FMR1 alleles with more than 200 CGG repeats bear chromosomal fragility when cells experience folate deficiency. CGG repeats were reported to be able to form secondary structures, such as hairpins, in vitro. When such secondary structures are formed, repeats can lead to replication fork stalling even in the absence of any additional perturbation. Indeed, it was recently shown that the replication forks stall at the endogenous FMR1 locus in unaffected and FXS cells, suggesting the formation of secondary repeat structures at the FMR1 gene in vivo. If not dealt with properly replication fork stalling can lead to polymerase slippage and repeat expansion as well as fragile site expression. Despite the presence of repeat structures at the FMR1 locus, chromosomal fragility is only expressed under replicative stress suggesting the existence of potential molecular mechanisms that help the replication fork progress through these repeat regions. DNA helicases are known to aid replication forks progress through repetitive DNA sequences. Yet, the identity of the DNA helicase(s) responsible for unwinding the CGG repeats at FMR1 locus is not known. We found that the human DNA helicase B (HDHB) may provide an answer for this question. We used chromatin-immunoprecipitation assay to study the FMR1 region and common fragile sites (CFS), and asked whether HDHB localizes at replication forks stalled at repetitive regions even in unperturbed cells. HDHB was strongly enriched in S-phase at the repetitive DNA at CFS and FMR1 gene but not in the flanking regions. Taken together, these results suggest that HDHB functions in preventing or repairing stalled replication forks that arise in repeat-rich regions even in unperturbed cells. Furthermore, we discuss the importance and potential role of HDHB and other helicases in the resolution of secondary CGG repeat structures.The fragile X syndrome (FXS) is caused by a CGG repeat expansion at the  FXS is caused by a CGG repeat expansion on the X chromosome in the 5’UTR of the FMR1 gene locus in FXS cells are characterized as rare fragile sites (RFS). While RFS are only found in some individuals, common fragile sites  at unwinding CGG repeat structures to aid replication fork progression. We present data that show HDHB localization to fragile sites specifically during S-phase even in unperturbed cells, suggesting that HDHB may help prevent or repair replication fork stalling. Furthermore, we discuss other possible DNA and RNA helicases that are capable of unwinding secondary CGG repeat structures.Here we discuss mechanisms that may prevent chromosomal fragility and repeat expansion at theDespite the threats to genome stability posed by the secondary structures adopted by repeats, replication most often proceeds through repeat regions without the expression of chromosomal fragility. This suggests the presence of molecular mechanisms that help replication fork progression through repeat regions. At the heart of these mechanisms are DNA helicase(s) responsible for unwinding the secondary structures adopted by repeats. Helicases separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases. DNA helicases translocate on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) to unwind dsDNA into ssDNA or remove proteins bound to DNA. There is a plethora of helicases encoded by the genome, each performing a specialized function dictated by their enzymatic properties as well as their interaction partners.Conserved among vertebrates, DNA helicase B (DHB) contains seven helicase motifs of superfamily 1 with sequence similarity to homologous recombination proteins prokaryotic RecD and bacteriophage T4 dda. It works with 5’ to 3’ polarity. The C-terminus of HDHB contains a phosphorylation-dependent subcellular localization domain  experiments. U2OS cells were blocked in G2/M-phase and released for 5 h (G1), or synchronized in G1/S-phase and released for 6 (S) or 9 h (G2/M), followed by formaldehyde treatment to crosslink chromatin. Chromatin was isolated, sheared and immunoprecipitated using purified polyclonal HDHB antibody or non-immune control antibody . In S-phase cells, HDHB was significantly enriched on chromatin in the repeat region of fragile sites relative to the distal regions. HDHB enrichment in both repeat regions was reduced in G1- and G2/M-phase. We found similar results at a second CFS, FRA3B (data not shown). These results show that HDHB is enriched in S-phase at the repetitive DNA sequences and suggest that HDHB is recruited to stalled replication forks at repetitive regions such as CGG repeats at the FMR1 gene.To investigate whether HDHB associates with replication forks stalled at repetitive DNA sequences, in particular to the CGG repeats at Upon recruitment to the fork, HDHB can unwind the secondary repeat structures formed by CGG repeats ahead of DNA-polymerase Figures . Repeat in vitro . HnRNP A2/B1 have a role in packaging nascent mRNA, alternative splicing and cytoplasmic RNA trafficking, translation and stabilization. Found in intranuclear inclusions of FXTAS patients (Iwahashi et al., Repeats present a challenge for the replication machinery and other cellular processes involving repetitive DNA and RNA. Therefore, mechanisms to resolve secondary structures that repeats might adopt are needed. Specialized helicases are able to unwind secondary DNA and RNA structures, which can otherwise lead to replication fork stalling or toxic RNA, respectively. Replication fork stalling at endogenous repeats is reported in cells derived from FXS and FRDA patients (Gerhardt et al., GG and JG performed the experiments. GG, JG and ZR wrote and edited the manuscript.GG is an employee of Celgene. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Covariates included PTSD diagnosis, sex, intracranial volume, ancestry, childhood trauma, SNP×PTSD diagnosis, and SNP×childhood trauma. We identified several genetic markers in high linkage disequilibrium (LD) with rs9373240  that were associated with caudate volume. We also observed a significant interaction between rs9373240 and childhood trauma , whereby increased trauma exposure produced a stronger association between SNPs and increased caudate volume. We identified several SNPs in high LD with rs34043524, which is downstream of the TRAM1L1 gene that were associated with right lateral ventricular volume  and were also associated with lifetime alcohol abuse or dependence . Finally, we identified several SNPs in high LD with rs13140180 , an intergenic region on chromosome 4, and several SNPs in the TMPRSS15 associated with right nucleus accumbens volume . Both TRAM1L1 and TMPRSS15 have been previously implicated in neuronal function. Key results survived genome-wide multiple-testing correction in our sample. Leveraging neuroimaging phenotypes may offer a shortcut, relative to clinical phenotypes, in mapping the genetic architecture and neurobiological pathways of PTSD.Depending on the traumatic event, a significant fraction of trauma survivors subsequently develop PTSD. The additional variability in PTSD risk is expected to arise from genetic susceptibility. Unfortunately, several genome-wide association studies (GWAS) have failed to identify a consistent genetic marker for PTSD. The heritability of intermediate phenotypes such as regional brain volumes is often 80% or higher. We conducted a GWAS of subcortical brain volumes in a sample of recent military veteran trauma survivors ( Thus, exposure to traumatic events is a necessary, but insufficient environmental risk factor for developing PTSD. The developmental timing of the traumatic events appears to be important since exposure to trauma or adversity in childhood increases the risk of developing PTSD following trauma in adulthood3. Family and twin heritability studies have demonstrated that genetic risk also contributes to the development of PTSD. In order to identify putative PTSD genetic loci, several groups have conducted unbiased genome-wide association studies (GWAS) based on the clinical diagnosis of PTSD8. These GWAS have had limited success. Among the loci that were implicated, the effect sizes on PTSD risk were relatively large for a complex disease, with odds ratios ranging from 1.4 to 3.7. However, despite these putatively large genetic effects, each GWAS identified different loci. None of the top hits (p < 10−5) for the studies overlapped except for RORA, which demonstrated a modest degree of replication9. The lack of replication in PTSD GWAS could be explained by differences among the study participants with respect to trauma type, trauma severity, ethnicity, and/or gender, among other distinctions. Additionally, it is expected that PTSD has a complex genetic architecture with polygenic and gene×environment influences. These underlying complexities likely have also contributed to the heterogeneity of the GWAS findings. A major component of the genetic heterogeneity is the clinical heterogeneity that is inherent in psychiatric nosology and the subjective approach to assessment of PTSD symptoms (patient-reported behaviors).Most individuals in the general population are exposed to at least one traumatic event in their lifetime13, but use of GWAS with brain measures in PTSD remains largely unexplored. A large neuroimaging GWAS of healthy  individuals  revealed four novel genetic variants that modulated subcortical brain volume in the putamen: (1) an intergenic locus 50 kilobases (kb) downstream of the KTN1 gene (rs945270) that encodes the protein kinectin involved in organelle transport; (2) an intronic locus within DCC (rs62097986) that encodes a netrin receptor involved in axon guidance and migration; (3) an intronic locus within BCL2L1 (rs6087771) that inhibits programmed cell death in immature neurons; and, (4) an intronic locus within DLG2 (rs683250) that encodes a protein that organizes postsynaptic density. Caudate volume was associated with an intergenic locus 80 kb from FAT3 (rs1318862) that influences neuronal morphology during embryonic development. Hippocampal volume was associated with two loci: (1) an intergenic locus near the HRK gene (rs77956314), and (2) an intronic locus in the MSRB3 gene (rs61921502)15. Brain connectivity assessed at the connectome-wide level with GWAS in healthy individuals also identified an association with SPON1 (rs2618516) that contributes to growth and guidance of axons in the spinal cord16.One approach to mitigating the clinical heterogeneity in PTSD and other neuropsychiatric disorders is to utilize quantitative brain-based neuroimaging phenotypes. A variety of brain measures from fMRI, DTI, and functional connectivity have been associated with candidate genes in PTSD14. In this way, genetic risk factors for psychiatric diagnoses such as PTSD may be more easily detected by examining intermediate phenotypes such as brain measures obtained from MRI17. Intermediate phenotypes, or endophenotypes, may have a simpler genetic architecture than the PTSD clinical diagnosis18. Brain measures also offer a more precise, objective, and reproducible phenotype than a clinical diagnostic scale19. Moreover, the quantitative aspect of most endophenotypes such as brain morphometry or volumetry provide a boost in statistical power compared with qualitative traits such as PTSD diagnosis. This is true for targeted candidate gene analyses, as well as for GWAS.Genetic variation is expected to exert an enduring effect on brain structures, which are ultimately associated with behavior and predisposition to disease22. The amygdala, perhaps the most strongly implicated structure in PTSD based in part on hypothesized mechanisms for PTSD, showed reduced volume in PTSD patients from a meta-analysis20 as well as a subsequent study we conducted in a large combat–veteran cohort23. The anterior cingulate, a medial prefrontal cortical structure, shows compromised voxel-based morphometry associated with PTSD26. These, and other successes associating genetic markers with variability in brain measures for other psychiatric disorders29, as well as parallel efforts establishing differences in subcortical brain volumes in PTSD, spurred our investigation of subcortical brain volumes on a genome-wide level in PTSD, which remain conspicuously unexplored. We hypothesized a modulation of subcortical brain volume, particularly hippocampus and amygdala, by genetic markers in PTSD patients and trauma-exposed controls23. We pursued a GWAS approach despite our small sample size because of the concerns with candidate gene approaches that can produce high rates of spurious associations31.We approached identifying a suitable endophenotype for PTSD by turning to the most consistently replicated neuroimaging finding, which is the association of PTSD with a smaller hippocampusn = 157) from a repository  of Iraq and Afghanistan era military service members who contributed blood for genotyping, clinical assessment data, and MRI scans were analyzed. Participants were screened for inclusion/exclusion criteria based on information available in the repository. Important exclusions included psychotic symptoms, high risk of suicide, contraindication to MRI, current substance abuse, neurological disorders, and age over 65 years. To reduce the effects of population stratification in a multi-racial sample, analyses were limited to non-Hispanic black  and non-Hispanic white  participants from these studies who consented to the genetic and imaging components and had data available at the time of analysis. All participants provided written informed consent to participate in procedures reviewed and approved by the Institutional Review Boards at Duke University and the Durham VA Medical Center. Participants completed questionnaires assessing traumatic life events , combat exposure , and depressive symptoms . PTSD diagnosis was ascertained with the Structured Clinical Interview for DSM-IV (SCID) and confirmed with the Clinician-Administered PTSD Scale35(CAPS) in 152 (97%) participants and with the Davidson Trauma Scale36(DTS) in 5 (3%) participants. Alcohol abuse and dependence was determined by the SCID.Participants  and fast spoiled gradient-recall (3D-FSPGR) were acquired axially for all participants. Image parameters were optimized for contrast between white matter, gray matter, and CSF .37  and its library tool recon-all . We applied standardized protocols for image analysis and quality assurance developed by ENIGMA, which are openly available online (http://enigma.ini.usc.edu/protocols/imaging-protocols/). All participants passed this inspection process without the need for manual adjustment.All T1 images were visually inspected  to assure appropriate quality. Automated segmentation and labeling of the left and right subcortical structures and estimation of total intracranial volume from participants’ T1 images were performed using the FreeSurfer image analysis suitemri_label2vol and a transformation matrix generated by tkregister2, the segmentation labels were returned to native space. The native-space segmentations were converted to LAS orientation and then the subcortical structures were extracted using the segmentation labels.Subcortical volumes for the left and right hemispheres were generated in each subject for the lateral ventricle, thalamus, caudate, putamen, pallidum, hippocampus, amygdala, and accumbens. Spatial normalization by affine registration to Talairach space and skull stripping were performed on the T1 images. Registration was checked visually for accuracy . FreeSurfer segmentation and labeling of subcortical structures was based on a combination of voxel intensity, probabilistic atlas location, and the spatial relationships of the voxels to the location of nearby subcortical structures. Using the FreeSurfer library function n = 157) obtained from a larger parent study of 2312 PTSD cases and controls were genotyped as previously described38. Briefly, DNA was extracted from whole blood and was genotyped in batches using three different Illumina BeadChips . Quality control was assessed separately in each batch and samples with low call rates were excluded from further analysis. A global reference panel from 1000 Genomes was used to impute missing genotypes in each batch separately; imputed SNPs with certainty <0.90 were excluded. Overlapping SNPs in the imputed NHB and NHW subsets were merged to create a final data set comprising 2,711,511 SNPs.Samples . To reduce redundancy in this imputed data set, we used the linkage disequilibrium (LD) clumping method in PLINK, choosing an r2 threshold of 0.25 and a 500 kb window, as reported previously41. False discovery rate (FDR) q-values were generated using PROC MULTTEST in SAS version 9.4 . The FDR correction was applied only across SNPs, but not across the eight subcortical regions and two hemispheres because of the inherent correlation between regional volumes. The known associations between these measures would produce a higher rate of significant associations than expected, and therefore an FDR correction would lead to overly conservative inferences. In addition, a multiple-comparison correction for brain structures previously implicated in PTSD that include amygdala42, caudate45 hippocampus23, lateral ventricles48, thalamus50, and nucleus accumbens52 would also contribute to overly conservative inferences. Post hoc interaction analyses were performed for the SNPs with significant main effects: SNP×childhood trauma and SNP×lifetime PTSD interactions were investigated for association with brain volume. Plots of significant SNP associations with LD structure by region were generated using LocusZoom53. Manhattan plots and Q–Q plots were produced using the R package qqman54  analysis was run using the 55. The analysis utilized nominally significant SNPs (p < 0.001) for the four brain regions showing FDR-significant results .We conducted gene ontology analysis to obtain a more comprehensive picture of the biological pathways that may be implicated in modulating subcortical volume following trauma exposure. We used the Genomic Regions Enrichment of Annotations Tool (GREAT), given that many of the SNPs associated with brain volume in our data set were intergenicp = 0.01).Important clinical and sociodemographic information is reported by diagnostic (PTSD and non-PTSD) and ancestry (NHB and NHW) groups in Table 3 . The SNPs on chromosome 6 also interacted with childhood trauma exposure to influence R-caudate . Further exploration of this interaction revealed that the associations of these SNPs with R-caudate volume became stronger with increasing exposure to childhood trauma . Among subjects with no childhood trauma exposure, rs9373240 is not associated with R-caudate volume (p = 0.132). Among subjects who endorsed one category of childhood trauma exposure, rs9373240 is associated with R-caudate volume (p = 0.007); however, among those subjects who endorsed two or more categories of childhood trauma, the association is much more robust (p = 6.36 × 10−8). None of the SNPs on chromosome 6 interacted significantly with PTSD diagnosis, and neither of the SNPs in NKAIN3 showed a significant interaction with childhood trauma or PTSD diagnosis.The R-caudate Fig. a and 2a uma Fig. . For exaTRAM1L1. The most significant SNP associated with R-lateral ventricle was rs34043524, such that for each additional G allele, R-lateral ventricle volume decreased. Figure TRAM1L1 with R-lateral ventricle. The TRAM1L1 locus in the 4q21-q32 region has been previously hypothesized to play a role in alcohol dependence, but prior investigation produced negative results56. This prompted us to conduct follow-up analysis for association with alcohol dependence (i) as an additional covariate in the association between genotype and caudate volume, and (ii) as a phenotype (main effect) associated with genotype56. We did, in fact, observe that the same SNPs downstream of TRAM1L1 that were associated with R-lateral ventricle volume were also associated with lifetime alcohol abuse or dependence in our data set . Importantly, inclusion of lifetime alcohol abuse or dependence as a covariate in the model of SNPs predicting R-lateral ventricle volume did not attenuate the association. None of the SNPs downstream of TRAM1L1 significantly interacted with either PTSD diagnosis or childhood trauma exposure to predict R-lateral ventricle volume.The R-lateral ventricle Fig. b and 2b ) volume was rs55886168, which is located on chromosome 20 in a long non-coding RNA (LINC01522). For each additional T allele, R-accumbens volume increased by 79.33 mm3 . There were also several correlated, intronic SNPs in TMPRSS15 on chromosome 21 associated with R-accumbens volume . A plot of these SNPs is shown in Fig. p = 2.58 × 10−7; FDR q = 0.016), and high LD with rs4053747 chromosome 6 . None of the SNPs associated with R-accumbens volume displayed significant interactions with either PTSD diagnosis or childhood trauma exposure.The most significant SNP associated with R-accumbens Fig. c and 2cp < 0.001) for the four brain regions showing FDR significance . We identified several significant gene ontology terms for SNPs associated with right lateral ventricle, right caudate, and right pallidum that are detailed in Supplementary Tables We performed gene ontology analysis with GREAT using nominally significant SNPs p < 0.00 for the NKAIN3 (rs34720850), which codes for a Na+/K+ transporting ATPase interacting 3 protein. The protein is a member of the NKAIN family of mammalian proteins that are neuronally expressed in multiple regions of the mouse brain. In particular, NKAIN3 is strongly expressed in the hippocampus57. Previously, NKAIN3 has been reported in bulimia nervosa and taste perception of salt59. Structural imaging volumetry of the caudate in PTSD has been limited to studies with small sample sizes, among other limitations, making it challenging to draw strong conclusions from the existing literature. Smaller caudate volume has been associated with early life stress45, with higher CAPS scores among combat-exposed veterans43. Conversely, larger caudate volumes have been associated with PTSD44, while many other studies including the present sample showed no such association61. Abnormal functioning of the caudate has been established in PTSD with generally lower caudate  activation to rewards and lower deactivation to losses (punishment) as compared to trauma controls64. The findings are consistent with hyporesponsivity to positive stimuli and anhedonia, which are common in PTSD65. Relatedly, abnormalities in D2 dopamine receptors located in the striatum have been observed in PTSD66. Diminished hedonic tone and reward responsivity are prominent features of depression, which has a more firmly established and extensively investigated association with caudate dysfunction67. However, depressive symptoms are highly comorbid in most PTSD samples and mounting empirical evidence questions a distinct nosology between the two disorders68.We performed high-density genome-wide association analysis of subcortical brain structures segmented by FreeSurfer in trauma-exposed US military veterans with and without a PTSD diagnosis. We identified several SNPs (in high LD with rs9373240) in an intergenic region on chromosome 6 that were associated with caudate volume. These SNPs also significantly interacted with childhood trauma, whereby increasing trauma exposure produced a stronger association between SNPs and caudate volume. While these SNPs do not reside within a gene, it is possible that they function as regulatory markers. Caudate volume was also modulated by SNPs in TMPRSS15. The accumbens  plays a major role in reward and reinforcement learning and a secondary role in processing fear, which are central to the prevailing behavioral models of PTSD69. The TMPRSS15 gene codes for a transmembrane protease serine 15, an enzyme that converts proenzyme trysinogen to trypsin. Trypsin-like serine proteases and trypsin play very important roles in neural development, neuroregeneration, and synaptic plasticity, particularly memory formation71. Accumulating evidence suggests that the brain has co-opted the activities of enteropeptidases, which are central to digestive function, to regulate various processes underlying synaptic activity and behavior. Enteropeptidases protect hippocampal neurons from death induced by glutamate toxicity72.Additionally, we found that nucleus accumbens volume was modulated by several SNPs in high LD with rs13140180 on chromosome 4, rs1577238 on chromosome 6, and TRAM1L1 gene on chromosome 4 that were associated with right lateral ventricular volume. Previously, an association of TRAM1L1 and alcohol dependence was hypothesized by Kalsi et al.56 based on LOD score from genetic linkage analysis. We identified a significant association (p = 1.9 × 10−7) between rs34043524 and clinical diagnosis of alcohol use disorder. Importantly, the association between the same SNP and ventricular volume remained significant after adding alcohol dependence as a covariate (p = 2.5 × 10−7), which suggests the association of this SNP with lateral ventricular volume and with alcohol dependence are independent of each other. Lateral ventricle volumetry lacks consistent results in PTSD. Early-life maltreatment that subsequently develops into PTSD was associated with larger lateral ventricular volume73. The presence of lateral ventricular volume differences reported in children with PTSD, but the lack thereof in adults deserves mention. Bipolar disorder74 and schizophrenia75 are consistently associated with enlarged lateral ventricle volume. The SNP associated with the lateral ventricle volume significantly interacts with PTSD diagnosis and was not identified in an extremely large normative sample  of subcortical volumes, suggesting the SNP could be a non-specific genetic marker for psychopathology.Finally, we identified several SNPs (in high LD with rs34043524) downstream of the NKAIN3, TRAM1L1, and TMPRSS15 have been previously implicated in neuronal function71, increasing the potential biological relevance of our findings. Indeed, the latest evidence implicates a common genetic architecture spans common neuropsychiatric disorders. A large GWAS  by the Psychiatric Genetics Consortium (PGC) found common neuronal, immune, and histone pathways across major depression, bipolar disorder, and schizophrenia. Risk variants for psychiatric disorders aggregate in particular biological pathways and these pathways are often shared between disorders suggesting a shared etiology among disorders and the co-heritability of multiple psychiatric disorders76. Calculations using common SNPs found high correlations between schizophrenia and bipolar disorder (h2 = 0.68), major depressive disorder and bipolar disorder (h2 = 0.47), as well as schizophrenia and major depressive disorder (h2 = 0.43)76. It is certainly possible that the current classification of psychiatric disorders as clinical syndromes with a constellation of behavioral symptoms may serve to inflate the apparent pleiotropy compared to an alternate nosology constructed on pathomechanistic markers of etiology77. Thus, genome wide associations of brain measures hold the promise of moving us one step closer to this new psychiatric nosology78. On the other hand, attempts to find disease-associated genetic variation that point to molecular mechanisms of pathogenesis has been further complicated by the polygenicity of clinical diagnoses and phenotypes79. Thus, the effects of an overall polygenic risk profile score on neuroimaging abnormalities might help to identify genetic susceptibility or genetically mediated traits of PTSD81.The three genes 10.It is important to consider the value of imaging genetics approach and its potential relevance to PTSD research, particularly in the translational and clinical domains. The first goal is simply looking for an association between genetic markers and neuroimaging phenotypes in the context of a PTSD sample. Any difference in the SNP associations with a particular neuroimaging measure could be explained by the presence of PTSD. It is possible that the neuroimaging phenotype is assessing an underlying dimension of PTSD not captured or accessible by its clinical diagnosis and may explain, at least in part, the lack of SNP×PTSD interaction finding in the present sample. However, a limitation of our analysis of interaction effects was that it was restricted to SNPs that demonstrated significant main effects because of concern that our sample was too small to pursue interaction effects at the genome-wide level. Alternatively, a significant interaction can be further explored by testing the association between genetic markers and brain imaging measures that are different in PTSD cases than controls82. The concern with accuracy introduces added variance that can be overcome by increasing the sample size in equal measure82. Factors contributing to negative results for the hippocampus include the heterogeneity of a structure comprising several subfields that are differentially affected by PTSD. Specific subfields, namely cornu ammonis 1 (CA1), cornu ammonis 3 (CA3), and dentate gyrus, show volume reduction in PTSD83, but including other subfields may confound associations with overall hippocampal volume, which was utilized in this study. Likewise, the amygdala contains several subregions (nuclei) that are differentially involved in PTSD based on their functional roles. Advanced methods that have recently become available for segmenting the hippocampus84 and amygdala85 into functional subunits may hold greater promise as phenotypes for identifying genetic markers associated with PTSD.Factors contributing to negative results for the amygdala include limited accuracy of FreeSurfer segmentation as compared to hand-tracing whereas the hippocampus and other structures are more accurateAnother limitation of the present study was the small sample size, rendering the results susceptible to type-1 error. The presence of two ancestry subgroups in our sample, which we controlled statistically with a single principal component, was considered as a minor limitation because separate analyses of these two racial groups yielded results consistent with the original analyses of the combined groups. Thus, we do not anticipate that population stratification is an explanation for the present results.79. Leveraging neuroimaging phenotypes may offer a shortcut over clinical phenotypes in identifying these elusive genetic markers and relevant neurobiological pathways87.Ultimately the promise of finding genetic determinants of PTSD is that they signal the presence of etiologic pathways for which targeted interventions may be devised and deployed. Attempts to find disease-associated genetic variation that point to molecular mechanisms of pathogenesis has proven challenging due to the polygenicity of clinical phenotypesSupplemental Figure 1Supplemental Figure 2Supplemental Tables


Ependyma have been proposed as adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after spinal cord injury (SCI). This proposal was based on small, midline stab SCI. Here, we tested the generality of this proposal by using a genetic knock-in cell fate mapping strategy in different murine SCI models. After large crush injuries across the entire spinal cord, ependyma-derived progeny remained local, did not migrate and contributed few cells of any kind and less than 2%, if any, of the total newly proliferated and molecularly confirmed scar-forming astrocytes. Stab injuries that were near to but did not directly damage ependyma, contained no ependyma-derived cells. Our findings show that ependymal contribution of progeny after SCI is minimal, local and dependent on direct ependymal injury, indicating that ependyma are not a major source of endogenous neural stem cells or neuroprotective astrocytes after SCI. Generating newly proliferated cells after tissue injury is a critical adaptation that limits damage, replaces lost tissue and sustains organ function234After CNS injury and disease, newly proliferated reactive astrocytes form glia-limitans-like scar borders around damaged tissue679101281418in vivo in adult transgenic mice by using inducible genetic recombination technology in which tamoxifen dependent Cre-recombinase (CreERT) activates reporter gene expression targeted by specific promoters222324Cell lineage tracing can be conducted Our laboratory has a longstanding interest in understanding the roles of scar-forming and reactive astrocytes in CNS injury and disease61012132025Foxj1CreERT2:GFP reporter based fate mapping strategy222324In the present study, we tested the generality of the proposal that ependymal cells represent a major source of adult neural stem cells that provide the majority of newly proliferated scar-forming astrocytes that protect tissue and function after SCI222324Foxj1CreERT2-tdT lineage analysis model, denoted henceforth as Foxj1-tdT, we determined which cells exhibited tdT reporter expression after tamoxifen induction in uninjured mice. In the absence of tamoxifen, there was no detectable tdT expression (not shown). In uninjured adult mice given tamoxifen and evaluated after drug washout, tdT was clearly expressed by essentially all ependymal cells defined as ciliated cells with apical surfaces contacting the central canal lumen2231313131To target CNS ependymal cells for fate mapping of progeny generated after SCI, we used mice with CreERT2 inserted into the Foxj1 locusFoxj1CreERT2-tdT mice were induced with tamoxifen and given a full transverse crush SCI at T10  after severe SCI involving the entire spinal cord, ependymal cell progeny contribute less than 2%, and more probably none, of the total newly proliferated molecularly validated astrocytes by staining for GFAP and Aldh1l1, (ii) ependymal cell contribution to other cell types after SCI is also minimal, (iii) ependymal progeny fail to migrate substantially into SCI lesions and remain primarily in the immediate vicinity of the damaged ependymal layer around the central canal, and (iv) ependymal progeny do not contribute any cells to nearby SCI lesions that do not directly damage ependyma. Thus, our findings do not support previous reports that after SCI, ependyma are a major source of endogenous neural stem cells that migrate extensively to and throughout lesions and provide the majority of neuroprotective astrocytes222324Foxj1 locusFoxj1CreERT2 linePrevious studies reporting ependyma as major neural stem cells after SCI used two transgenically-targeted fate mapping strategies with an unusual Nestin-CreERT line and a human FOXJ1-CreERT line22232446222324Choice of SCI models can influence interpretation and extrapolation of experimental observations. Small stab SCI can be useful to study blood-spinal cord-barrier damage or to assess immediately local cellular and molecular responses, but not all observations may be generalizable to larger SCI lesions generated by full transverse crush or contusion. The previous proposal that ependyma are a major source of endogenous neural stem cells after SCI was extrapolated from studies using a specialized SCI model of stab injuries that were placed longitudinally along the spinal cord midline and that penetrated radially into the ependymal layer, thereby directly injuring ependymal cells, as evident from the photomicrographs provided22232422222324Molecular markers can be useful signatures to identify cell phenotypes. To identify specific cell types, we used multiple well-established markers to identify ependyma, Sox9, CD133, vimentin3132363864142434422232438Foxj1CreERT2-tdT used here. We show that small numbers of ependymal progeny are generated only after direct damage, and these remain locally in the immediate peri-ependymal area. Ependyma make no contribution to SCI lesions by which they are not directly injured. Instead our findings suggest that ependymal cells may proliferate in particular to repair and seal off the central canal when it has been damaged. Our findings provide evidence that the proposal that ependyma are major contributors of neural stem cells after SCI is based on the use of (1) small radial SCI lesions that directly injure ependyma and exaggerate the contribution of ependymal progeny to the wound response, and (2) inappropriate claims regarding molecular markers used to identify putative astrocytes. Lastly, our findings strongly suggest that putative ependymal progenitors are not likely to be good candidates for endogenous manipulation or transplantation after SCI because of their restricted differentiation potential and limited migration capacity.The observations of our study have implications for others attempting to identify potential cellular manipulations to improve neural repair and functional outcome after SCI. Severe human SCI lesions have large cores of non-neural tissue and cysts23422232451Foxj1CreERT2 All transgenic and non-transgenic mice used were derived from in house breeding colonies maintained on C57/BL6 backgrounds. All mice were between two and five months of age at the time of SCI. Transgenic mouse lines have been well-characterized previously: (1) Tamoxifen was dissolved in corn oil and administered as subcutaneous injections. Uninjured mice were given tamoxifen, 75 mg/kg, once a day for 7 days followed by a 2-week drug washout period to avoid potential confounds of residual tamoxifen remaining during the first two weeks after deliveryAll surgeries were performed under general anesthesia with isoflurane in oxygen-enriched air using an operating microscope , and rodent stereotaxic apparatus . Laminectomy of a single vertebra was performed and severe crush SCI were made at the level of T10 to expose the spinal cord. For severe spinal cord injury (SCI), No. 5 Dumont forceps  without spacers and with a tip width of 0.5 mm were used to completely compress the entire spinal cord laterally from both sides for 5 seconds7Bromodeoxyuridine  was dissolved in saline plus 0.007 N NaOH. For mice with 2 weeks survival times after SCI, BrdU was administered by intraperitoneal injections given twice daily at 100 mg/kg per injection, on days 2 through 7 after SCI, which is the well-established peak period of astrocyte proliferation after SCI in rodents733After terminal anesthesia by barbiturate overdose, mice were perfused transcardially with a phosphate buffered saline rinse followed by either 4% paraformaldehyde or 10% formalin. Spinal cords were removed, post-fixed overnight, and cryoprotected in buffered 30% sucrose for 48 hours. Frozen sections (30 μm) were prepared using a cryostat microtome (Leica) and processed for immunofluorescence as described712Cell counts were performed on 3-dimensional image stacks collected using x40 or x63 objectives to generate cells per volume values by investigators blinded to experimental groups and given only randomly assigned animal numbers. Areas or boxes counted are shown in schematics associated with specific graphs. At least one hundred cells were counted per animal for every discrete area-box evaluated. Means were generated per animal and these used to generate group means to compare for statistical analyses. Statistical evaluations were conducted either by t-tests for pair comparisons, and by ANOVA with post hoc, independent pair wise analysis as per Newman-Keuls for evaluations of repeated measures . Group sizes were determined based on previous experience and post hoc power analysis was performed for all experiments using G * Power Software V 3.1.9.2How to cite this article: Ren, Y. et al. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury. Sci. Rep.7, 41122; doi: 10.1038/srep41122 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined.To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses.Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood.in vitro cell culture.CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells  with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone.These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4. We studied CXCL4 in endometrial tissue and cells and found that expression was maximal at menses, regulated by cortisol in endothelial cells, and reduced in HMB, consistent with a role in endometrial repair. The human endometrium displays a remarkable ability to break down and fully repair each month in the absence of pregnancy or lactation. Menstruation is triggered by the withdrawal of the ovarian steroid hormones estrogen and progesterone as the corpus luteum regresses. This results in a local inflammatory response, including leukocyte influx and edema, which culminates in tissue breakdown by matrix metalloproteases and bleeding . Much lein situ (Macrophages have a well-established role in the repair process at multiple tissue sites . They enin situ . Recent in situ ].α and granulocyte macrophage colony-stimulating factor–independent fashion (CXCL4 (PF4) is a member of the CXC family that has a role in chemotaxis of neutrophils and monocytes . It is u fashion . CXCL4-s fashion . Notably fashion , cannot  fashion , and do  fashion  but prod fashion . In addi fashion . CXCL4 h fashion  and has  fashion .in vitro cell models to examine the regulation of CXCL4 by steroid hormones, including cortisol, because this steroid is thought to play a key role in regulating the local endometrial environment during menstruation. Next, we investigated the effect of CXCL4 on endometrial cells and macrophages. Our results highlight a potential role for this cytokine in the physiologic processes of menstruation and endometrial repair.Because CXCL4 is thought to have a key role in the regulation of angiogenesis, recruitment of monocytes, and wound healing, we hypothesized that it has a key role in endometrial repair at the time of menstruation . Therefoin vitro culture. Cycle stage was determined by (1) histologic dating  in RPMIt tests were used for immunoscore data. GraphPad Prism software, version 6, was used . P < 0.05 was considered to represent a statistically significant difference.For cell culture, mRNA results are expressed as fold increase, wherein relative expression of mRNA after treatment was divided by the relative expression after vehicle treatment. For tissue data, results were expressed as a quantity relative to a comparator, a sample of placental cDNA. Data are presented as mean ± standard error of the mean and statistically significant differences among raw data  were determined by using Kruskal–Wallis nonparametric test with Dunn multiple comparison posttest. Statistical analysis between women with HMB and NMB at different stages of the cycle was performed by using a two-way analysis of variance with Bonferroni posttest analysis. Student CXCL4-encoded mRNAs were detected in human endometrial tissue biopsy samples throughout the cycle [CXCL4 mRNA concentrations were significantly higher in menstrual biopsy specimens than those from the proliferative (P < 0.05), early secretory (P < 0.01), and mid-secretory (P < 0.05) phases.Immunohistochemistry detected CXCL4 protein in the cytoplasm of epithelial and stromal cells throughout the menstrual cycle, with an increase in staining intensity noted in endometrium collected from women during the secretory and menstrual phases [CXCL4 mRNA compared with those treated with vehicle, estradiol, or cortisol [After confirming the presence of CXCL4 in endometrial stromal and endothelial cells, we examined its regulation by steroids using primary hESCs and an HEEC cell line. Treatment with 10 nM estradiol mimicked the proliferative phase; 1 µM progesterone, the secretory phase; and sequential progesterone treatment and subsequent removal, the late secretory/menstrual phase. Mounting evidence suggests that cortisol has an important role in the local endometrial environment at menses ; therefocortisol .CXCL4 mRNA [P < 0.01).Interestingly, CXCL4 regulation in HEECs was different from that detected in hESCs. Cortisol treatment of HEECs displayed maximal increases in concentrations of CL4 mRNA , which wCXCL4 mRNA was maximal in endometrium from the late secretory and menstrual phases of the cycle, we compared mRNA concentrations in endometrial tissue homogenates from these two phases, taken from women with objectively measured MBL. Using a blood loss of >80 mL to define HMB, we found no significant differences in mRNA concentrations when comparing women with HMB and NMB [Because in vitro, we hypothesized that cellular levels of CXCL4 may differ in women with HMB and NMB, despite no significant differences in global endometrial CXCL4 mRNA concentrations. We examined CXCL4 protein by immunohistochemistry in the endometrium of the late secretory and menstrual phases from women with NMB and HMB. Semiquantitative immunoscoring of the stromal compartment and endothelial cells revealed no significant changes during the late secretory phase between women with NMB and HMB [P < 0.05) [Because we determined that regulation of CXCL4 varied in stromal and endothelial cells  < 0.05) . In contγ–pretreated), and M2 (cortisol-pretreated) macrophages or macrophages exposed to a proliferative phase environment (estradiol-pretreated) or to a secretory phase environment (progesterone-pretreated). These pretreated macrophages were plated into wells opposite CXCL4 on a multichanneled microslide. Cortisol-exposed macrophages migrated toward CXCL4 at a significantly higher rate than any of the other macrophage subtypes [Because CXCL4 increased at menses and colocalized to macrophage cells, we investigated the effect of CXCL4-induced chemotaxis on different macrophage subtypes. Peripheral macrophages were pretreated to induce different subtypes: M0 (macrophage colony-stimulating factor–pretreated), M1 (granulocyte macrophage colony-stimulating factor– and interferon subtypes .This report details the presence of CXCL4 in the human endometrium across the menstrual cycle and reveals that maximal levels are present during menstruation. Steroid regulation of CXCL4 occurs in hESCs, with significant increases after withdrawal of progesterone. In contrast, endometrial endothelial cells do not display an increase in CXCL4 on progesterone withdrawal but do demonstrate significant increases in response to cortisol treatment. Furthermore, we reveal that women with HMB have significantly reduced CXCL4 in endothelial cells in the menstrual phase, consistent with a defective cortisol response at menses . MacrophCXCL4 is present in the human endometrium during menstruation, with both RT-qPCR and immunohistochemistry being consistent with maximal detection during the menstrual phase. The functional layer of the endometrial breaks down during menses, with repair occurring simultaneously in adjacent areas . Therefoin vitro studies revealed that progesterone withdrawal resulted in a significant increase of CXCL4 expression within endometrial stromal cells, consistent with maximal levels during menstruation. HEECs, however, do not express the progesterone receptor (Next, we investigated the regulation of CXCL4 in the human endometrium. Because of the dramatic variations observed across the menstrual cycle, we examined steroid regulation of this cytokine. A series of receptor ; hence, receptor , and trereceptor . TherefoCXCL4 mRNA concentrations were detected between these two groups of women during the late secretory or menstrual phases.Because CXCL4 is a putative endometrial repair factor, we examined mRNA concentrations in endometrial tissue sample homogenates from women with HMB and NMB. We hypothesized that women with HMB would have reduced CXCL4 induction during menstruation, leading to inefficient endometrial repair and prolonged, HMB. However, no significant differences in β-hydroxysteroid dehydrogenase-2 is significantly increased in endometrium from women with HMB versus NMB, thereby creating a local glucocorticoid deficiency (There are two potential explanations for these findings. First, there may be no deregulation of CXCL4 in women with HMB. However, our results suggested an alternative explanation: that different cell types within the human endometrium have differential regulation of CXCL4 induction, with progesterone withdrawal having a substantial impact on stromal cells and cortisol regulating CXCL4 in endothelial cells. Examination of homogenized whole endometrial biopsy specimens may mask differential expression of CXCL4 within different cell types in women with heavy versus normal menstrual blood loss. Therefore, we examined CXCL4 protein in stromal cells and endothelial cells in women with NMB and HMB during the late secretory and menstrual phases. This revealed that endothelial cell CXCL4 protein was significantly reduced in women with HMB versus NMB during menses, which might be consistent with a defective cortisol microenvironment . Our labficiency . Therefoficiency , but itsCXCL4 is known to be a chemoattractant in many tissues, triggering migration of monocytes and macrophages to sites of inflammation . Herein In summary, we have identified that CXCL4 is increased in the human endometrium during menstruation, a time consistent with involvement in endometrial repair. Mechanistically, we have revealed that endometrial CXCL4 is regulated by progesterone withdrawal and cortisol. In addition, we reveal that CXCL4 is reduced in endothelial cells of women with HMB at menses. Functionally, CXCL4 appears to have an important role as a macrophage chemoattractant, particularly for macrophages pre-exposed to cortisol. These data implicate CXCL4 as a key player in the physiologic process of endometrial repair after menses.


Excess alcohol use is known to promote development of aggressive tumors in various tissues in human patients, but the cause of alcohol promotion of tumor aggressiveness is not clearly understood. We used an animals model of fetal alcohol exposure that is known to promote tumor development and determined if alcohol programs the pituitary to acquire aggressive prolactin-secreting tumors. Our results show that pituitaries of fetal alcohol-exposed rats produced increased levels of intra-pituitary aromatase protein and plasma estrogen, enhanced pituitary tissue growth, and upon estrogen challenge developed prolactin-secreting tumors (prolactinomas) that were hemorrhagic and often penetrated into the surrounding tissue. Pituitary tumors of fetal alcohol-exposed rats produced higher levels of hemorrhage-associated genes and proteins and multipotency genes and proteins. Cells of pituitary tumor of fetal alcohol exposed rat grew into tumor spheres in ultra-low attachment plate, expressed multipotency genes, formed an increased number of colonies, showed enhanced cell migration, and induced solid tumors following inoculation in immunodeficient mice. These data suggest that fetal alcohol exposure programs the pituitary to develop aggressive prolactinoma after estrogen treatment possibly due to increase in stem cell niche within the tumor microenvironment. Moreover, International Association of Research on Cancer’s 2009 survey identify that around 3.5% of cancer deaths in USA are caused by alcohol consumption3. A number of reports have now demonstrated that alcohol abuse promotes development of aggressive tumors of breast, prostate, pancreas, and colon tissues in human patients6. The cause of alcohol promotion of tumor aggressiveness is not clearly understood, but recent evidence suggests that enhanced estrogen signaling might be involved in alcohol promotion of tumor aggressiveness. Using an animal model of fetal alcohol exposure, Hilakivi-Clarke and colleagues have shown elevated plasma estrogen levels may increase the risk for mammary tumor development in fetal alcohol exposed offspring7. Polanco and colleagues have also connected increased plasma estrogen levels with the tumor promotion in fetal alcohol exposed rats9. The ovarian steroid estradiol is also known to increase pituitary prolactin (PRL)-producing lactotropic cell proliferation and to induce prolactinoma development in humans as well as in laboratory animals14. Prolactinomas account for approximately 40% of all pituitary tumors and are an important cause of hypogonadism and infertility in human16. While most are benign and can be controlled by current therapies, some show a lack of sensitivity to a combination therapies or recur during follow-up and are considered as aggressive with unclear epidemiology17. Whether environmental imprinting may influence the development of aggressive prolactinomas has not been studied. We therefore determined if fetal alcohol exposure, which causes stable alcohol epigenetic marks on the genome20, promotes development of aggressive prolactinomas using a Fischer-344 (F344) rat animal model.The National Cancer Institute has established that alcoholic beverage is a human teratogen and human carcinogen21, pituitary PRL immunoreactivity was increased in fetal alcohol-fed (AF) animals  mRNA levels, were elevated in AF offspring as compared to control-fed (AD and PF) offspring  are used to identify the tumor aggressiveness in clinics24. By employing immunohistochemical methods we determined the changes in these biochemical marker proteins in pituitaries of fetal alcohol-fed (AF) and control-fed (AD and PF) rats. As shown in Fig. As noted earlier, pituitary tumors are generally adenomas but some may show aggressive and/or malignant behaviors26. To determine if pituitary tumors of fetal alcohol-exposed rats contains PSCs, we measured stem cell marker proteins SOX2, CD133 and OCT4 by immunocytochemistry and their precursor genes by q-PCR in the tumor tissues. As shown in Fig. Sox2  have been proposed to play a role in pituitary tumorigenesis, particularly in the formation of aggressive pituitary tumorsox2 Fig. , Cd133  mRNAs -treated rats were grown in ultra-low attachment plates, a large number of spheres developed from AF pituitary tumor cells while a small number of spheres developed from AD pituitary tumor cells Fig. . AF pituNAs Fig.  and mostNAs Fig. . These rin vitro techniques that examine the ability of the cells to form colonies in semisolid agar. AF pituitary tumorispheres formed increased number of colonies in soft agar under normal growth factors as compared to AD pituitary tumorispheres  that characterize invasive adenoma33. Also, the pituitary tumors of fetal alcohol exposed rats had elevated levels of oncogenes PTTG, FGF4 and MMP-9, which are known to be upregulated in the invasive prolactinoma and pituitary carcinoma35.Our conclusion that fetal alcohol-exposed rat develops aggressive pituitary tumors is based on anatomical, histological, biochemical evidence. Pituitary tumors of fetal alcohol-exposed offspring were large, hemorrhagic and appeared to penetration to the neighboring tissue. In human, some aggressive macroprolactinomas remain within the sella, while others extend beyond its confines and extensively invade the bones comprising the base of the skullPttg and Fgf genes and promote aggressiveness of pituitary tumors36. It should be noted that heavy alcohol drinking in the adult promotes development of aggressive cancer in various tissues including mammary glands37. In the case of mammary gland, sex hormones, such as estrogen is also considered to play an important role in growth factors expression abnormalities and the etiology of alcohol promotion of aggressive cancer38. We therefore propose that elevated production of estrogen in the pituitary may be a cause for the development of aggressive pituitary tumors in fetal alcohol exposed rats.The data presented here connected elevated estrogen levels with the tumor promotion in the pituitary of fetal alcohol-exposed rats. We showed increased levels of pituitary aromatase and plasma estrogen in AF rats. Also, exogenous estrogen was more effective in increasing pituitary weight, plasma levels of PRL and tumor aggressiveness in the pituitary of AF rats. It has previously been shown that locally produced estrogen through aromatization might enhance tissue expression of 39, are expressed in pituitary tumors40 and are known to be overexpressed in cells with high tumoriginicity41. A recent study also showed that forced up-regulation of the SOX2-positive pituitary stem cells by transgenic approaches in mouse stimulates a transient burst of proliferation, and subsequently induce tumorigenesis in a non-cell autonomous manner42. The pituitary tumors of fetal alcohol-exposed rats also developed tumor spheres in ultra-low attachment plate and expressed increased levels of stem cell marker proteins and genes, suggesting that pituitary tumors of AF rats have an increased stem cell niche. It is well known that cancer malignancy and metastatic ability of the tumor cells depends on the “stemness” phenotypes such as self-renewal, proliferation and migration features44. Recent evidence also suggest that cancer stem cells play an important role in alcohol-induced tumor initiation, progression and metastasis of breast and other cancers45. Our data showed that pituitary tumor cells of AF rats grew rapidly, showed high colony formation, cell migration, and successfully induced tumors in xenotransplanted mice. Together these data provide strong evidence to support that the pituitary gland of fetal alcohol exposed rat develops aggressive and invasive adenoma, possibly due to an increase in estrogenicity and an expanded stem cell niche within the tumor microenvironment. Further investigation is need to identified the molecular mechanism(s) underlying fetal alcohol promotion of aggressive prolactinomas.The data presented here also implicates an increased stem cell niche within the tumor microenvironment with the tumor aggressiveness. AF tumors overexpressed multipotency proteins . These proteins are established markers of pituitary stem cells22. On postnatal day 2 (PD2), AF and PF pups were cross-fostered to untreated lactating AD dams to prevent any compromised nurturing by the AF and PF moms. Litter size was maintained at 8 pups per dam to minimize any nurturing effect on the body growth. Pups were weaned on PD21 and housed by sex. In each experimental group only one female offspring from a rat litter was used.Animal surgery and care were performed in accordance with institutional guidelines and compiled with policies of the National Institutes of Health. Rutgers institutional animal care and use committee (IACUC) approved this research protocol. Fisher-344 rats were obtained from Harlan Laboratories  and housed under controlled condition of 12 h light/dark cycle at a constant temperature 22 °C throughout the study. Rats were housed in pairs in Open-type Shoe Box Cages with Bedcob bedding, and they were fed ad libitum with rat chow and tap water in a conventional facility. Health status of animals was checked regularly by determining body weight, feeding and general behaviors, and university veterinarians were consulted to address any special health needs. Some of these rats were bred, and on gestational day 7 through 21 they were fed either rat chow ad libitum (AD), a liquid diet containing ethanol  ad libitum, or pair-fed  an isocaloric liquid control diet . The concentration of ethanol in the diet was increased over the first 4 days from 1.7 to 5.0% v/v to habituate the animals to the alcohol diet. After this habituation period, rats were fed the liquid diet containing ethanol at a concentration of 6.7% v/v. We have previously shown that the liquid-diet paradigm in pregnant rats elevates the blood ethanol concentration in the range of 80–90 mg/dl at 2 h after the beginning of the dark period46. After surgery, animals were observed for pain and suffering or infection for 3 days. Estrogen-treated rats were kept in cages fitted with HEPA filter for prevention of estrogen contamination. At 120 days after the estradiol implants the rats were killed and the pituitary tissues were fixed for microscopic examination or frozen for further biochemical evaluation.We determined whether fetal alcohol exposure changed pituitary lactotropic cell growth and hormone production during adulthood. For this, a group of AD, PF and AF female rats at 90 days of age on the day of estrus were sacrificed by decapitation . Pituitary tissues were collected for pituitary weight and protein and gene measurements, and plasma samples were obtained for PRL measurement. We also determined if fetal alcohol exposure altered the mitogenic effects of estrogen on lactotropes. For this, a group of animals at 60 days of age was ovariectomized under sodium pentobarbital anesthesia  as a general anesthesia and 2.5% Bupivacaine (sc) as a local analgesia, and sc implanted with an estradiol-17β  filled 1-cm silastic capsule  or an empty 1-cm silastic capsule. These estradiol capsules were shown to maintain plasma levels of estradiol-17β between 120 and 150 pg/mlFixed pituitary tissues were embedded in Paraplast and sectioned at 6 µm. Some of these sections were stained with hematoxylin and eosin then mounted with Permount. A toxicological pathologist, who was blind to treatment, viewed tumor slides to determine whether the tumors were adenomas or invasive adenoma. Representative images were taken using a Nikon photo microscope at 10X and 20X. Other sections were used for immunocytochemical detection of proteins. All the antibodies vendor and information is listed in Supplement Table 1. In general, tissue sections on a slide were blocked in normal horse serum  for 60 minutes at room temperature. Sections were incubated overnight at 4 °C with either primary antibody or rabbit primary antibody isotype control , which served as a negative control. The following day the tissues sections were incubated with secondary antibody  for 60 minutes at room temperature, counterstained with DAPI  then mounted with Permount prolong media. For all immunocytochemical detection, tumor sections were viewed and five representative images were captured at random from one section per tumor tissue using an Olympus FSX100 microscope at 20X (Olympus). The pictures were taken with the same exposure settings for all samples. The dark brown staining is counted as positive staining while the counter blue staining (nuclear staining) counted as negative staining. The percentage stained cells were calculated by dividing the number of positive stained cells with the number of negative stained cells multiply by 100.Plasma PRL and estradiol levels were measured using rat PRL EIA kit  and rat estrogen ELISA kit  respectively, using the instructions from the manufacturer.Gapdh and ribosomal protein 19 (Rpl-19) were used as internal controls. Gene expression levels as GAPDH ratios are only presented in the figures. Gene expression levels as RPL-19 ratios are presented in Supplemental Fig. 1.Expression levels of various genes in rat pituitaries were measured by quantitative real time PCR (SYBR green assay). Total RNA from the pituitary gland was extracted using the All in One Purification Kit . Total RNA (1 μg) was converted to first strand complementary DNA (cDNA) using a high capacity cDNA reverse transcription kit . All the primer sequences used for the study are given in Supplemental Table 2. Real-time quantitative PCR was performed at 95 °C for 5 min followed by 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec, 72 °C for 40 sec in an Applied Biosystems 7500 Real-time PCR system. The quantity of target gene expression was measured using standard curve method. Data were analyzed using Prism 5.0 (GraphPad Software). The data shown in the figures are mean ± SEM. Significant differences between different treatment groups were assessed with one-way analysis of variance (ANOVA) with post-hoc analysis using the Newman Keuls posttest. P < 0.05 was considered significant. Additional information on materials and data are available upon request.Supplementary Information


CNS cryptococcal meningoencephalitis in both HIV positive (HIV+) and HIV negative (HIV−) subjects is associated with high morbidity and mortality despite optimal antifungal therapy. We thus conducted a detailed analysis of the MR imaging findings in 45 HIV− and 11 HIV+ patients to identify imaging findings associated with refractory disease. Ventricular abnormalities, namely ependymitis and choroid plexitis were seen in HIV− but not in HIV+ subjects. We then correlated the imaging findings in a subset of HIV− subjects (n = 17) to CSF levels of neurofilament light chain (NFL), reflective of axonal damage and sCD27, known to best predict the presence of intrathecal T-cell mediated inflammation. We found that ependymitis on brain MRI was the best predictor of higher log(sCD27) levels and choroid plexitis was the best predictor of higher log(NFL) levels. The availability of predictive imaging biomarkers of inflammation and neurological damage in HIV− subjects with CNS cryptococcosis may help gauge disease severity and guide the therapeutic approach in those patients. The prevalence of the infection in previously healthy, HIV-negative (HIV−) patients, on the other hand, has been persistent2 even in developed countries where it accounts for almost a third of the cases3 and is associated with 30% mortality despite optimal therapy5. In contrast to HIV+ patients who have defective cell immunity, previously healthy, HIV− patients with CM and clinically refractory disease show a strong intrathecal expansion and activation of innate and adaptive immunity cells despite effective microbiological control. Activated T-cells include primed CD4+ T-cells that recognize cryptococcal antigen and express high levels of IFN-γ, accompanied with increased cerebrospinal fluid (CSF) levels of neurofilament light chain (NFL), reflective of axonal damage5.Central nervous system (CNS) cryptococcal meningoencephalitis (CM) remains a common opportunistic infection in HIV positive (HIV+) patients, especially in developing countries with limited access to antiretroviral therapies although its prevalence has been declining in the US and other developed countries6 creates a need for readily available clinical biomarkers of inflammation and neuronal damage that can be used in combination with microbiological biomarkers, such as early fungicidal activity8, to individualize and guide therapy. The imaging findings of CM have been described in HIV+15, and HIV− patients23, including meningitis, meningoencephalitis, hydrocephalus, isolated cryptococcomas and disseminated cryptococcosis. Additional findings in HIV− subjects include choroid plexitis and ependymitis as well as pachymeningeal enhancement and ischemic infarcts26. There has been however no direct comparison of imaging findings between the two populations and no attempts at correlating MR imaging findings with CSF indicators of inflammation and lymphocyte activation.Assessing responsiveness to immunosuppressive agents including corticosteroids27. To provide a broader clinical context, we also compared these imaging manifestations to those of HIV+ subjects presenting with CM to better understand differences in disease pathophysiology between the two populations.The present studies seek to identify imaging biomarkers that would be most predictive of CSF lymphocytic activation and inflammation patterns in the HIV− population, by correlating MRI findings with cerebrospinal fluid (CSF) levels of NFL and a soluble marker of T-cell activation, sCD27, recently shown to best predict the presence of intrathecal T-cell mediated inflammation compared to other biomarkersC. neoformans at the time of the imaging.The mean age for the previously healthy HIV− subjects was 49.1 +/− 12.8 years at the time of scanning while the mean age for HIV+ subjects was 39 +/− 9.9 years. In the HIV− group, only four out of 45 patients were on steroids at the time of the scanning  and viral load varied from <50 to 1.1 million copies/ml. Five out of 11 subjects were on Fluconazole treatment and 5 were on antiretroviral therapy.In the HIV+ group, 10 out of 11 patients had positive cryptococcal antigens titers in the CSF (range from 1:2 to 1:1024) with no titer measured for one patient  analysis with natural log(sCD27) as the y-variable, and considering the group and all seven imaging findings as x-variables, the best set of predictors was documented CM , ependymitis and hydrocephalus with respective p-values of 0.034, 0.012 and <0.003. Interestingly, when only assessing patients with documented CM (eliminating group), and excluding prominent perivascular spaces  and hydrocephalus (cannot be appropriately assessed in patients with ventricular shunts) from the list of x-variables, the best predictor of higher sCD27 levels was found to be ependymitis with p-value of 0.0017.For natural log(NFL) values in patients and controls, on the other hand, the “best” set of predictors was found to be a history of CM (group) and hydrocephalus with respective p-values of 0.007 and 0.002. When evaluating the patient group separately and excluding prominent perivascular spaces and hydrocephalus, as above, choroid plexitis became the best predictor of higher NFL levels although this did not reach statistical significance .3 and is associated with significant (~30%) mortality despite optimal therapy5. MR Imaging of the brain is usually one of the earliest diagnostic examinations in this setting and many therapeutic decisions are based on the intracranial imaging findings.CNS cryptococcosis in previously healthy HIV− subjects accounts for almost a third of the cases in developed countries5. Similarly, sCD27 levels have been recently shown to best predict the presence of intrathecal T-cell mediated inflammation compared to other biomarkers27 and suppression of sCD27 has been associated with clinical improvement in CNS cryptococcal disease6. Therefore, to better gauge the pathophysiology of this inflammatory reaction using non-invasive biomarkers, we used MRI to identify the imaging findings most related to elevations in NFL and sCD27 levels in the CSF. Using a GLM analysis with imaging variables as x-variables and cytokine levels as y-variables, ependymitis and hydrocephalus were the strongest predictors of high log(sCD27) levels in the presence of symptomatic CM disease while hydrocephalus and a history of overt CM best predicted higher log(NFL) levels. However, since hydrocephalus cannot be appropriately evaluated post shunting and since prominent basal ganglia perivascular spaces are generally irreversible, and could sometimes reflect a normal variant, we performed a second GLM analysis excluding the two factors as potential predictors (x-variables). This assessment would potentially apply for patients being followed up after shunt insertion. In this analysis we found ependymitis to be the best predictor of higher sCD27 levels and choroid plexitis to be the best predictor of higher NFL levels  rather than microbiological failure since the majority of the patients had negative fungal CSF cultures. Those inflammatory changes have been previously seen in association with elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damageels Fig. . These r5. Despite effective microbiological control, the expanded CSF T-cells included a majority of cryptococcal-antigen specific CD4 + T-cells, expressing high levels of IFN-γ, a pro-inflammatory cytokine. At the same time, while macrophage recruitment appeared intact, M2 macrophage polarization resulted in poor phagocytosis of fungal cells5. The ventricular involvement in HIV− subjects with CM is probably related to the mechanism of pathogen spread to the brain. As the interface between the CSF and systemic circulation, the choroid plexus is suggested as an important portal for entry of Cryptococcus. Anatomically, the papillary fronds of the choroid plexus protrude into the ventricles and have an epithelial lining that is continuous with the ependymal surface29. This could explain the co-occurrence of choroid plexitis and ependymitis in our HIV− patient population. Historically, a similar ependymal reaction has been reported experimentally in reaction to cryptococcal polysaccharide30. In patients, the ependymal reaction may be happening because of release of cellular material after treatment with antifungals. Along the same lines, higher rates of hydrocephalus in the HIV− population upon presentation can also be potentially explained by the inflammatory exudates obstructing CSF drainage at the level of the arachnoid granulations.Besides showing higher rates of ventricular manifestations, more HIV− patients presented with hydrocephalus (51.1%) compared to HIV+ patients (9.1%). The reason for this discrepancy in imaging findings between HIV+ and HIV− patients could potentially reflect a different mechanism of CNS spread although a more likely explanation is a difference in the immune reaction induced at the site of spread, in this case the blood-CSF barrier. While T-lymphocyte defects in HIV+ patients may explain vulnerability to cryptococcal (and other opportunistic) infections, the mechanism of susceptibility to CM in the HIV− population is not fully understood. A recently published detailed immunological study of HIV− infected patients with normal CD4 counts and refractory CM demonstrated that rather than showing T-cell immunity deficits, there was strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR + CD4 + and CD8 + T-cells and NK cells31 although the effectiveness and cost-benefit ratio remain debatable34. Irrespective of the treatment strategy, however, gauging treatment for PIIRS requires diagnostic certainty and repeated lumbar punctures can be prohibitive especially in clinically unstable patients. In addition, research markers such as sCD27 and NFL may not be readily available in most centers. Our study suggests enhanced brain MRI as a surrogate non-invasive imaging approach for following up HIV− patients with cryptococcal meningitis, which might prove to be of use in specific clinical scenarios. Besides commonly recognizable prognostic features such as hydrocephalus, increased lesion load or worsening meningeal enhancement, ependymitis and choroid plexitis appear to be especially reflective of immune activation patterns and could guide treatment in complex follow-up cases.The management of cryptococcal meningitis in HIV− patients is not straightforward, and controlling the ensuing PIIRS can be very challenging. One suggested treatment approach is the use of corticosteroids Limitations of our study include a small number of HIV+ patients, as well a slightly heterogeneous HIV− patient population as far as CSF antigen titers, microbiological control and treatment. We did however exclude HIV− patients who had received immunosuppressive regimens in order to better control for patient heterogeneity. Another limitation is possible referral bias with more severe cases evaluated in this cohort, which makes our results not fully applicable to all HIV− subjects with cryptococcal meningitis. However, this is exactly the cohort of subjects that would benefit most from our findings considering the inherent high morbidity and mortality figures associated with severe disease. In addition, we were not able correlate MRI findings to clinical outcome because of the few numbers of adverse events (1 death) in the HIV− cohort, perhaps due to the routine use of corticosteroids and immunosuppressive therapy in refractory cases. Larger validation cohorts may thus be required for further validation.41), in the correct clinical setting, their presence on imaging should raise the suspicion for CM in HIV− patients. After diagnosis, such findings may provide an effective and readily available biomarker of inflammation in the setting of microbiological control that could help manage the subset of patients with clinical deterioration.Finally, even though choroid plexitis and ependymitis are not specific for cryptococcal CNS involvement . All methods were carried out in accordance with relevant guidelines and regulations.A total of 56 patients diagnosed with CM were evaluated. Forty five patients were previously healthy HIV− and 11 were HIV+. In addition, 5 HIV− patients with pulmonary but no CM  were evaluated with imaging and CSF cytokine levels.C. neoformans were recorded. In the HIV+ group, CD4 cell counts and plasma viral loads were measured at the time of imaging.Treatment information for antifungals (either Amphotericin B or Fluconazole), antiretroviral therapy and steroids at the time of scanning was collected for both the HIV+ and HIV− groups. In addition, CSF cryptococcal antigen titers and culture results for MR scans were either performed at NIH using 1.5T or 3T Philips MR scanner or on outside basis using a variety of different scanners. All the scans included at least T1, T2, FLAIR, diffusion weighted and enhanced T1 weighted scans. Scans at NIH included post contrast FLAIR images. Only scans with good diagnostic quality were included.Brain MRI findings were stratified into three main categories: parenchymal Fig. , ventricCSF levels of sCD27 and NFL were measured in a subset of the HIV− CM patients at presentation (n = 17), as well as in 5 patients with pulmonary but no documented CM. None of the 17 patients included in this analysis was being treated with corticosteroids at the time of CSF collection.Imaging findings of HIV+ and previously healthy HIV− patients were evaluated using comparison of proportions of the seven main MRI characteristics (dichotomous variables): enhancing basal ganglia lesion(s), non-enhancing prominent perivascular spaces, other parenchymal enhancing lesions , ependymitis, choroid plexitis, pial meningeal enhancement and hydrocephalus. The comparisons were performed using fisher’s exact tests (two-sided).Regression analysis of two CSF cytokines (sCD27 and NFL) and MR imaging characteristics were performed in 17 previously healthy HIV− patients with documented CM and 5 previously healthy HIV− patients with pulmonary but no documented CM. Since the distribution of the measured cytokines (sCD27 and NFL) was skewed, the distributions of their natural log-transform were evaluated and were found to be closer to normal. With the natural log transformation, statistical model assumptions were met. Hence, all subsequent statistical models were based on the log-transform values rather than the absolute values.We explored the association between log-cytokine levels (sCD27 and NFL), and several factors including the group (CM versus no CM) and seven imaging characteristics: enhancing basal ganglia lesion(s) (yes/no), non-enhancing prominent perivascular spaces (yes/no), other parenchymal enhancing lesions (yes/no), ependymitis (yes/no), choroid plexitis (yes/no), pial enhancement (yes/no) and hydrocephalus (yes/no). A repeated-measures mixed model was applied with the log-cytokine level as the response variable (Y-variable) and the factors listed above as potential predictors. The same analysis was repeated in the group of patients with documented CM (n = 17) for each cytokine, using five rather than 7 variables .The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


Acne vulgaris is a common skin disease. Pharmacological modalities for treatment are proven to be efficacious but have limitations. Light therapy for acne vulgaris has shown promise in previous studies. This case report and its accompanying images show how a novel approach of visually augmented high fluence light therapy has been used to good effect.A 26-year-old Caucasian woman with acne vulgaris resistant to treatment with topical therapy underwent three sessions of combination potassium titanyl phosphate laser (532 nm)/neodymium-doped: yttrium aluminum garnet laser (1064 nm) light therapy with visually augmented narrow spot size and high fluence. A 73% reduction in total inflammatory lesions was evident 6 months after the initial treatment.This case report illustrates that there may be utility in this novel approach of narrow spot size, magnification-assisted, high fluence targeted combination laser therapy for inflammatory acne. Acne vulgaris is a common skin disease affecting 90% of people at some time in their lives, to varying degrees. It can have a profound psychological impact on the patient, often out of proportion from the perceived physical effect.Cutibacterium acnes organism that contributes to inflammation in acne [Established medical treatment for acne includes topical application of benzoyl peroxide, topical and oral antibiotics, and systemic retinoids for severe or resistant acne. Although proven to be efficacious, these treatments have disadvantages that limit their use. Systemic retinoids are teratogenic and have systemic side effects, while non-compliance is an issue with topical treatment. There is also the problem of growing antibiotic resistance of the  in acne .There has been recent interest in the role of light therapy in acne. A recent Cochrane review concluded that high quality evidence was lacking, but that treatment with green light was more effective than placebo . There hThe following case illustrates an approach to using phototherapy for acne that uses a novel combination of narrow spot size wavelengths with high energy level phototherapy under visual magnification to achieve a 73% reduction in inflammatory lesions. A literature search of acne phototherapy indicates that this is a unique approach that merits further investigation.A 26-year-old Caucasian woman with Fitzpatrick skin type II presented with facial acne vulgaris. She had a slim build, no evidence of hirsutism, and no significant past medical or family history. Multiple inflammatory lesions , predominantly papules, were present bilaterally, consistent with severe grade 4) acne on the US Food and Drug Administration (FDA) Investigator’s Global Assessment (IGA) scale for acne severity [ acne on Three treatments in total of combination non-ablative laser therapy were applied; each session was held 2 to 3 weeks apart. We used a combination of treatment with high fluence potassium titanyl phosphate (KTP) and neodymium-doped: yttrium aluminum garnet (Nd:YAG) laser.2, 20 millisecond pulse width, 1 mm2 spot size, and 2 Hz pulse rate were used superficially, targeting the oxyhemoglobin within the pilosebaceous unit. Nd:YAG laser settings of 1064 nm, 150 to 250 J/cm2, 55 to 75 millisecond pulse width, and 1 mm2 spot size were used to eradicate the base of a hair follicle on a deeper level of the dermis along with any des-oxyhemoglobin resulting from the more superficial treatment. No topical sensitizer was used. Polarized visual magnification with a 5× headset magnification was used to assist with identification of lesions. External sclera shields were applied to our patient during treatment.KTP laser settings of 532 nm, high fluence 20 J/cmThe procedure was painless. On each occasion following treatment, erythema of the treated area was apparent for approximately 3 days but subsided uneventfully with no blistering. There were no late complications. A 73% reduction in total inflammatory lesions (from 37 to 10) was evident on completion of the three applications when our patient was reviewed 6 months after the initial treatment Fig. . This reThe method of phototherapy used in this case aims to precisely target the infected pilosebaceous unit with a narrow spot size green laser using visual augmentation, thus limiting damage to adjacent tissue. An infrared laser is used to target melanin in a deeper layer and resolve the mottled appearance of des-oxyhemoglobin following superficial treatment.2 fluence treatment of the entire surface of the designated treatment side [et al. used 5 to 12 J/cm2 fluence with a 4 mm spot size. Neither used magnification aid [Studies on the use of green light (495 to 570 nm) in the treatment of acne have treated the entire surface of the designated treatment side, or with a notably larger spot size, no visual augmentation, and lower fluence. Baugh and Kucaba demonstrated efficacy with 12 J/cmtion aid .Combination laser therapy of different wavelengths of the green and infrared spectrum have been shown to be safe and effective in pulsed dye lasers (PDL)  with a pThis case study represents a unique instance of combination KTP laser (532 nm)/Nd:YAG laser (1064 nm) light therapy with visually augmented narrow spot size and high fluence. The outcome of this case indicates that this treatment method is a candidate for further research.Optimal parameters in phototherapy for acne are yet to be established. This case illustrates that there may be utility in this novel approach of narrow spot size and magnification-assisted targeted combination laser therapy for inflammatory acne that merits further investigation.


BRCA2 and rs17879961 in CHEK2 were associated with lung cancer. However, the associations between these two variants and urinary tract cancers risk remain largely unexplored. We applied imputation of three genome-wide association studies published in the database of Genotypes and Phenotypes (dbGaP). Unconditional logistic regression analysis and meta-analysis were performed to assess the association between these two variants and the risk of urinary tract cancers. Our results showed that rs11571833[T] had an effect on urinary tract cancers predisposition , especially associated with increased the risk of bladder cancer . Moreover, rs17879961[C] had a protective effect on the urinary tract cancers  and was mostly associated with a lower incidence of renal cell carcinoma . Together, our study indicates that BRCA2 and CHEK2 play an important role in the genetic susceptibility to urinary tract cancers.Previous studies have shown that two rare variants, rs11571833 in  According to statistics, approximately 429,800 newly diagnosed cases of bladder cancer occurred and caused 165,100 deaths in 2012 worldwide156Genome-wide association study (GWAS) and its corresponding meta-analysis have successfully identified several risk loci and regions, which had small or moderate effect to increase the risk of diseases89111213815BRCA2  and rs17879961 (c.470T > C) in CHEK2 , which have large effect on lung cancer in populations of European ancestryBRCA2 and CHEK2, two important DNA repaired genes, acted a crucial role in response to DNA alterations and involved in maintenance of genome stability181921BRCA2 and CHEK2 that changes amino acid may alter their function of DNA damage repair, which may influence the risk of diseasesBRCA2 and CHEK2 confer susceptibility to multi-organ cancers, such as breast, lung, prostate and bladder cancer2425262728In recent, an imputation-based genome wide association study had identified two rare variants rs11571833 (c.9976A > T) in A total of three GWAS studies including 6,064 cases and 8,661 controls were enrolled in our study. The detailed characteristics of GWAS studies are shown in P = 4.5 × 10−3). Simultaneously, rs17879961[C] was related with decreased the risk of renal cell carcinoma in Illumina 610 array and bladder cancer in Illumina 610 array .Our logistic regression results are applied in combined = 1.70, 95%CI = 1.19–2.42 and Pcombined = 3.6 × 10−3), but showed non-significant association between renal cell carcinoma and prostate cancer . There were no remarkable associations between rs17879961 and the other two cancers  and rs17879961[C] was associated with a significantly lower risk of urinary tract cancers . In conclusion, rs11571833 may play a risk factor in urinary tract cancers predisposition, especially be associated with the risk of bladder cancer. Meanwhile, rs17879961 is associated with decreased risk of urinary tract cancers and mostly associated with the risk of renal cell carcinoma.In consideration of the relative small sample, we attempted to combine the original GWAS data to implement joint analyses. We merged genotype data across each cancer  to increase the sample size. Totally, the sample size is 3591 cases and 4132 controls for bladder cancer, 1322 cases and 3428 controls for renal cell carcinoma, 1151 cases and 1101 controls for prostate cancer. Our results indicated that rs11571833[T] was significantly associated with increased the risk of bladder cancer . In addition, a significant association between rs11571833[T] and bladder cancer was observed in the subgroup meta-analysis  and had a protective effect on renal cell carcinoma  plays an important role in encoding a pluripotent kinase which can induce cell cycle arrest or apoptosis in response to unrepaired DNA damage34CHEK2 and injures binding of principal substrates. The rs17879961 (CHEK2 p.Ile157Thr) substitution may alter its ability to bind p53, BRCA1 and Cdc25A proteins436BRCA2 (breast cancer early onset 2) is a widely known anti-oncogene and associated with the risk of breast cancer and ovarian cancerBRCA2 also involves in the maintenance of genome stability through interacting with RAD51 recombinase, specifically in the homologous recombination pathway for DNA repair394041423444Recently, an imputation study identified two rare variants rs11571833 in In this study, we investigated the associations between rare genetic variants of rs11571833 and rs17879961 and the risk of urinary tract cancers  and found that the rare variant rs11571833 (c.9976A > T) was associated with increased risk of urinary tract cancers, especially associated with bladder cancer. Concurrently, rs17879961 (c.470T > C) played a protective role in the urinary tract cancers carcinogenesis and mostly decreased the risk of renal cell carcinoma. Our meta-analyses result show highly consistency with joint analyses, which strengthen our conclusion that the rs11571833 is associated with increased the risk of urinary tract cancers and rs17879961 reduces urinary tract cancers predisposition. Interestingly, association signals of these two variants in urinary tract cancers  are in the same direction to lung cancer . It is noteworthy that the rare variants rs17879961 may impair function of CHEK2, but it was associated with decreased the risk of renal cell cancer. A speculation for the possible protective mechanism is that CHEK2 can have two opposite effects on damaged stem cells and it impedes stem cell division until DNA damage has been repaired or actives apoptosis if unrepaired DNA damage happened. Accumulating evidence has shown that in the circumstance of continued DNA damage by long-term exposure of tobacco, the normal stem cell defenses that involve CHEK2 can be attenuated by reducing the CHEK2 activity as a result of rs17879961 (p.Ile157Thr)1732There are several limitations in our study. Firstly, the available GWAS data published in dbGaP database provides relatively little samples, which may influence the power to identify the association between the rare variants and diseases. Furthermore, our analysis based on public database is short of relevant demography information applied in stratification analysis. Additionally, our study is an imputation-based analysis from public database and the ORs of two rare variants are relatively modest. Besides, the sample sizes of controls are unevenly matched with cases in some cohorts, which may result in sample bias. Further large well-designed studies in other independent populations and functional studies are needed to validate our findings. Meanwhile, the interaction between environmental exposures and genetic susceptibility should be also considered in the future researchBRCA2 and CHEK2 play an important role in the genetic susceptibility to urinary tract cancers.In conclusion, our imputation analysis results indicated that the rare variant rs11571833 (c.9976A > T) showed an effect on urinary tract cancers predisposition, especially associated with increased the risk of bladder cancer. However, rs17879961 (c.470T > C) may play a protective role in the urinary tract cancers carcinogenesis and notably decrease the risk of renal cell carcinoma. Association signals of these two variants in urinary tract cancers are in the same direction to lung cancer. These results suggest that GWAS data from three cancer studies were available, which were requested from dbGaP. Two genotype datasets were excluded because of low-density genotype panel and low sample size . In the final analysis, we included three bladder cancer studies, three renal cell carcinoma studies and one prostate cancer study, totally including 6,064 cases and 8,661 controls.P < 1.0 × 10−6; (4) A/T or C/G SNPs with MAF >0.45.First, we applied the LiftOver tool to convert study data from earlier genome builds (NCBI build 36) to NCBI build 37. Few SNPs that failed in the LiftOver were excluded from the imputation. Before performing imputation, we filtered the data to remove low-quality variants and individuals to increase the accuracy of the results. For individuals, (1) we excluded those missing rate per person >5%; (2) excluded samples with ambiguous gender; (3) additional duplicates or probable relatives were excluded according to PI_HAT > 0.5; SNPs filter criterion : (1) completion rate per locus <95%; (2) minor allele frequency <0.05; (3) Hardy-Weinberg Proportion for autosomal SNPs in control population with We used SHAPEIT, a highly accurate phasing algorithm, to infer the haplotypes underlying genotype dataI2 statistics. I2 values ≥ 75% were considered to be significant heterogeneity. All statistical analyses were two-sided, and a P value < 0.05 was considered statistically significant.An unconditional logistic regression model was applied to calculate the odds ratio (OR) and 95% confidence interval (CI) for each SNP in an additive model using SNPTEST software. We adjusted for some covariates such as age, gender, study and significant principle components (PCs). The principal component analysis (PCA) was carried out to assess for population stratification of study cohorts. Meta-analysis was conducted by Stata v.10 . Heterogeneity was evaluated by Cochran’Q and How to cite this article: Ge, Y. et al. Rare variants in BRCA2 and CHEK2 are associated with the risk of urinary tract cancers. Sci. Rep.6, 33542; doi: 10.1038/srep33542 (2016).