolar and venular tortuosity, arteriolar branching angle, cpVD and cpFD. Decreased venular caliber, arteriolar and venular branching angles, cpVD and cpFD were associated with thinner average RNFL thickness. Decreased arteriolar and venular branching angles, cpVD and cpFD were also associated with worse standard automated perimetry measurements . Compared with retinal arteriolar and venular metrics, regression models based on OCT-A capillary metrics consistently showed stronger associations with NTG and structural and functional measurements in NTG. We concluded that NTG eyes showed generalized microvascular attenuations, in which OCT-A capillary metrics attenuations were more prominent and strongly associated with NTG. While elevation of intraocular pressure (IOP) is a major risk factor of glaucoma3, it is increasingly recognized that many patients with primary open angle glaucoma (POAG) have IOP within the normal range4. Such phenomenon of normal tension glaucoma (NTG) constituting the majority of POAG cases (77–92%) is particularly common among Asian populations6. The etiology of NTG remains largely unknown and current literature suggests a role of vascular dysfunction in the pathogenic process8. The prevailing hypothesis is that in the absence of elevated IOP, compared with high-tension glaucoma, microcirculatory insufficiency may play a more pivotal role in NTG pathogenesis12.Glaucoma, a major cause of irreversible blindness in the world, is a progressive optic neuropathy characterized by gradual degeneration of retinal ganglion cells (RGCs)15, and eyes with thinner retinal nerve fiber layer (RNFL) have decreased retinal vessel caliber and fractal dimension18. Increasingly, capillary network (5–6 µm in diameter) can be analysed using optical coherence tomography angiography (OCT-A)20. OCT-A studies have similarly reported decreased circumpapillary vessel density (cpVD), quantified from radial peripapillary capillary network, in eyes with POAG23. Nevertheless, previous studies have focused on and utilized only either of these modalities, and a comprehensive assessment of global microvascular changes  in glaucoma is lacking. Furthermore, studies specifically on NTG vasculature remain scarce. There is one study (n = 28) reporting no differences in retinal vessel caliber measured from retinal photographs in NTG eyes as compared with normal subjects24, which conflicts results of previous studies from comparison between POAG and normal eyes showing reduced vessel caliber in POAG14, and only a few OCT-A studies with relatively small sample sizes (n ≤ 31) reporting reduced cpVD in NTG, compared with normal subjects28.The non-invasive assessment of microvascular changes in the retina has evolved. Previously, quantitative retinal microvasculature analysis was confined to arterioles and venules (100–300 µm in diameter) based on fundus photography. Studies using this technique have reported that eyes with POAG have decreased retinal vessel caliber, tortuosity, branching angle and fractal dimensionThe objective of this study was two-fold. First, we aimed to offer a comprehensive global assessment of microvascular changes  in NTG, and the associations between such vascular changes with glaucoma-related structural and functional changes in NTG. Second, we aimed to compare the relative associations of arteriolar and venular changes with capillary changes in NTG eyes with the underlying structural and functional changes. The capillary network is the site of tissue perfusion for oxygen delivery and waste removal. Any vascular insufficiency and hypoxia–ischemia injury in NTG should therefore have direct impacts to the capillary network which is adjacent to the site of primary insults and hence be best reflected by changes and dysfunction of the capillary network. We therefore hypothesized that NTG eyes have global microvascular attenuations and that capillary metrics are more prominent and important microvascular markers.P < 0.05). NTG patients were also more myopic (− 3.19 D vs. − 0.69 D), had longer axial length (25.53 mm vs. 24.08 mm), worse SAP MD (− 6.73 dB vs. 0.18 dB), worse SAP PSD (7.41 dB vs. 1.62 dB), worse SAP VFI (82.42% vs. 99.29%) and thinner average RNFL thickness (72.41 µm vs. 95.04 µm) compared with normal control subjects . There were no significant differences in age, history of hypertension, cerebrovascular accident and ischemic heart disease, SBP, DBP, IOP and CCT between NTG patients and normal control subjects . The comparison of retinal photographic arteriolar and venular metrics and OCT-A capillary metrics between NTG and normal eyes were shown in Supplementary Table At baseline, 17 study subjects  were excluded because 6 of them had ungradable retinal photographs and 11 of them had ungradable OCT-A images. The remaining 168 subjects were included in the final analysis. The characteristics of included study subjects were shown in Table Table P = 0.04), 2.78 µm (P = 0.011), 2.69 µm (P = 0.01), 4.95 µm (P < 0.001) and 5.52 µm (P < 0.001) decrease in average RNFL thickness, respectively.The relationships between the microvascular imaging metrics and average RNFL thickness were summarized in Table P = 0.007) and 2.85% decrease in VFI (P = 0.014), respectively. Each SD decrease in venular branching angle was associated with 0.96 dB decrease in MD (P = 0.015) and 2.67% decrease in VFI (P = 0.017), respectively. Each SD decrease in cpVD was associated with 2.34 dB decrease in MD (P < 0.001) and 6.46% decrease in VFI (P < 0.001), respectively. Each SD decrease in cpFD was associated with 2.48 dB decrease in MD (P < 0.001) and 7.36% decrease in VFI (P < 0.001), respectively.The relationships between the microvascular imaging metrics and SAP measurements (MD and VFI) were summarized in Table A sensitivity analysis was also performed by excluding subjects with systemic cardiovascular diseases  and subsequently determining the odds of NTG in relationship to decreased measurements in the microvascular metrics and the relationships between the microvascular metrics and glaucoma-related structural and functional changes (Supplementary Tables P < 0.05 in Z-test and Steiger’s Z-test).Finally, we evaluated the strength of associations between retinal photographic arteriolar and venular and OCT-A capillary metrics with NTG and the glaucomatous changes in terms of average RNFL thickness, SAP MD and VFI  were associated with NTG and the structural (average RNFL thickness) and functional (SAP) glaucomatous measurements. We further demonstrated that OCT-A capillary metrics have stronger associations with NTG than that of photographic metrics.22. However, inclusion of POAG eyes in which high-tension glaucoma constitute a vast majority, if not all, of the study subjects made it impossible to distinguish between worse microvasculature as a cause of ischemia resulting in RGC loss and glaucoma or in reverse elevated IOP causing structural damage and neural tissue loss in glaucoma to drive attenuations in microvasculature through decreased metabolic demands29. Studies in the past revealed a higher prevalence of disc hemorrhage in NTG eyes compared with other glaucoma eyes and the association between disc hemorrhage in NTG and systemic vascular risk factors30. These altogether suggest vascular factors may be of a determinant role in NTG pathogenesis compared to other forms of glaucoma and merits independent investigation. Our study therefore sought to understand the relationship between retinal microvasculature and this non-high-tension glaucoma subtype which has a long-standing hypothesis that microvascular dysfunction plays a key role in its pathogenesis in the absence of elevated IOP10. Our findings of the associations between microvascular attenuations and NTG are consistent with the widespread vascular alterations in the ONH, retina, choroid, and retrobulbar circulations in glaucoma33. This demonstrates the strong relationships between microvascular factors and NTG and supports the hypothesis of their potential roles in the pathogenic processes of NTG where a background of elevated IOP is absent. Further longitudinal studies to evaluate the temporal and causal relationships between generalized microvascular attenuations and NTG development or progression to ascertain such hypothesis are warranted.Previously, studies reported attenuations in retinal microvasculature  in POAG eyes compared with normal eyes15. The summary findings in the current study and these previous studies were compared and outlined in Supplementary Table 36. Previous studies showed that narrower arteriolar branching angle is associated with systemic vascular pathologies such as hypertension37. If microcirculatory insufficiency at all has a role in NTG pathogenesis, the arteriolar branching angle has potential utility as a sentinel microvascular marker for NTG. Further studies are warranted to evaluate the research and clinical utility of this parameter.In the past, the only easily available non-invasive measurement of retinal microvascular changes was confined to arterioles and venules measured from fundus photographs using computer-assisted software. This has been applied to large population-based studies to evaluate the vascular associations in POAG, such as the Blue Mountains Eye Study (n = 3654) and the Singapore Malay Eye Study (n = 2789)38. In our study, retinal arterioles and venules on the original OCT-A images were hence removed to provide more accurate assessment of the capillary network in NTG eyes using OCT-A. We found that NTG eyes had decreased cpVD compared with normal subjects and decreased cpVD was associated with thinner RNFL, lower SAP MD and VFI. These are comprehensive evidences to support the associations of capillary network attenuations and NTG which corroborate with findings in previous OCT-A studies using relatively small samples of NTG patients  in which similar findings were reported39.OCT-A now allows measurement of capillary network changes beyond retinal arteriolar and venular changes. A recent study using OCT-A reported that the removal of larger vessels  avoids the measurement of cpVD being confounded by the presence of beta-zone parapapillary atrophy (PPA) which may be present and increase with the severity of glaucoma42, in the current context being retinal capillary vasculature, has not been quantified using OCT-A in previous studies on glaucoma nor applied to study NTG . The retinal FD is a measure of the complexity of the retinal vasculature and a reflection of its optimality and the efficiency of blood flow distribution with the least amount of energy43. Our results revealed that cpFD was significantly decreased in NTG as compared with normal eyes, and decreased cpFD was also associated with thinner RNFL, lower SAP MD and VFI. This further supported an association between microcirculatory dysfunction indicated by worse cpFD and NTG, and suggested the possible utility of cpFD in future NTG microvasculature assessments. All in all, these findings utilizing OCT-A capillary metrics offered evidence to support the relationships between capillary rarefaction and NTG, and the possible role of microcirculatory insufficiency in the pathogenesis of NTG as hypothesized in the vascular theory of glaucoma44.Meanwhile, previous studies only focused on the use of cpVD to quantify the peripapillary capillary network. The fractal dimension (FD), a mathematical measure that quantifies complex geometric patterns in objects that are self-similar in their scaling patterns46. Previous in-vitro studies have shown that formation of peroxynitrite (OONO-) and increased expression of vascular endothelial growth factor (VEGF) in the hypoxic retina resulted in increased retinal vascular permeability and disruption of the blood-retina barrier (BRB)48. The dysfunctional BRB with hyperpermeability and increased expression of monocyte chemoattractant protein-1 (MCP-1) in retinal hypoxia–ischemia attract monocytes from circulation and resident microglia to infiltrate hypoxic areas49, resulting in their activation and the subsequent inflammatory response51. The hypoxia-activated macrophages and microglia release proinflammatory molecules such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), intercellular cell adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2)53. These inflammatory molecules released in such state of neuroinflammation are known play a critical role in degeneration of retinal capillaries and RGC death56. Peripapillary capillaries were observed to atrophy proportionally with retinal nerve fibers in glaucoma57. These evidences support the intimate relationship between concomitant degeneration of retinal capillary microvasculature and RGC death in the complex pathologic processes with retinal hypoxia–ischemia ultimately culminating in glaucoma. Although current evidence is insufficient to indicate a direct causal relationship between capillary vasculature atrophy and RGC degeneration, degeneration in capillary vasculature is theoretically an ideal surrogate marker of vascular dysfunction, RGC degeneration and glaucoma. Our findings in this study with NTG patients lend support to such hypothesis by demonstrating the strong and consistent associations of NTG and the underlying glaucomatous changes with OCT-A capillary metrics. OCT-A capillary metrics may therefore be of utility in future studies of NTG vasculature and can potentially be assimilated into the standard of care to aid diagnosis and monitoring of NTG progression given their strong associations58.In this study, we also provided the first evidence to demonstrate that the associations of NTG and glaucoma-related structural and functional changes with OCT-A capillary metrics were stronger and more consistent than that with photographic arteriolar and venular metrics. It is thought that vascular dysfunction and hypoxia lead to oxidative injury of retinal ganglion cells and contribute to the pathogenesis of glaucomaThe strength of our study is the inclusion of only NTG patients instead of any types of POAG patients, which should better reflect and support the hypothesis of the role of vascular factors in the pathogenesis of glaucoma in the absence of elevated IOP. Furthermore, the current study presents a comprehensive assessment of both retinal photographic arteriolar and venular and OCT-A capillary metrics in NTG. To our knowledge, this is the first glaucoma study documenting assessments in both sets of vascular metrics and their associations with both structural and functional glaucomatous changes. Limitations should also be noted. Firstly, because of the cross-sectional design, we could only report the associations of generalized microvascular metrics attenuations with NTG but unable to establish any causal relationships. The microvasculature atrophy could either be a triggering event of retinal ischemia, RGC loss and NTG development or as a direct consequence driven by reduced metabolic demands following structural and functional degeneration in NTG. The results should be interpreted in consideration of both scenarios. Longitudinal studies are therefore warranted to evaluate the temporal relationships between microvascular metrics attenuations and NTG development and progression as reflected by structural and functional glaucomatous measurements to ascertain the underlying causal relationships. Secondly, the use of retinal fundus photographs and SIVA as a semi-automated computer-assisted software for evaluation of retinal vasculature was operator-dependent and human variability were inevitable in the estimation of the photographic vascular metrics. In addition, the measured areas between retinal photographs used for generating arteriolar and venular metrics and OCT-A images used for generating capillary metrics were different, which may account for the discrepancies between the strength of associations of the two sets of vascular metrics with NTG and the underlying glaucomatous changes. This however reflects the intrinsic nature of the two imaging modalities in reality settings, hence their respective efficacy and accuracy in NTG microvasculature assessment. Finally, our study population comprised of only subjects of Chinese ethnicity, which may limit the generalizability of our findings. Nonetheless, since NTG represents the majority of POAG cases in Asia, these findings are best suited for clinical practice and future NTG studies in which patients of such ethnicity would likely constitute a significant majority.In conclusion, we have demonstrated generalized changes in retinal arteriolar, venular and capillary metrics in NTG eyes. We further demonstrated that OCT-A capillary metrics showed stronger associations with NTG than fundus photographic arteriolar and venular metrics. These findings provide additional insights into the role of vascular factors in NTG and the potential utility of OCT-A capillary metrics in NTG assessments, diagnostics and progression monitoring, given its strong associations with NTG and the underlying structural and functional glaucomatous changes.This is a clinic based, case–control study. A total of 115 patients with NTG and 70 normal subjects were consecutively recruited from the Hong Kong Eye Hospital and the CUHK Eye Center of The Chinese University of Hong Kong from 2016 to 2018. For inclusion in this study, participants have to be diagnosed with NTG according to the definition below and of 18 years of age or above. Control subjects had to meet their respective criteria as listed below. One eye from each subject was selected for the current analysis. All participants had visual acuity of 20/40 or better. Subjects with diabetes mellitus, maculopathy, other optic neuropathies, history of ocular surgery (except cataract surgery), neurologic disease, or major systemic illness were excluded. This study was conducted in accordance with the 1964 Declaration of Helsinki and was approved by the Research Ethics Committee  of Hospital Authority (Ref: KC/KE-17-0099/ER-3). Written informed consent was obtained from all subjects.All participants underwent a comprehensive ophthalmologic examination at baseline, including measurements of best-corrected visual acuity, refractive error by an autorefractor , axial length , intraocular pressure (IOP) by Goldmann applanation tonometry, central corneal thickness (CCT) by a noncontact tonopachymeter , slit-lamp biomicroscopy examination of the optic disc and retina, dilated fundus examination, simultaneous stereophotography of the optic disc, standard automated perimetry  were performed. Peripapillary retinal nerve fiber layer (RNFL) thickness was measured with Cirrus HD-OCT . Systolic and diastolic blood pressure (SBP and DBP) and pulse rate were measured thrice with an Omron automatic blood pressure instrument , and the mean values were used in the analysis.59. At least 2 readings were obtained at different times of the day from the rest. (2) Drainage angle of Shaffer grade II or above on dark room gonioscopy. (3) Glaucomatous optic disc cupping and loss of neuroretinal rim. (4) Visual field defect according to the Collaborative NTG Study59. (5) Glaucoma hemifield test being ‘outside normal limits’. (6) Pattern standard deviation with a P value < 0.05. (7) A cluster of 3 points or more in the pattern deviation plot in a single hemifield with a P value < 0.05, one of which must have a P value < 0.01. Any one of these criteria, if repeatable, was considered to be sufficient evidence of a glaucomatous VF defect. (8) Absence of secondary causes for the glaucomatous optic neuropathy . Normal controls had anterior chamber angle of Shaffer grade II or above, no optic disc abnormalities in clinical examination, no visual field abnormalities on visual field testing, and no family or personal history of glaucoma, or other ocular diseases except visually insignificant senile cataract, or myopia or hyperopia of less than 3 diopters.NTG was diagnosed based on: (1) Six median untreated IOP readings consistently less than 21 mmHg, with no more than 1 reading equal to 23 or 24 mmHg, and no single measurement more than 24 mmHg, as per the Collaborative NTG Study60. The quality of visual field tests was also reviewed by study investigators to identify and exclude visual fields with evidence of inattention or inappropriate fixation, artefacts such as eyelid and lens rim artefacts, fatigue effects, and abnormal results caused by diseases other than glaucoma.All participants underwent visual field testing using the 24-2 pattern Swedish interactive threshold algorithm (SITA) on the Humphrey Field Analyzer  within 6 months of imaging. Only reliable tests  were included61, according to a standardized grading protocol by three trained graders  who were masked to the study subjects applied SIVA to each image  after pharmacological pupil dilation. A series of retinal photographic vascular metrics  were measured by a semi-automated computer-assisted program  from the disc-centered retinal photographs (ETDRS field 1)62. In the current analysis, we selected radial peripapillary capillary network from the ONH scan which comprises of capillary beds within RNFL layer predominantly.Prior to OCT-A imaging, pupils were dilated with tropicamide and phenylephrine (0.5% each). All participants then underwent OCT-A with a swept-source OCT , which contained a swept-source with a wavelength of 1,050 nm light source and a speed of 100,000 A-scans per second. A volumetric OCT scan centred on the optic nerve head was obtained from each eye, with a scan area of 3 mm by 3 mm containing 320 × 320 A-scans for each volumetric scan. An image quality score ranging from 0 to 100 was given by the software for each volumetric OCT scan. We used the built-in software (IMAGEnet6) to generate horizontal depth-resolved OCT-A slabs to provide improved detection sensitivity of low blood flow and reduced motion artefacts without compromising axial resolutionTwo readers  carefully evaluated each OCT-A image in the CUHK Ocular Reading Centre. The readers were masked to all study subjects. OCT-A images with significant image artefacts and poor image quality were excluded from analysis, including (1) quality score of below 40; (2) motion artefacts ; (3) inaccurate segmentation of tissue layers or slabs; (4) blurry images ; (5) signal loss (e.g. due to eye blinking) or (6) poor centration (i.e. ONH not at center). All the OCT-A scans met the image quality requirement for inclusion in the current analysis.The OCT-A images were exported in grayscale from the built-in software and imported into a semi-automated customized MATLAB program  for image analysis. The circumpapillary vessel density (cpVD) and circumpapillary fractal dimension (cpFD) within a region defined as a circular annulus with 3 mm outer diameter and inner boundary fitted to the optic disc based on 360° global area were analysed Fig.  online.F63. The NLM filter is a non-linear neighborhood filter in which the pixel/voxel value to be restored is replaced by a weighted average of the pixel intensities in the entire noisy image. The denoised image was then binarized, using phansalkar adaptive local thresholding method with white pixels representing the blood vessels and black pixels representing the background (non-perfusion regions), or skeletonized64, respectively.A non-local means (NLM) denoising filter was first applied to the grayscale images in order to reduce the background noise, sharpen the blood vessels and improve signal-to-noise ratio while preserving the image features after the OCT-A images were exported in grayscale and imported to an automated customized MATLAB programRetinal arterioles and venules were removed before subsequent analyses on circumpapillary vessel density (cpVD) and fractal dimension (cpFD) were conducted. The detection of the retinal vessels was based on the original OCT-A image extracted from RPC layer (defined by built-in software of Triton OCT). The vessel removal mask was generated by combining masks processed from two different approaches. For the first approach, the RPC layer image was denoised with averaging filter and median filter. The denoised image was subsequently enhanced with contrast stretching. Part of the retinal vessels was then extracted by binarization with global thresholding on the contrast-stretched image with reference to the overall brightness of the image. For the second approach, the RPC layer image was first processed with contrast-limited adaptive histogram equalization to adaptively enhance the regional contrast. The processed image was subsequently denoised with averaging filter and median filter, and further enhanced with contrast stretching. Another part of the retinal vessels was then extracted by binarization with 2-D Frangi filter. Both the first mask and the second mask were further processed with morphological opesrations to remove artefacts. A final mask representing all retinal vessels (arterioles and venules) was computed by union of the two detected masks and applied to the original grayscale OCT-A image for vessel removal.64 ONH OCT-A image using “box-counting method”66 in the same region defined above, after removal of the retinal arterioles and venules.cpVD was calculated as the percentage of area not defined as non-perfusion regions over the total area in the region defined above from the binarized ONH OCT-A image, after removal of the retinal arterioles and venules. cpFD was calculated from the skeletonizedCirrus HD-OCT  was used to measure the average RNFL thicknesses from the optic disc cube scan (200 × 200 pixels), generating the RNFL thickness map at the peripapillary region (6 mm × 6 mm). Average RNFL thickness was used for evaluation of the associations with photographic arteriolar and venular and OCT-A capillary metrics. All OCT scans had signal strength of 6 or above.t test was used. For categorical variables, the chi-square test was performed. Logistic regression models were constructed to determine odds ratios (ORs) and 95% confidence intervals of NTG in relation to each standard deviation change in the microvascular imaging metrics, adjusting for age, sex and axial length (AL), systolic blood pressure (SBP), fellow caliber  and OCT-A quality score . Multivariable linear regression models adjusting for the aforementioned variables were used to estimate changes in average RNFL thickness , MD and VFI for each standard deviation change in the microvascular metrics. Finally, the different regression models were compared to determine if photographic arteriolar and venular or OCT-A capillary metrics reflect stronger associations with NTG and the glaucomatous structural and functional alterations using Z-test for comparison of logistic regression models and Steiger’s Z-tests for comparison of linear regression models.Statistical analysis was performed using SPSS version 25 . Photographic arteriolar and venular metrics  generated from SIVA and OCT-A capillary metrics (cpVD and cpFD) were analyzed as continuous variables. Clinical characteristics, photographic arteriolar and venular metrics and OCT-A capillary metrics were compared between NTG patients and normal controls. For continuous variables, independent sample Supplementary Information


Archival tissue samples collected longitudinally from a patient who died from HPV16-induced high-grade anal intraepithelial squamous cell carcinoma with vertebral HPV16–positive metastasis were retrospectively analyzed by the Capture-HPV method (Capt-HPV) followed by Next-Generation Sequencing (NGS). Full length nucleotide sequences of the same HPV16 were identified from the initial and second anal biopsy samples, from plasma sample and from vertebral metastasis biopsy. Remarkably, HPV was episomal in each sample. The HPV genome sequence was closest to the HPV16 Qv18158E variant subtype (A1 lineage) exhibiting base substitutions and deletions in 7 and 2 HPV loci, respectively. In conclusion, the powerful Capt-HPV followed by NGS allows evidencing the detailed cartography of tumoral and circulating HPV DNA, giving rise to a unique and unexpected episomal virus molecular status in a context of aggressive carcinoma, underlying the importance of HPV status and its association with clinical features for further prospective studies. Although the natural history of human papillomavirus (HPV) infection in ASCC remains unclear, it has been shown that high-risk HPV16 was by far the most carcinogenic genotype for ASCC1.Despite being a rare cancer accounting for approximately 20,000 new cases each year globally, the incidence of anal squamous cell carcinoma (ASCC) has been increasing dramatically over the past decade in HIV-positive patientsversus integrated) as well as the integration sites in both HPV and human genomes2.New tools and biomarkers to assess the prognosis and to improve patient stratification as well as identifying therapeutic targets are needed. Recently, sensitive double capture-HPV method (Capt-HPV) followed by Next-Generation Sequencing (NGS) allowed the capture of 208 variants from 88 HPV genotypes and thus determining at once HPV genotype and nucleotide sequence, the molecular status , the patient hospitalized at Hôpital Européen Georges Pompidou (HEGP), Paris, presented vertebral HPV16–positive metastasis, associated with a paravertebral collection. The patient received first line palliative chemotherapy with docetaxel, cisplatin, and 5FU (DCF), for 6 months, leading to stability according to RECIST criteria4. Due to grade 3 asthenia, a second line chemotherapy with weekly paclitaxel and carboplatin was started. After 2 months, CT-scan imagery showed progression according to RECIST criteria, and the patient presented with an ECOG performance status of 3, leading to discontinuation of chemotherapy. He received supportive care for 4 additional months and died 12 months after the first round of chemotherapy. Witnessing this very aggressive and unexpected clinical presentation prompted us to evaluate the molecular status of the HPV16 detected in the collected samples from different origins using Capt-HPV and NGS, including the initial anal biopsy that diagnosed HGAIN, a 2-year post-diagnosis follow-up anal biopsy showing HGAIN recurrence, the vertebral metastasis biopsy, and the plasmatic HPV16 ctDNA.We previously reported the contribution of digital droplet polymerase chain reaction for early detection of HPV-induced aggressive metastatic anal cancer in an HIV-positive MSM Fig. 3. In bri2, but the present study is the first one to describe episomal HPV16 ctDNA in peripheral blood.We identified similar HPV16 sequences with full genome coverage in the initial and second anal lesions, the plasma sample, and the metastasis biopsy. This HPV sequence was classified into the A1 lineage. The closest variant in our reference data base was HPV16 Qv18158E (GenBank accession number: AY686583.1). The virus clearly appeared episomal in each sample, without any chimeric HPV-human genome reads potentially resulting from HPV integration in the human genome. De novo assembly of the NGS reads allowed circularizing the genome, confirming its episomal status  and 5140 (L2 gene) were silent at the protein level while the nucleotide deletions at positions 4184 and 4215 were located in the same intergenic region. No mutations in the E7 and E4 genes nor in the long control region were identified.Compared to the closest HPV16 Qv18158E genome reference, the present HPV genome analysis revealed 7 mutations. Namely, 2 base substitutions at nucleotide positions 176 and 350 were located in the Our observations report the comprehensive longitudinal molecular characterization of HPV DNA sequences from initial aggressive anal tumor to ctDNA extracted from plasma and deadly metastasis. The present study is the first one to describe episomal HPV16 ctDNA in peripheral blood. Indeed, and unexpectedly, the HPV status of each full length HPV16 sequence evidenced in tumor tissue samples as well as plasma ctDNA from study patient was episomal.. showed that the HPV16 E7 protein leading to cervical cancer was invariant5. Accordingly, we did not find any of the 32 mutations they identified in the E7 ORF from their control subjects. However, the E6-G350T found in our patient was associated with good prognosis in cervical cancer6, and none of the other mutations we found were previously described, underlying the need to aggregate HPV complete sequences associated with clinical features in order to correlate the presence of specific mutations with prognosis.Mirabello et al7. Using Capt-HPV, we also recently showed that 2 out of 4 patients with metastasis after an initial ASCC presented episomal HPV in their initial tumor, but no data were available regarding ctDNA nor metastasis, and de novo assembly was not performed to confirm the episomal status neither8. Our observation that episomal HPV can lead to metastatic progression challenges the widespread claim that tumors with integrated HPV are of worse prognosis than the ones with episomal HPV. Furthermore, HPV variants remained unchanged over time in this very aggressive metastatic ASCC, raising the central issue to which extent the cancer-associated HPV virus was functionally active during the advance of the disease and the therapeutic treatments or remained passive once the oncogenic process had started.Capt-HPV was used previously to confirm the metastatic nature of a tongue carcinoma in a patient but no ctDNA data were provided and the same signature for HPV integration was found in both the tumor and the metastasisThe present case report highlights that the availability of robust, affordable and reliable technologies such as Capt-HPV followed by NGS should facilitate the design of prospective studies where HPV status and its complete DNA sequence at baseline and/or during the follow-up would be associated with clinical data, especially to anticipate overall and progression-free survival and to offer the best adapted personalized treatment.9. Capt-HPV was performed using the SeqCap EZ library reagents (Roche NimbleGen)2. Illumina MiSeq systems were used to sequence the post capture libraries (150 bp paired-end reads). NGS data were automatically analyzed using a specific pipeline developed by the HEGP biomedical informatics team. HPV genotyping, HPV full length sequence, HPV molecular status, and positions of HPV-human junctions when present were obtained. To confirm the assumption that HPV was episomal when no integrations sites were found, we performed de novo assembly of the virus using the software Geneious . This software was also able to circularize the HPV genome if episomal, confirming its structure. The assembled genome was aligned against our 208 reference HPV sequences in order to identify the closest variant. The closest variant lineage was assessed by building a phylogenetic tree including the assembled genomes and 10 HPV16 A, B, C and D variant lineage reference sequences10.DNA extracts were obtained from formalin-fixed and paraffin-embedded anal and vertebral biopsies as well as from HPV16 circulating tumoural positive plasma, as describedhttps://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000032719520&categorieLien=id).Our report is an observational study, in which the biological analyses were carried out with the patient approval before his death. Their post-mortem interpretation and publication are not in the scope of an ethical committee, according to the French Jardé’s law on biomedical research (No animal was done for the study.


In April 2016, a resolution by all members of the 68th World Health AssemblyTo complement CDC’s ongoing technical assistance, the U.S. CDC’s Emergency Operations Center, which activated a Polio Response in December 2011, initiated a “polio surge” in September 2019 to assist country programs. This surge consisted of recruiting CDC volunteersWith increasing restrictions on CDC international deployments because of the coronavirus disease 2019 (COVID-19) pandemic, CDC’s Emergency Operations Center first recalled deployed staff members back to CDC country offices in capitals of the supported countries to allow for contingency planning. By March 23 however, all 32 polio surge staff members deployed during March had ended their missions early and returned to the United States. On March 26, the GPEI recommended delaying OPV SIAs until at least June 2020Disruptions in routine immunization and SIAs because of the COVID-19 pandemic have elevated the risk for increases in vaccine-preventable diseases, including polio (


Lung cancer is one of the leading causes of cancer-related deaths. Radiotherapy, either alone or with chemotherapy, is still the primary treatment for patients with non-small-cell lung cancer (NSCLC). There are variations in how patients with NSCLC respond to radiotherapy and how toxic the therapy is. DNA repair gene polymorphisms are related to cancer development; however, their association with radiotherapy outcomes remains unknown. We hypothesized that gDNA repair gene variation could affect the efficacy and toxicity of radiotherapy in patients with NSCLC. XRCC1, XRCC2, XPD, and MSH2) were genotyped in these patients. A multiple factor logistic regression analysis was used to assess the association between these SNPs and the efficacy and toxicity of radiotherapy.  A total of 486 histologically confirmed patients with NSCLC were recruited from the Shengjing Hospital of China Medical University from July 2015 to September 2019. Eleven potentially functional single nucleotide polymorphisms (SNPs) in four DNA repair genes (XRCC1), rs3218556 (XRCC2), and rs13181 (XPD), were all significantly associated with the efficacy of radiotherapy. The allele frequencies of the rs25487 CC genotype  and the rs3218556 AG or AA genotypes  were both significantly higher in the response group than in the nonresponse group. For rs13181, the radiotherapy efficacy was associated with the heterozygous genotype GT . Statistically significant associations between radiation-induced toxic reactions and rs25487 (XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) were also observed. The rs13181GT genotype was associated with lower toxic reactions than the TT genotype .  Three SNPs, rs25487 (XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) all contribute to the efficacy and toxicity of radiotherapy in patients with NSCLC. Our findings may clarify the predictive value of DNA repair genes for prognosis in patients with NSCLC after radiotherapy. Further investigation of more genes and samples should be performed to confirm our findings. The variants rs25487 ( Lung cancer accounts for a large proportion of cancer patients worldwide and is the leading cause of cancer-related deaths . There wERCC1 [XRCC1 [XPA [XRCC1 and XRCC2 are involved in the BER and HR pathways, respectively, and have been associated with the occurrence of cancer [MutS homolog 2 (MSH2), a key component of the MMR pathway, plays an important role in the development of neoplastic diseases [Xeroderma pigmentosum group D (XPD) gene, another important DNA repair gene, has been reported to contribute to the risk of human cancer [Ionizing radiation leads to cell death mainly by creating double-strand breaks (DSB)  or throuERCC1 , XRCC1 [1 [XRCC1 , and XPACC1 [XPA , play a f cancer , 13. Mutdiseases . The Xern cancer .XRCC1, XRCC2, XPD, and MSH2).Although the relationship between polymorphisms in DNA repair genes and the development of cancer has been well explored in previous studies, few reports have investigated the interrelationship between gene polymorphisms and radiosensitivity or radiotherapy toxicity in patients with NSCLC. To explore the possible association between DNA repair gene variations and radiotherapy sensitivity and associated toxicity, we evaluated eleven single nucleotide polymorphisms (SNPs) in four DNA repair genes  or intensity modulated radiation therapy (IMRT), with a total radiation dose of 50–70 Gy. Followups were conducted on all patients three months after radiotherapy, and their response to treatment was assessed using computed omography (CT) according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. There were four categories of response defined: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) . In the XRCC1, XRCC2, MSH2, and XPD, which had previously been shown to be positively associated with the development of cancer, were selected for analysis. SNP genotypes were downloaded from the 1000 Genomes project  and analyzed using Haploview 4.2 software (https://www.broadinstitute.org/haploview/haploview). Candidate SNPs which met the following criteria were included: (1) SNPs that had a minor allele frequency (MAF) > 0.1 in Han Chinese in Beijing (CHB); (2) SNPs with a potential function, such as missense variations causing amino acid changes or were present in the 5′ or 3′ untranslated regions (UTRs) that could affect transcription factor binding site (TFBS) activity; (3) SNPs that have been reported in previous association studies. As a result of using these selection criteria, a total of 11 potentially functional SNPs in DNA repair genes were selected. These included 4 XRCC1 SNPs (rs25487 (exon 10), rs25489 (exon 9), rs1799782 (exon (6) and rs3213245 (5′ UTR)), 3 XRCC2 SNPs (rs3218556 (3′ UTR), rs3218544 (3′ UTR), rs3218385 (5′ UTR)), 2 MSH2 SNPs (rs2303424 (exon 16) and rs2303425 (5′ UTR)), and 2 XPD SNPs (rs13181 (exon 10) and rs238419 (3′ UTR)).Four DNA repair genes, μL of master mix (Applied Biosystems), 10 μL of DNA, and 15 μL of ddH2O. Amplification was performed under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min. Three negative controls (no DNA) and three positive controls in each 96-well plate were used to ensure the accuracy of the PCR amplification. In addition, 10% of randomly repeated samples were used for quality control.Genomic DNA from all patients was extracted using a TIANamp Genomic DNA Kit . Genotyping was performed using the TaqMan methodology and an Applied Biosystems 7500 FAST Real-Time PCR System  according to the manufacturer's instructions. The predesigned SNP-genotyping assay mixture, containing the PCR primers and probes, were supplied by Applied Biosystems. The PCR amplification mix was prepared as follows: 25 p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 22.0 software . The Hardy-Weinberg equilibrium was used to assess that all SNPs met the group representation. A multiple factor logistic regression was applied to assess the association between SNPs and efficacy of radiotherapy as well as radiation-induced toxicity reaction after adjusting for age, gender, smoking history, cancer histology, family history, and chemotherapy. A The clinical characteristics and demographics of the 486 NSCLC patients are described in p > 0.05, data not shown). The SNPs rs25487 (XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) all showed a significant association with the efficacy of radiotherapy. The allele frequency of rs25487 CC genotype  and the rs3218556 AG or AA genotypes  were significantly higher in the response group than in the nonresponse group. For rs25487, although the response rate between the CT and CC genotypes was not statistically significant (p=0.078), a higher response rate was also observed when comparing the CT + CC genotype with the CC genotype, using a dominant model. For rs13181, a better radiotherapy efficacy was associated with the heterozygotic genotype GT . There were no significant associations between the other SNPs and the efficacy of radiotherapy.The associations between candidate SNPs and the response to radiotherapy in NSCLS patients are shown in XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) was observed. For rs25487, the genotypes CT, TT, and CT + TT were associated with a severe toxic reaction compared to the CC genotype . For rs3218556, the ORs for cases with the AG, AA, and AG + AA genotypes compared with homozygous CC genotype were 0.605 , 0.279 , and 0.540 , respectively. The rs13181GT genotype was associated with lower toxic reactions compared with the TT genotype .The associations between candidate SNPs and radiation-induced toxic reactions are shown in XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) were associated with the efficacy and toxicity of radiotherapy in patients with NSCLC.NSCLC is the leading cause of cancer-related death. Radiotherapy is an important treatment for NSCLC patients, especially for advanced NSCLC patients. However, there are significant differences in the efficacy of radiotherapy as well as in the incidence rate for radiation-induced toxic reactions. Identification of the key determinants that affect efficacy and toxicity is of paramount importance for the efficacy of radiotherapy in patients with NSCLC. Although numerous studies have reported that genetic polymorphisms in DNA repair genes are related to the development of cancer, their association with the outcomes of radiotherapy remains unknown. In the present study, we demonstrated that rs25487  of MSH2 gene, and rs13181  of XPD gene cause amino acid changes and then affect the biological function of the protein. Also, other variations located in the 5′ or 3′ UTRs may affect the transcription factor binding site (TFBS) activity and then influence the DNA repair gene expression. All the variants may impact the clinical outcome of radiotherapy through these two ways.The potential functional variations of the four DNA repair genes were listed in Supplementary XRCC1, an important component of BER, has been reported to be associated with an increased risk of NSCLC in nonsmoking female patients with a history of exposure to cooking oil mist [XRCC1-194 and XRCC1-399 are also related to the risk of NSCLC [XRCC1 rs25489 had a significant impact on primary tumor efficacy at the end of radiotherapy and may act as a biomarker for the curative effect of radiotherapy. Zhai et al. [XRCC1 codon 399 Gln/Gln genotype had a higher rate of tumor regression after radiotherapy. Another study showed that, in 114 patients with NPC, the XRCC1 rs25487 GA genotype was related with grade 3 dermatitis and grade 3 mucositis. In this study, a significant association was also observed between the rs25489 CC genotype and a higher response rate to radiotherapy as well as lower toxic reaction. Although three other SNPs  in XRCC1 were also genotyped, no significant associations were observed. Due to the fact that XRCC1 rs25487 is clearly important in radiation sensitivity and the resultant toxic reactions, it could be considered as a biomarker that can be used to predict the clinical outcomes of radiotherapy.Many genetic variants that are involved in DNA damage repair and the regulation of oxidative stress are associated with radiotherapy outcomes . XRCC1, oil mist . Geneticof NSCLC . Wang eti et al.  found thXRCC2, which is involved in the HR pathway, were also evaluated for a possible association with the efficacy and toxicity of radiotherapy in patients with NSCLC. Of these, only rs3218385 showed a significant association with both the efficacy and toxicity of radiotherapy. Popanda et al. [XRCC2 variations in patients with breast cancer receiving radiotherapy, which is inconsistent with our findings. Nevertheless, Yin et al. [XRCC2 R188H SNPs was independent prognostic factor for overall survival. Qin et al. [XRCC2-deficient cancer cells were more sensitive to irradiation in vitro and speculated that the inhibition of XRCC2 expression or activity represents a potential therapeutic strategy for improving preoperative radiotherapy responses in patients with locally advanced rectal cancer. These results strongly suggest that XRCC2 plays an important role not only in the development of the cancer but also in radiotherapy outcomes.Three SNPs  in a et al.  suggesten et al.  also fouMSH2 is an important MMR gene and several studies have suggested that variations in MSH2 are associated with sensitivity to radiotherapy and disease progression in rectal cancer patients [MSH2 gene expression levels in patients with NSCLC who were treated with chemotherapy, which is consistent with our results. SNPs in the XPD gene have been widely studied and suggested to be genomic markers to predict the response to radiation dose and potentially guide personalized radiotherapy [XPD rs13181 was associated with the progression and outcomes of patients with NSCLC after radiotherapy. Patients carrying the rs13181 GT genotype who were treated with radiotherapy had a high level of toxic reaction coupled with low efficacy.patients , 24. Howpatients  also fouotherapy , 26. ThiMSH2 and NSCLC patients. This could be due to the genetic heterogeneity in different population and a small number of samples included. Fourthly, gene–gene and gene–environment interactions were not analyzed. Additionally, some other important genes, such as ATM gene of DNA-damage sensing [ATG16L2 gene related to autophagy [RUNX3 [Despite the positive findings, there are also some limitations in this study. First, we were unable to explain how the DNA repair gene variations influenced the outcomes of radiotherapy. Secondly, too few SNPs were genotyped. We selected the potentially functional SNPs based on three criteria so that the SNPs did not cover all SNPs in the entire gene. Thirdly, our sample size is still too small. In this study, we failed to replicate the association between  sensing , ATG16L2utophagy , and RUNy [RUNX3  gene in XRCC1), rs3218556 (XRCC2), and rs13181 (XPD) SNPs, which appear to contribute to the efficacy and toxicity of radiotherapy. Our findings may be helpful in understanding the predictive value of examining DNA repair genes in the prognosis of patients with NSCLC after radiotherapy. Further investigation of more genes and samples should be carried out to confirm our findings. In addition, more studies should be implemented to explain the underlying molecular mechanisms to explain how these polymorphisms affect the response to radiotherapy and prospective clinical trials in patients with NSCLC.In conclusion, we have identified patients with NSCLC carrying the rs25487 (