CHSY1, we identified a candidate causal variant (rs9920291) overlapping an open chromatin region. Reporter assays demonstrated that rs9920291 shows allelic regulatory activity, and CRISPR/Cas9 targeting of human chondrocytes demonstrates that the region regulates CHSY1 expression. Thus, integrating biologically relevant epigenetic information  with genetic association results can identify biological mechanisms important for human growth.GWAS have identified hundreds of height-associated loci. However, determining causal mechanisms is challenging, especially since height-relevant tissues (e.g. growth plates) are difficult to study. To uncover mechanisms by which height GWAS variants function, we performed epigenetic profiling of murine femoral growth plates. The profiled open chromatin regions recapitulate known chondrocyte and skeletal biology, are enriched at height GWAS loci, particularly near differentially expressed growth plate genes, and enriched for binding motifs of transcription factors with roles in chondrocyte biology. At specific loci, our analyses identified compelling mechanisms for GWAS variants. For example, at  Humans vary considerably in height, a trait that is partly inherited from each individual's parents. Studies have identified hundreds of small changes in DNA that contribute to differences in human height. These small changes often swap out just one of the four letters that make up the DNA code. Some changes occur within genes, yet most occur in stretches of DNA that do not contain genes. These stretches of DNA likely control whether genes important for the growth of the skeleton are switched on or off.Cartilage cells, also known as chondrocytes, are important for height. These cells are found near the end of bones in the growth plates, where the bones grow during childhood and adolescence. The on and off switches for growth genes in chondrocytes are unknown. Identifying these genetic switches could help scientists understand how hundreds of small changes in DNA help determine how tall a person will be.Now, Guo, Liu, Willen et al. identify thousands of switches that turn on and off genes in chondrocytes. In their experiments, chondrocytes were removed from mouse bones and a type of genetic sequencing called ATAC-seq was used to identify the stretches of DNA that act as on/off switches for genes in these cells. Further analysis revealed that many of these same on/off switches occur in human chondrocytes too. Importantly, the experiments showed that many of the small changes in DNA that contribute to differences in human height are also found within these DNA switches.Guo, Liu, Willen et al. next tested how these switches and height-linked genes interact, and found, for example, that one switch acts to shut off the gene for a protein called chondroitin sulfate synthase 1. People with mutations in this gene can have unusually short stature. However, people with DNA changes in its nearby switch can be taller or shorter than average based on how the DNA influences the switch. More studies might help scientists understand the evolutionary significance of these DNA changes. They will also help determine if the genetic switches in chondrocytes contribute to diseases that affect the bones and joints, like bone cancer or arthritis. Human height, a main outcome of skeletal growth, is the product of many biological and environmental interactions spanning numerous cell and tissue types acting during pre- and post-natal development. The growth plate, located at the distal ends of long bones such as the femur, is among the most important tissues influencing height. In the growth plate, condensations of mesenchymal cells differentiate into chondrocytes in the process of endochondral ossification. Chondrocytes in the growth plate reside in longitudinally oriented columns divided in zones  each with important functions in allowing the bone to elongate and mature see . This prHeight has been studied for centuries as a model genetic trait, as it is easily measured and highly heritable  . Height Recently, genome-wide association studies (GWAS) have been used to study the genetics of height, as well as many other traits and disorders . This apHowever, as with all GWAS, moving from genetic associations to biological mechanisms at any individual locus can be challenging. There are two primary reasons for this difficulty. First, there is extensive linkage disequilibrium (LD) in the human genome, meaning that the genotypes at nearby genetic variants are often tightly correlated . The conAlthough the large number of associated variants offers advantages in making biological inferences for the set of associated variants as a whole , defininIn this study, we profile the epigenetic landscape of mouse growth plate chondrocytes using a new approach called ATAC-seq , 2013, wIn order to identify open chromatin regions involved in long bone development, we performed ATAC-seq , 2013 onSince a main goal was to understand the gene regulatory functions of variants influencing human height, we next mapped the locations of each mouse femur open chromatin region to the human genome . Our liftover resulted in 24,805 proximal femur peaks and 24,788 distal femur peaks, representing approximately 87.7% and 88.6% of sites identified in mouse, respectively. These sites that were capable of liftover to human likely reflect relatively conserved genes involved in vertebrate musculoskeletal development . The uniTo determine whether ATAC-seq peaks are enriched near gene sets/pathways with specific functions, we performed an enrichment analysis using Genomic Regions Enrichment of Annotations Tool (GREAT) , which iCol2a1, Fgfr1, and Pth1r   Ohba et. We founWe also wanted to determine if our ATAC-seq peaks reflect regulatory elements involved in human limb development and chondrocyte biology. First, we used a ChIP-seq dataset comprised of H3K27ac peaks found in E47 human limb buds  that are shared with mouse limb buds . We founOur ATAC-seq data were also able to identify 14 of 26 previously-discovered active regulatory elements residing near key cartilage genes detected at different time-points and using different assays e.g. see . This su2 >0.5 to the lead SNP based on the European subset of 1000 Genomes Phase 3  contained at least one variant that overlapped with an ATAC-seq peak. To test the significance of the observed overlap, we applied GoShifter , which performs a shuffling-based approach to evaluate the overlap between an epigenetic data set and GWAS data. We found strong enrichments for the ATAC-seq peak set and height GWAS data (p=0.0059) . We repeTo further demonstrate the specificity of our ATAC-seq peaks for capturing height GWAS signals, we repeated the GoShifter analysis by examining the enrichment of GWAS results for other complex traits/diseases at our ATAC-seq peaks. These other traits/diseases were chosen as they have no obvious connection to height and included age of menarche , body maAs a second approach to evaluate overlap between our ATAC-seq peak set and height GWAS data, we generated 1000 sets of random loci which were matched to the actual height GWAS loci based on factors including SNP density, minor allele frequency, number of proxy SNPs in the locus, and gene density  . For eacNotably, we observed that the actual number of SNPs that overlap an ATAC-seq peak for each set was higher than the number of represented loci. In total, there were 928 variants that overlapped with an ATAC-seq peak; these 928 variants were distributed across 317 loci . This fi2 >0.5 to the lead variant based on the European subset of 1000 Genomes Phase 3  and found that 46/688 loci (or ~6.7%) contained at least one GWAS variant that overlapped with an ATAC-seq peak and resided near a differentially expressed gene , transcription factors (e.g. RUNX2), extracellular matrix factors , among other factors involved in the growth plate in both humans and mice.Of the 46 loci in the ATAC-seq femur comparison that fall near genes differentially expressed in growth plates, 44 different genes have been identified as possibly being modulated by these putative regulatory variants  . These gWe next examined some of the most compelling loci from a genetic and mechanistic perspective. We filtered for loci where a height GWAS variant in the PICS 95% credible set overlapped an ATAC-seq peak, and was near a differentially expressed growth plate gene (±100 kb). In total, 59 variants distributed across 26 loci met these criteria. These 59 variants thus have strong evidence for being potentially causal variants and might be prioritized for downstream study .CHSY1 locus, four variants  met these criteria. Disruption of Chsy1 in mice and humans leads to severe skeletal phenotypes including long bone length reductions   and a 3’UTR SNP (rs11433), we demonstrate that rs9920291 displays allelic skew in expression  . In addiCHSY1, we performed CRISPR-Cas9 targeting of a 135 bp sequence spanning a conserved portion of the regulatory element containing rs9920291 (guides sg1 and sg4), or an 17 bp sequence immediately surrounding rs9920291 (guides sg6 and sg8). Either deletion led to a significant upregulation of CHSY1 expression in vitro in T/C-28a2 cells  at the h plates . We have activity. Thus, oAtf3 . First, we performed a de novo motif analysis on the union of distal and proximal femoral ATAC-seq peaks in mouse to identify enriched transcription-factor-binding motifs. We identified a number of highly enriched motifs relevant to chondrogenesis and other skeletal developmental processes, including Atf3 , Msx2 , will be important factors shaping height variation in humans.While our datasets capture aspects of general chondrocyte biology, we also found that ATAC-seq peaks acquired on femoral tissues show enrichment in human phenotypes related to the anatomy of femur. This finding points to a fine-grained, modularized control of skeletal element-specific biology. This is also supported by our independent identification of a number of previously published long bone growth plate chondrocyte enhancers in mice and humans, as well as the growing evidence that a number of important skeletal development genes, such as Bmp5 , Gdf6  locus. Human mutations in CHSY1 result in Temtamy preaxial brachydactyly syndrome, characterized by short stature, preaxial brachydactyly, and hyperphalangism of digits 1–3 , as well as a number of additional soft and hard tissue disorders. rs9920291 is in the fine-mapped credible set, resides in regulatory elements active in human adult chondrocytes and embryonic limb tissues, and displays evidence of binding by Sox9 in mouse chondrocytes. rs9920291 additionally modifies a critical nucleotide (C to T) and disrupts the experimentally determined in vitro binding motif of HOXD13, a homeodomain transcription factor that is expressed in chondrocytes  resides in the drocytes  and has drocytes . We perflidation .A number of other variants, listed in the browsable All experiments were performed following protocols approved by the Harvard University IACUC committee. Timed matings were established between FVB mice, and pregnant females were euthanized at E15.5 in order to acquire embryonic long bone growth plates. Embryos were dissected in PBS1X on ice under a dissection scope and the proximal and distal growth zones of the right and left femur were stripped clear of soft tissues. Each proximal or distal cartilaginous end was then micro-dissected from the bony diaphysis  and sepaBuenrostro et al., 2015Next, samples were subjected to PCR amplification and barcoding following Buenrostro et al., 2015http://homer.salk.edu/homer/motif/).Sequencing yielded approximately 400 million reads per lane and an average of 100 million per sample. Quality control statistics are presented in Raw sequencing fastq files and processed peak bed files have been deposited on NCBI GEO (GSE100585).http://great.stanford.edu/) were used. GREAT v3.0.0 was used.GREAT (Genomic Regions Enrichment of Annotations Tool)  identifiMouse chondrocyte data: ChIP-seq profiles Sox9 binding in mouse rib chondrocytes was obtained from http://egg2.wustl.edu/roadmap/web_portal/processed_data.html  (ENCSR000COE), liver male adult (8 weeks) (ENCSR000CNI), and lung male adult (8 weeks) (ENCSR000CNM). Processed NarrowPeak bed files were downloaded for each tissue and then lifted over to hg19 as described above.Mouse ENCODE data: DNA-seq data were downloaded from the mouse ENCODE project . All sam2 >0.5 to lead SNP).All intersection analyses of ATAC-seq peaks with GWAS loci, genes, or ChIP-seq peaks were performed using BEDTools v2.18  and custFor overlap of ChIP-seq datasets, any overlap (>=1 bp) was considered to represent the same feature. To evaluate the significance of overlap, a permutation-based procedure was implemented in the R package regioneR . We used2 >0.5 to the index SNP was used. A window size of 100 kb was used to find LD SNPs (‘window’ option). All other parameters were set to the software default. 100,000 permutations were run. GoShifter analyses for additional traits/diseases were performed in the same fashion.GoShifter v0.2 was used to evaluate the statistical enrichment of the overlap between height GWAS loci and ATAC-seq peaks (or other epigenetic peaks) . The Eur2 > 0.5 to the index SNP. Loci were matched using default criteria: MAF ± 5%, gene density ± 50%, distance to nearest gene ±50% and LD buddies ± 50%. SNPSNAP was able to generate matched gene sets for 676 out of the 697 loci from SNPSNAP  was usedA list of 427 differentially expressed mouse genes was downloaded from Using the set of 427 differentially expressed growth plate genes (see above), we identified 157 genes that were also genes for cis-eQTLs in whole blood (FDR < 0.05) as identified by The Genotype-Tissue Expression (GTEx) Project  was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 10/27/2017.ATAC-seq peak centers from proximal/distal tissues were aggregated and padded outwards to a fixed length of 200 bp (based on the distribution of ATAC-seq peak sizes). HOMER  de novo Lifted over human ATAC-seq peaks were then intersected with height GWAS variants from To assess enrichment of predicted transcription factor binding near variants intersecting ATAC-seq peaks, a set of hg19 DNA sequences was first constructed using 30 bp windows centered on each variant, generating an ATAC-intersected subset and whole GWAS superset. A utility from HOMER (findmotifs.pl) was then used to scan this sequence set using its provided vertebrate motif library to search for motif hits. Motifs from JASPAR 2016 were filtered (386 motifs) and counted within each of the two sets. Hypergeometric tests were performed for all motifs using Benjamini-Hochberg FDR correction .CRISPR-Cas9 PX458 vector was obtained from Addgene . pGL4.23 luciferase and pGL4.74 renilla vectors were acquired from Promega . Plasmids encoding HOXD13-FLAG and HOXD13-HA were generously gifted from Dr. Vincenzo Zappavigna (University of Modena and Reggio Emilia) .2 at 37°C in Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (P/S). Media was replaced every 2–3 days and the cells were sub-cultured every 5 days.Human embryonic kidney (HEK-293FT) cells were acquired from Dr. Pardis Sabeti (Harvard University and Broad Institute). T/C-28a2 human chondrocyte cells were acquired from Dr. Li Zeng (Tufts University) courtesy of Dr. Mary Goldring . Both cell lines were cultured at 5% COCHSY1 regulatory element or rs9920291 were designed using MIT CRISPR Tools (http://crispr.mit.edu), synthesized by Integrated DNA Technologies, Inc , and cloned into the PX458 vector following published protocols  with a modification efficiency of approximately 10% and smaller CHSY1 regulatory region around rs9920291  with a modification efficiency of approximately 8.5%. RNA was also extracted from control and CRISPR-Cas9 targeted T/C-28a2 cells. RNA was DNase-treated and converted to cDNA for qPCR (see below).After confirmation that sgRNAs worked in HEK-293FT cells, we performed in vitro deletion in the T/C-28a2 chondrocyte cell line . The samTotal RNA was extracted from HEK-293FT and T/C-28a2 cells and prepared using the Trizol Reagent  and Direct-zolTM RNA Miniprep kit (ZYMO). Three micrograms of total RNA were used to synthesize first-strand cDNA using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). qRT-PCR analysis was then performed with specific primers and Applied Biosystems Power SYBR master mix (Thermo Fisher Scientific) with GAPDH as a reference gene. Primers used for qRT-PCR are listed in CHSY1 regulatory sequences into HEK-293FT and T/C-28a2 cells (see below), primers containing KpnI/HindIII linker sequences were used to amplify the putative regulatory element surrounding rs9920291.  at a Lipofectamine:DNA ratio of 4:1 according to manufacturer instructions. Luciferase activity was measured 48 hr after transfection using the 96-welll Dual-Glo Luciferase Assay System (Promega E2940) following manufacturer protocols on a SpectraMax L Microplate Reader  with a 1 min dark adapt, 5 s integration, and max range settings. For cell culture experiments, to compare expression between the reference and alternate allele CHSY1 constructs (HEK-293FT cells and T/C-28a2 chondrocytes were passaged using standard conditions (see above) prior to reporter gene transfection and HOXD13 over-expression experiments. Prior to transfection, cells were first cultured in DMEM with 10% FBS for 24 hr in 96-well dishes at a seeding density of 3 × 10nstructs , we perfhttp://hek293genome.org/v2/) that HEK-293FT cells were heterozygous at rs9920291, unlike T/C-28a2 cells which were homozygous for the reference C allele. Thus, only HEK-293FT cells could be used for allele-specific expression. Next, using the HEK-293 browser with follow-up verification using Sanger sequencing, we identified two heterozygous transcript variants (exon 3 coding variant [rs28364839] and 3’UTR variant [rs11433]).Both HEK-293FT cells and T/C-28a2 chondrocytes were screened using Sanger sequencing to identify genotype rs9920291. Sanger sequencing results confirmed preliminary findings using the HEK-293 browser . Samples were then run on a bioanalyzer to achieve RNA Integrity Numbers greater than 8. These RNA samples were then reverse transcribed using a SuperScript IV First Strand cDNA Synthesis Reaction kit  following manufacturer’s recommendations.For allelic skew analyses on untreated HEK-293FT samples, five independent biological replicates were grown and harvested. RNA was isolated separately from each sample using the Trizol Reagent  and the RNA Clean and ConcentratorCHSY1 (rs11433 and rs28364839), were validated by EpigenDx . Pyrosequencing for SNP genotyping  is a real-time sequencing-based DNA analysis that quantitatively determines the genotypes of single or multiple mutations in a single reaction. Briefly, 1 ng of sample cDNA was used for PCR amplification. PCR was performed using 10X PCR buffer  at 3.0 mM MgCl2, 200M of each dNTP, 0.2 µM each of the forward and reverse primers (available through EpigenDx), and 0.75 U of HotStar DNA polymerase (Qiagen Inc.), per 30 µl reaction. PCR cycling conditions were: 94°C 15 min; 45 cycles of 94°C 30 s; 60°C 30 s; 72°C 30 s; 72°C 5 min. One of the PCR primer pairs was biotinylated to convert the PCR product to single-stranded DNA sequencing templates using streptavidin beads and the PyroMark Q96 Vacuum Workstation. Ten microliters of the PCR products were bound to streptavidin beads and the single strand containing the biotinylated primer was isolated and combined with a specific sequencing primer (available through EpigenDx). The primed single stranded DNA was sequenced using the Pyrosequencing PSQ96 HS System (Qiagen Pyrosequencing) following the manufacturer’s instructions (Qiagen Pyrosequencing). The genotypes of each sample were analyzed using Q96 software AQ module (Qiagen Pyrosequencing). Pyrosequencing results for each CHSY1 exonic or 3’UTR SNP were used to calculate the allelic ratios in the heterozygous state.cDNA samples were then sent to EpigenDx for allelic skew analysis assay design and execution. SNPs in the coding regions of For regulatory element transfection experiments, CRISPR-Cas9 deletion experiments, and HOXD13 over-expression experiments, the means ± SEM of multiple independent measurements were calculated. The unpaired two-tailed Student’s t test was used to determine the significance of differences between means. P values smaller than 0.05  were considered to be statistical significant.CHYS1 we used UniProbe (http://the_brain.bwh.harvard.edu/uniprobe/browse.php) and identified the corresponding z-score for each 8-mer.To find upstream transcription factors (TF) predicted to bind at rs9920291 near UniProbe . UniProbUniProbe . For twoUniProbe . To idenhttp://genome.ucsc.edu/cgi-bin/hgVisigene), Eurexpress (http://www.eurexpress.org/ee/), Genepaint (http://www.genepaint.org/Frameset.html), and the Mouse Genome Informatics expression and phenotypic databases (http://www.informatics.jax.org). HOXD13 met the above criteria. HOXD13 had enrichment scores below the recommended UniProbe threshold of 0.4 for the REF sequence but much higher (above 0.4) for the ALT sequence, and this corresponded to a significant change in z-score  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your article "Epigenetic profiling of growth plate chondrocytes sheds insight into regulatory genetic variation influencing height" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:All the reviewers were enthusiastic about your comprehensive generation of epigenetic data for the growth plate and the potential utilization for future studies on genetic regulatory regions. The ATAC-seq approach and the overlay of GWAS data will provide significant insights into murine and possibly human growth determinants.There were three major concerns that need to be addressed before a final decision can be made.First delineation of sex from the E15.5 murine chondrocytes and justification for the E15.5 time point are essential for understanding the framework for growth variants and for future studies by other investigators.Second, the manuscript still lacks a strong connection to chondrocyte biology as none of the links established by the data directly address (or explain) chondrocyte proliferation or hypertrophy and genes well known to regulate them positively or negatively .Third, although different genes have been identified as possibly being putative regulatory variants there are no experimental or computational evidence implicating the ATAC-seq peaks in regulating the nearby growth plate genes, nor altering transcriptional regulatory activity.These issues underlie a perception that, independent of your Nature Genetics paper, there is a lack in this manuscript of mechanistic evidence that the epigenomic profiles can be linked to functional correlates. For example, there is a suggestion that rs9920291 disrupted HOXD13 binding, but there is no evidence that HOXD13 actually binds this site at the endogenous locus. As computationally predicted TF motifs are prevalent in the genome, it is hard to assess whether this SNP/motif overlap is biologically meaningful. This requirement for a mechanistic/functional connection may require additional work.These major points will need to be addressed in the revision along with several other points imbedded in the three reviewers’ comments that have been included below in the comments, and should be considered carefully.Reviewer #1:In this paper, Capellini and colleagues utilize a novel method (ATAC-seq) to read the open chromatin regulatory landscape of growth plate chondrocytes in neonatal mice. They were then able to analyze height GWAS data in the context of femur growth to gain new insights into how height GWAS variants act at the growth plate. Briefly they showed that ATAC-seq datasets generated from mouse growth plate chondrocytes overlap with previously identified signals of chondrocyte biology in the mouse, as well as in humans. They identified locations of active regulatory enhancers and Sox9 binding sites as assessed using ChIP seq on mouse rib chondrocytes and provided datasets that overlap with evidence of regulatory control during chondrogenesis and human limb embryogenesis. Their work is a nice supplement to their Nature Genetics paper identifying an ancestral regulatory variant in 'Grow'1 enhancer in mouse limbs that also affects human chondrocyte biology. Finally, the authors detected a variant in the chondroitin sulfate synthase 1 gene which might influence binding of HOXD13, a trans acting regulator of chondrocyte growth.Strengths: There are several strengths to this paper: First the authors were able to provide very comprehensive data sets on the epigenetic landscape of mouse, and human chondrocytes, an important breakthrough in an otherwise very difficult organ to characterize. Second, they have provided evidence that these regions are critically important for growth of the chondrocyte and thus provide support from previous studies of candidate genes in chondrogenesis such as GDF5, 6, Bmp5 etc. that others have defined. Third they have been able to overlay height GWAS variants that are also significantly enriched in growth plate chondrocyte regulatory regions, particularly for those that are intergenic. Finally, this is state of the art technology in epigenetics; i.e. the use of ATAC methodology, importantly it requires fewer chondrocytes to peruse the chromatin landscape thus breaking new ground.Limitations: – there are some concerns that need to be fully addressed:1) The authors used E15.5 embryos from FVB mice to study the epigenetic regions for growth plate regulation. But inbred strains often exhibit numerous nucleotide variants; since C57BL6J mice are the most frequently utilized strain for in vitro and in vivo chondrocyte (and osteoblast) studies, have the authors determined whether the specific nucleotide sequence in the CHYS1 variant, rs9920291, is also present in B6 mouse chondrocytes?2) Sex is a major determinant of height particularly early in life and during puberty. The early influences of sex steroids likely begins during late embryonic development; i.e. after E11.5 when SRY is expressed in the males, but not females. It would be important to fully understand the epigenetic landscape in respect to sex differences in vivo. Hence, the embryos should have been typed for sex, and both male and female embryos should be examined to more fully understand the transcriptional regulation of chondrocyte differentiation.3) Can the authors justify why they did not follow up with functional studies on the CHYS1 variant rs9920291 which they identified as being a cis acting site for HOXD13.4) Reviewer #2:In this study, the authors have performed epigenetic profiling of active chromatin regions in E15.5 mouse embryo femurs to establish possible links with GWAS variants associated with human height previously described by other groups. The regions were found to positively associate with known genes regulating chondrocyte and skeletal biology, particularly those encoding growth plate transcription factors. These and other data lead the authors to conclude that integration of epigenetic information with genetic association data can identify mechanisms important for human height.The study comes from a research group with recognized expertise in genetics and genomics and for important contributions to the field. It also addresses human height that can be severely affected by congenital conditions, thus introducing an element of translational medicine relevance to the work. The extensive data combining epigenetic profiling using ATAT-seq with several bioinformatics and analytical tools provide a wealth of possible links with specific genes and transcriptome data sets from previous studies. Despite these positive assets, the study raises several concerns.While the study is technically strong, it is deficient in skeletal cell and developmental biology, raising questions about the major conclusions reached and specific relevance to human height.The growth plate is certainly the key structure determining long bone and vertebral elongation and thus, body height. It is clearly established that the rates of skeletal growth are determined by chondrocyte proliferation (minor contribution) and chondrocyte hypertrophy (major contribution) in the growth plate. It is also well established that different growth plates within the same organism at any given time "grow" at very different rates, such as proximal and distal growth plates in radius and ulna. None of the links established by the data presented here directly address (or explain) chondrocyte proliferation or hypertrophy and genes known to regulate them positively or negatively . The only links established are to general aspects of chondrocyte and skeletal biology, thus making it very difficult to understand how the links have anything to do with "height". The behavior of the growth plate is controlled by intrinsic mechanisms (see above) as well as systemic factors such as growth hormone. Again, none of the links related to this or other systemic cues.The authors made use of the proximal and distal portions of E15.5 mouse embryo femur and associated perichondrium as starting material for the epigenetic analysis. The tissues were digested into single cell suspension enzymatically before processing for ATAT-seq. There is no explanation of why this developmental stage was chosen and why both ends of the femur were considered. Also, the lengthy enzymatic digestion could have altered gene expression and chromatin configuration.To establish stronger links, the authors considered the most compelling GWAS variants overlapping ATAT-seq peaks, finding 59 variants distributed over 26 loci. The two examples mentioned are CHSY1 and HOXD13 neither of which has established roles in growth plate function. Indeed, most evidence indicates that HOXD13 regulates autopod skeletal patterning and morphogenesis, but not rates of growth.The authors make a strong case that the data can provide insights into the meaning and functional relevance of GWAS correlations, but I do not see how they provide any direct insights into what regulates human height. It may be worth considering an overall change in rationale for the study.Reviewer #3:The article by Guo et al. describes the epigenetic profiling of mouse growth plate chondrocytes to elucidate potential molecular mechanisms through which genetic variants contribute to human height. The authors perform ATAC-seq profiling to identify open chromatin regions from murine femoral growth plates and demonstrate that femur open chromatin are (i) enriched nearby skeletal development genes, and (ii) enriched for human height GWAS loci, above other epigenomic datasets available from mouse and human. While it has previously been shown that height GWAS loci are enriched nearby musculoskeletal genes . Given that r2>0.5 is not a strict LD threshold , is it still meaningful to report overlap statistics using the r2>0.5 set when the PICS 95% credible set is available?1) Substantially fewer GWAS loci overlap ATAC-seq peaks when using the fine-mapped 95% credible variants, instead of all SNPs with r2) The authors state "44 different genes have been identified as possibly being modulated by these putative regulatory variants". However, in the present manuscript the authors do not provide experimental or computational evidence implicating the ATAC-seq peaks in regulating the nearby growth plate genes, and provide limited evidence that any of these overlapping variants can alter transcriptional regulatory activity.3) Following point #2, and to support the authors' claim of identifying "prime targets for functional testing", it would be helpful to know how many GWAS SNP overlaps with femur open chromatin regions the authors believe to be biologically meaningful versus coincidence.4) Similar to point #2, it is premature to state "59 variants thus have strong evidence for being potentially causal variants". For the authors' example of the CHSY1 locus, while there are 4 SNPs in the PICS 95% set that overlap an ATAC-seq peak nearby CHSY1, 5) Subsection “Known and novel targets of human height variants in putative chondrocyte regulatory regions”, first paragraph: The authors suggest rs9920291 disrupts HOXD13 binding, but do not provide evidence that HOXD13 actually binds this site at the endogenous locus. As computationally predicted TF motifs are prevalent in the genome, it is hard to assess whether this SNP/motif overlap is biologically meaningful.6) Wood et al. (2014) found that height GWAS loci were more likely to be eQTLs (in blood) for genes expressed in cartilage. Did the authors examine whether the 468 credible SNPs overlapping femur open chromatin peaks are enriched for eQTL signals? This analysis may strengthen the authors' mechanistic claims. […] There were three major concerns that need to be addressed before a final decision can be made.First delineation of sex from the E15.5 murine chondrocytes and justification for the E15.5 time point are essential for understanding the framework for growth variants and for future studies by other investigators.We thank the Reviewing Editor for summarizing these two main concerns and address each below separately.While we agree that sex is a major determinant of height in humans particularly during puberty, our method of cell collection for ATAC-seq is not yet conducive to examining this aspect of biology. We have optimized our protocol to acquire large numbers of viable chondrocytes that have not been removed from their in vivocontext for extended periods of time.First, we perform our ATAC-seq on freshly acquired chondrocytes, extracted from the embryonic growth plates, and we must pool embryonic long bone ends across a litter to collect enough for each specific growth plate end . In the process, any significant delay in extraction can significantly impact chromatin state, as the cells are no longer fresh and instead are subjected to prolonged exposure to artificial media and in vitro conditions. Genotyping for sex constitutes a major delay in the process.Second, we perform our ATAC-seq on E15.5 embryos, a stage when sex differences are not readily apparent in the lengths of the skeletal elements . However, we intentionally chose E15.5 for a number of reasons (see next point below), most notably our ability to easily extract large numbers of viable chondrocytes from the limited extracellular matrix environment using a brief collagenase digestion step (<2 hours). Our current attempts to extract chondrocytes at much later stages  require at least 12 hours of collagenase digestion, but result in an increased ECM content that interferes with ATAC-seq and more importantly a significant increase in cell death (>25%), which results in low quality and biased ATAC-seq libraries. In addition, the prolonged duration of exposure to collagenase and artificial media in vitro that comes hand-in-hand with studying these later stages likely impacts chromatin profiles. In time, when chondrocyte extraction techniques improve, we plan to extract chondrocytes from single early post-natal mice, genotype each embryo for sex while we perform ATAC-seq, and explore sex-specific differences in growth plate chondrocyte chromatin biology.We chose E15.5 as a stage to explore chondrocyte growth plate biology for a number of reasons:First, growth plates at this stage show a very limited amount of extracellular matrix, which can interfere with the ATAC-seq protocol. For example, extensive extracellular matrix, characteristic of later fetal and post-natal stages, requires long periods of collagenase digestion  to isolate chondrocytes, which in turn has a significant impact on the nature of open chromatin profiles in the resultant extracted cell populations. Increased collagenase digestion also increases cell death which also influences chromatin state biology.Second, growth plates are readily dissected at E15.5 since soft tissues, such as tendon, ligament, and muscle, strip easily off the growth plate using simple mechanical force rather than enzymatic digestions that can alter chromatin state profiles. At later stages, increased enzymatic concentrations and processing times, as well as more mechanical force are necessary to strip soft tissues off of growth plates, and the effects of these treatments on ATAC-seq libraries remainproblematic and unclear.Third, growth plates at E15.5 display all major chondrocyte differentiated cell types  and at the same time lack evidence in and around the epiphysis of secondary ossification center formation. Thus, we when we extract at this stage, we are less concerned about introducing additional cell types .Fourth, we chose to separate both long bone ends because the growth plates of the proximal and distal femur mature at different stages and under different processes, with the former undergoing complex chondrogenesis and osteogenesis patterns not evident in the latter , and because proximal and distal growth plates show evidence of differential growth across a number of species, including humans. Thus, by separating growth plates, we are able to identify ATAC-seq peaks unique to each region and likely important for the marked distal femur elongation seen in humans.Second, the manuscript still lacks a strong connection to chondrocyte biology as none of the links established by the data directly address (or explain) chondrocyte proliferation or hypertrophy and genes well known to regulate them positively or negatively .Col2a1 (encoding a collagen subunit), Pth1r (parathyroid hormone receptor), and Fgfr1 (fibroblast growth factor)new . Our GREctor)new . We now Third, although different genes have been identified as possibly being putative regulatory variants there are no experimental or computational evidence implicating the ATAC-seq peaks in regulating the nearby growth plate genes, nor altering transcriptional regulatory activity.CHSY1 locus.We thank the reviewers for their suggestions of including experimental validation. To this end, we have included a variety of experiments aimed at validating our experimental predictions at the 1) First, using a luciferase reporter assay, we demonstrated that rs9920291 demonstrates allelic regulatory activity in the T/C-28a2 human chondrocyte cell line new .CHSY1 expression. We chose to use HEK-293FT because it is heterozygous “C/T” for rs9920291. We had genotyped several human chondrocyte cell lines, and unfortunately they were all homozygous “C” for this variant (new CHSY1 in human transformed fibroblasts (new 2) Using a human cell line (HEK-293FT), we demonstrate that there is allelic skew in iant new . This isasts new .CHSY1 expression, suggesting that rs9920291 marks a regulatory element with repressive activity (new 3) Next, we used two separate pairs of CRISPR guide RNAs to delete the region surrounding rs9920291 in the T/C-28a2 cell line. We deleted either a large 135 bp region, or a smaller 17 bp region. Deletion of either region enhances vity new .CHSY1 expression, providing evidence to support our computational predictions that HOXD13 may bind at the open chromatin region marked by rs9920291 and regulate CHSY1 expression (new 4) Finally, we demonstrate that overexpression of HOXD13 can increase sion new .GDF5 from our group) with experimental validation, again highlighting the novelty of our paper.Together, we believe these new results provide strong evidence that our approach can pinpoint plausible candidate causal GWAS variants. Additionally, to our knowledge, this is one of the few height GWAS loci  The authors used E15.5 embryos from FVB mice to study the epigenetic regions for growth plate regulation. But inbred strains often exhibit numerous nucleotide variants; since C57BL6J mice are the most frequently utilized strain for in vitro and in vivo chondrocyte (and osteoblast) studies, have the authors determined whether the specific nucleotide sequence in the CHYS1 variant, rs9920291, is also present in B6 mouse chondrocytes?Thank you for raising this important point regarding mouse strains and the potential for there to be genetic and epigenetic differences across strains. We chose the FVB strain for this study given the consistently large number of embryos that the FVB strain produces, which allowed us to increase the number of mice from which we could collect difficult to extract tissue. We also wish to clarify that rs9920291 is a human variant that, to our knowledge, is not present in mouse. We note that the reference “C” allele is the ancestral allele that is present on the mouse sequence.2) Sex is a major determinant of height particularly early in life and during puberty. The early influences of sex steroids likely begins during late embryonic development; i.e. after E11.5 when SRY is expressed in the males, but not females. It would be important to fully understand the epigenetic landscape in respect to sex differences in vivo. Hence, the embryos should have been typed for sex, and both male and female embryos should be examined to more fully understand the transcriptional regulation of chondrocyte differentiation.Thank you for raising this important point regarding sex of the mice used for analyses. We have addressed this point in our response to the Editor’s first comment (see above).3) Can the authors justify why they did not follow up with functional studies on the CHYS1 variant rs9920291 which they identified as being a cis acting site for HOXD13.CHSY1. We describe the new experiments we have performed in our response to the Editor’s third comment (see above).As suggested by multiple reviewers, we have pursued functional studies of our candidate causal variant rs9920291 at 4) GDF5 experiment from our paper. Additionally, we have moved the epigenetic view of the GDF5 locus to the new We thank the reviewer for this comment. We wish to clarify that the lacZ reporter used in this paper is from a stable line, and thus different from the original Nature Genetics paper. Given that the results are very similar, we have decided to remove the Reviewer #2:[…] While the study is technically strong, it is deficient in skeletal cell and developmental biology, raising questions about the major conclusions reached and specific relevance to human height.Thank you for your careful review of our manuscript. We are pleased that you found our paper “technically strong” and with “positive assets”. Below, we address the criticisms that you raise. We hope that our revised manuscript and new experiments address these gaps and make our manuscript suitable for publication.The growth plate is certainly the key structure determining long bone and vertebral elongation and thus, body height. It is clearly established that the rates of skeletal growth are determined by chondrocyte proliferation (minor contribution) and chondrocyte hypertrophy (major contribution) in the growth plate. It is also well established that different growth plates within the same organism at any given time "grow" at very different rates, such as proximal and distal growth plates in radius and ulna. None of the links established by the data presented here directly address (or explain) chondrocyte proliferation or hypertrophy and genes known to regulate them positively or negatively . The only links established are to general aspects of chondrocyte and skeletal biology, thus making it very difficult to understand how the links have anything to do with "height". The behavior of the growth plate is controlled by intrinsic mechanisms (see above) as well as systemic factors such as growth hormone. Again, none of the links related to this or other systemic cues.Ihh, Pth1r, and Fgfr1. We believe that these revisions help more directly connect our data to chondrocyte biology.Thank you for raising these points about linking our epigenetic data with biological insights into chondrocyte and skeletal biology. In our revision, we have included several improvements to strengthen the connection between our ATAC-seq data and chondrocyte biology. First, we revised our GREAT analysis by removing ATAC-seq peaks that are also seen in the mouse brain. By doing so, we are limiting ourselves to more chondrocyte-specific regions. When repeating the GREAT analysis, we find many chondrocyte-relevant gene sets, such as “Cartilage condensation”, “Positive regulation of chondrocyte differentiation”, and “Chondrocyte proliferation”. Second, we have included an additional figure new  where weThe authors made use of the proximal and distal portions of E15.5 mouse embryo femur and associated perichondrium as starting material for the epigenetic analysis. The tissues were digested into single cell suspension enzymatically before processing for ATAT-seq. There is no explanation of why this developmental stage was chosen and why both ends of the femur were considered. Also, the lengthy enzymatic digestion could have altered gene expression and chromatin configuration.Thank you for raising this important point regarding our choice of using E15.5 embryos. We have addressed this point in our response to the Editor’s first comment (see above).To establish stronger links, the authors considered the most compelling GWAS variants overlapping ATAT-seq peaks, finding 59 variants distributed over 26 loci. The two examples mentioned are CHSY1 and HOXD13 neither of which has established roles in growth plate function. Indeed, most evidence indicates that HOXD13 regulates autopod skeletal patterning and morphogenesis, but not rates of growth.CHSY1 as one example relevant to both human height and human health, as it has previously published roles in chondrocyte biology and mutations in CHSY1 cause a variety of skeletal dysplasias (see main text). We identified HOXD13 as a potential transcriptional regulator of CHSY1 at the rs9920291 enhancer variantusing the UniProbe Database . While it is well-known that Hoxd13 plays important roles in autopod patterning, it is also expressed in growth plates  and has been known for some time  to be growth promoting factor . As Hox proteins act redundantly during limb bud development and growth plate function, deciphering the specific functions of Hoxd13 or its targets has been extremely difficult. However, our newly added data reveal that HOXD13 is expressed in chondrocytes and can increase CHSY1 expression when overexpressed in chondrocyte cell lines  may act to influence human height. Unfortunately, despite claiming the identification of "compelling mechanisms for GWAS variants" (Abstract), the authors do not provide sufficient experimental or computational evidence in support, except for rs4911178 that references data in Capellini et al. 2017 (PMID 28671685). In summary, the authors present an important epigenomic dataset that can act as a first step for future study of height GWAS SNPs, but do not provide "strong evidence"  for compelling mechanisms at any height GWAS loci beyond referencing their previously published paper.CHSY1 locus. We have also made many other changes and performed new experiments as suggested by the reviewer.We thank the reviewer for his/her careful review our paper and are pleased that the reviewer recognizes the value of our work. To strengthen our connections between ATAC-seq peaks at GWAS loci with biological mechanisms, we now provide extensive functional work at the 2>0.5 . Given that r2>0.5 is not a strict LD threshold , is it still meaningful to report overlap statistics using the r2>0.5 set when the PICS 95% credible set is available?1) Substantially fewer GWAS loci overlap ATAC-seq peaks when using the fine-mapped 95% credible variants, instead of all SNPs with r2>0.5 is not a universally agreed upon LD threshold. We have now included GoShifter analyses at several different r2thresholds ranging from 0.2 to 0.8 (new 2 cutoff of 0.5 demonstrated the strongest enrichment (p=0.0059).We thank the reviewer for raising these important points regarding proxy SNPs at GWAS loci. We agree with the reviewer that r 0.8 new . Among t2>0.5 in our new The reviewer makes an excellent point regarding whether we should just report PICS credible set overlaps. While the reviewer’s point is valid and well-taken, we have decided to be more inclusive in our approach and report both PICS overlaps and variants overlaps with r2) The authors state "44 different genes have been identified as possibly being modulated by these putative regulatory variants". However, in the present manuscript the authors do not provide experimental or computational evidence implicating the ATAC-seq peaks in regulating the nearby growth plate genes, and provide limited evidence that any of these overlapping variants can alter transcriptional regulatory activity.CHSY1 locus.Thank you for this comment. We now provide evidence that differentially expressed growth plate genes that are also eQTLs genes in whole blood are enriched at ATAC-seq peaks (see response to point #6). Additionally, as detailed in our response to the Editor’s third comment, we now provide extensive experimental evidence for rs9920291 as a causal variant at the 3) Following point #2, and to support the authors' claim of identifying "prime targets for functional testing", it would be helpful to know how many GWAS SNP overlaps with femur open chromatin regions the authors believe to be biologically meaningful versus coincidence.CHSY1 locus to demonstrate the value of our approach.We thank the reviewer for raising this important point. Unfortunately, without a “truth” set, it is difficult to evaluate what proportion of our nominated SNPs are likely to be biologically meaningful as opposed to coincidence. However, we do note that using both GoShifter and random matched GWAS loci, we detect very strong enrichments of our height GWAS SNPs at ATAC-seq loci, suggesting that a large proportion of our nominated SNPs are meaningful. We also point out the work of Ulirsch et al., Cell, 2016, which performed MPRA for red blood cell traits and demonstrated that a large proportion of variants nominated by similar approaches as used in our paper demonstrated functional activity in the MPRA assay. As an additional approach to demonstrate that our observed overlaps are not simply coincidence, we also performed GoShifter analyses for enrichments of GWAS associations of several traits  at our ATAC-seq peaks. For most of these additional traits, we did not detect significant associations (p>0.05) of enrichment new . Lastly,4) Similar to point #2, it is premature to state "59 variants thus have strong evidence for being potentially causal variants". For the authors' example of the CHSY1 locus, while there are 4 SNPs in the PICS 95% set that overlap an ATAC-seq peak nearby CHSY1, CHSY1, intersection with ATAC-seq data prioritized the 87 SNPs in the 95% credible set down to just four variants. In our revision, we now provide extensive functional evidence for rs9920291, providing further credence for our claim that our approach can nominate likely causal variants  Subsection “Known and novel targets of human height variants in putative chondrocyte regulatory regions”, first paragraph: The authors suggest rs9920291 disrupts HOXD13 binding, but do not provide evidence that HOXD13 actually binds this site at the endogenous locus. As computationally predicted TF motifs are prevalent in the genome, it is hard to assess whether this SNP/motif overlap is biologically meaningful.CHSY1 , there are limited ChIP-seq datasets in relevant tissues to assess whether HOXD13 truly binds at rs9920291, in part because of the known difficulty of generating and using HOXD13-specific antibodies for ChIP-seq. We have toned down the language in the manuscript regarding this HOXD13 connection . Additionally, we do now provide evidence that overexpression of HOXD13 increases expression of HSY1 new .6) Wood et al. (2014) found that height GWAS loci were more likely to be eQTLs (in blood) for genes expressed in cartilage. Did the authors examine whether the 468 credible SNPs overlapping femur open chromatin peaks are enriched for eQTL signals? This analysis may strengthen the authors' mechanistic claims.We thank the reviewer for this excellent suggestion. We have now included additional analyses, which confirm the observation in Wood et al., that height GWAS loci are more likely to be eQTLs in blood for genes expressed in cartilage. To do this, we used a set of eQTLs from whole blood from Westra et al., 2014 and subsetted for eQTLs genes that are differentially expressed in the growth plate. We then ran GoShifter on this set of corresponding eQTLs variants and indeed detected a significant enrichment when compared with our ATAC-seq peaks (p=0.0004). This is discussed in the third paragraph of the subsection “A subset of human height variants in femoral open chromatin regions reside near genes 261 differentially expressed in the growth plate”.


To investigate the effect of maternal MitoQ treatment on renal disorders caused by maternal cigarette smoke exposure (SE). We have demonstrated that maternal SE during pregnancy increases the risk of developing chronic kidney disease (CKD) in adult offspring. Mitochondrial oxidative damage contributes to the adverse effects of maternal smoking on renal disorders. MitoQ is a mitochondria-targeted antioxidant that has been shown to protect against oxidative damage-related pathologies in many diseases. Female Balb/c mice (8 weeks) were divided into Sham (exposed to air), SE (exposed to cigarette smoke) and SEMQ (exposed to cigarette smoke with MitoQ supplemented from mating) groups. Kidneys from the mothers were collected when the pups weaned and those from the offspring were collected at 13 weeks. Maternal MitoQ supplementation during gestation and lactation significantly reversed the adverse impact of maternal SE on offspring’s body weight, kidney mass and renal pathology. MitoQ administration also significantly reversed the impact of SE on the renal cellular mitochondrial density and renal total reactive oxygen species in both the mothers and their offspring in adulthood. Our results suggested that MitoQ supplementation can mitigate the adverse impact of maternal SE on offspring’s renal pathology, renal oxidative stress and mitochondrial density in mice offspring. Human studies have shown that intrauterine exposure to cigarette smoke (SE) is closely linked to impaired fetal renal growth2. Maternal smoking is associated with a 1.24-times increased risk of child proteinuria compared with offspring of non-smoking mothers3. These phenomena have also been confirmed in our mouse model of maternal smoking, which demonstrated that maternal SE leads to renal underdevelopment in offspring at birth and renal dysfunction in adulthood4.It has been increasingly recognised that maternal programming during fetal development predisposes the offspring to future disease. Maternal smoking imposes a significant adverse impact on fetal renal development that determines the future risk of chronic kidney disease (CKD) in adulthood5, which is subsequently cleared by the endogenous antioxidants, such as manganese superoxide dismutase (MnSOD). Mitochondrial abnormalities, such as the accumulation of mitochondrial DNA mutations and damaged mitochondria structure due to metabolic stress, can overconsume the antioxidant enzymes or impair the production of antioxidants leading to oxidative stress which in turn triggers pro-inflammatory response7. Renal tubular cells contain abundant mitochondria, therefore mitochondrial density plays a fundamental role in the pathogenesis of kidney diseases. Growing evidence suggests that mitochondrial damage is implicated in the pathophysiology of renal diseases9. It has been reported that nicotine can accumulate in the kidney10. Several studies indicated that maternal smoking is closely related to increased levels of oxidative stress in the mothers, infants and newborns12, and reduced levels of the antioxidant enzymes superoxide dismutase (SOD) in the arteries of offspring from nicotine treated rats13. Moreover, we also demonstrated that oxidative stress and mitochondrial dysfunction are closely associated with the adverse effects of maternal smoking on the kidney pathology in the male offspring15.Mitochondria are intracellular organelles that generate the energy required for cellular functions through oxidative phosphorylation, which involves a series of oxidation-reduction reactions. During this process, reactive oxygen species (ROS) are released as a by-product. Thus, mitochondria are the major source of ROS during energy synthesis16. Amniotic fluid CoQ10 levels are significantly lower among women delivering preterm babies, a risk which is increased by maternal smoking18. In addition, plasma CoQ10 levels are reduced in smokers19. However CoQ10 is not a viable treatment option due to poor bioavailability and delayed mitochondrial uptake20. Mitoquinone mesylate, also known as MitoQ, is a mitochondria-targeted antioxidant. It consists of a ubiquinone moiety, the same structure to the ubiquinone found in CoQ10, which allows its rapid uptake and accumulation in the mitochondria to restore the antioxidant efficacy of the mitochondrial respiratory complex21. As such, it has been reported that MitoQ has a protective role against oxidative damage-related pathologies in metabolic22 and neurodegenerative diseases23. Moreover, our previous study demonstrated that maternal MitoQ supplementation during pregnancy and lactation is beneficial in reducing lung inflammatory and oxidative stress responses caused by maternal SE in the adult offspring24. Therefore, this study aimed to investigate whether maternal MitoQ supplementation can also mitigate the adverse impact on renal disorders caused by SE and whether the benefits of MitoQ administered to the SE mother are transmitted to the fetus and result in reduced future risk of CKD.Coenzyme Q10 (CoQ10) is a mitochondrial endogenous antioxidant. It has been shown that CoQ10 supplementation in mice can lower hepatic oxidative stress and inflammation associated with diet-induced obesity in mice4), and SEMQ (SE mothers supplied with MitoQ (500 µM in drinking water27) during gestation and lactation). Male breeders and suckling pups stayed in the home cage when the mothers were exposed to normal air or cigarette smoke. Pups were weaned at postnatal day 20 and maintained without additional intervention.The animal experiments were approved by the Animal Care and Ethics Committee at the University of Technology Sydney (ACEC#2014-638 and #2016-419). All protocols were performed according to the Australian National Health & Medical Research Council Guide for the Care and Use of Laboratory Animals. Female Balb/c mice (8 weeks) were housed at 20 ± 2 °C and maintained on a 12:12 hour light/dark cycle with ad libitum access to standard laboratory chow and water. After the acclimatisation period, mice were divided into three groups: SHAM (exposed to air), SE  at adulthood (13 weeks). A terminal urine collection was undertaken via direct bladder puncture and the blood was collected via cardiac puncture after mice were anesthetized. The kidney tissues were collected and stored at −80 °C for later analysis.Since we have previously demonstrated that maternal SE have a greater impact on the male offspringThe levels of urinary albumin and creatinine were measured using Murine Microalbuminuria ELISA kit (Albuwell M) and Creatinine Companion kit, respectively  following the manufacturer’s instructions.4 and quantitated using Image J software .Kidney structure was examined in the male offspring at 13 weeks as previously described. Briefly, fixed kidney samples were embedded in paraffin and sectioned. Kidney sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Glomerular number and size were assessed as we have previously describedConfocal laser scanning microscopy images of frozen kidney sections were acquired using Leica SP2 confocal laser scanning microscope . Data was generated from 5–6 animals/group. Four to 6 Images were collected from each kidney and averaged before the analysis. All imaging parameters including laser intensities, Photomultiplier tubes voltage and pinholes were kept constant during imaging. For total reactive oxygen species (ROS) detection, CellROX Deep Red  was used at 5 µM final concentration, images were acquired at 633 nm excitation wavelength and detected in the 640–680 nm emission range. MitoTracker Green  was used for staining the mitochondria at 200 nM final concentration, Images were acquired at 488 nm excitation wavelength and detected in the 510–550 nm emission range.Kidney tissues were homogenized in lysis buffer with phosphatase inhibitors . Protein concentrations were measured using DC Protein assay . Equal amount of proteins (20 μg) were separated on 4–12% Criterion™ XT Bis-Tris Protein Gel  and transferred to PVDF membranes. The membranes were blocked with TBS-0.05% Tween 20 (TBS-T) containing 5% BSA or skim milk for 1 h, before incubation with primary antibodies against endogenous antioxidant Manganese superoxide dismutase , translocase of the outer membrane-20 , fibronectin , phospho-extracellular signal-regulated kinase-1/2 , Erk1/2 , phospho-JNK , c-JUN N-terminal kinase , phospho-p38 Mitogen-activated protein kinase , p38 MAPK , transcription factor nuclear factor-κ-light-chain-enhancer of activated B cells , phospho-NFκB , F4/80 , Collagen I , Collagen III  and Collagen IV  overnight at 4 °C, then followed by secondary antibodies . The blots were then incubated in Super Signal West Pico Chemiluminescent substrate  and the membranes were then visualized by an Amersham Imager 600 . Protein band density determined using ImageJ software  was used for densitometry, and β-actin  was used as the control.Total mRNA was isolated from kidney tissues using TRIzol Reagent . First strand cDNA was generated using M-MLV Reverse Transcriptase, RNase H, Point Mutant Kit . Genes of interest were measured using pre-optimized SYBR green primers (Sigma-Aldrich) and RT-PCR master mix . The primers used in real-time RT-PCR experiments were as follows: macrophage chemoattractant protein (MCP)-1 forward primer: 5′-GTTGTTCACAGTTGCTGCCT-3′, and reverse primer: 5′-CTCTGTCATACTGGTCACTTCTAC-3′. Interleukin (IL)-1α, IL-6 and cluster of differentiation (CD) 68 mRNA expressions were measured using Taqman probe . The average expression of the control group was assigned as the calibrator against which all other samples were expressed as fold difference. The 18S rRNA was used as the house-keeping gene for all gene of interest.15.Genomic DNA was extracted from renal tissue using the DNeasy blood and tissue kit (Qiagen). The content of mtDNA was calculated using real-time quantitative PCR by measuring the threshold cycle ratio (∆Ct) of the mitochondrial-encoded gene cytochrome c oxidase subunit 1 (COX1)  vs. the nuclear-encoded gene cyclophilin A  was used to extract ATP according to manufacturer instructions. In brief, kidney tissues (15–20 mg) were homogenized in 0.5 ml ice-cold Phenol-TE . Chloroform (200 μl) and de-ionised water (200 μl) were added and followed by twenty seconds shaking. Aqueous phase was extracted and ATP was determined with luciferin-luciferase assay.Results are presented as the mean ± S.E.M. The differences between the groups were analysed by one-way ANOVA followed by post hoc Bonferroni test . The differences were considered statistically significant at P < 0.05.Results in Table MitoQ supplementation during gestation and lactation significantly reversed the impact of SE on mitochondrial density  and total ROS level  which may be induced by increased work load for ATP synthesis.Several studies have indicated that maternal cigarette smoking during pregnancy was the most common cause of fetal growth restriction and reduced size of the fetal organs37. Several studies have suggested that RAGE can also influence the pathogenesis of renal disorders39. Our previous study demonstrated that maternal SE can increase RAGE and its signalling elements, as well as promoting oxidative stress and inflammatory responses in offspring’s lung24. However in this study, none of the RAGE signaling elements including RAGE, p38 MAPK, ERK1/2, JNK, and NFκB, were changed in the SE offspring’s kidney. These findings suggest that the RAGE pathway does not seem to be involved in maternal SE induced inflammatory response in the offspring’s kidney. There is also evidence suggesting that inflammatory cell infiltration correlates with both the extent of renal fibrosis and the severity of renal damage in CKD40. Irreversible renal fibrosis is a common consequence after renal injury and leads to a gradual loss of kidney function, which is a hallmark of CKD. In this study, there was a significant increase in fibronectin level in the SE offspring’s kidney. We have previously demonstrated that maternal SE induced subtle pathological changes in the offspring’s kidneys at 13 weeks. Increased risk of CKD may prevail if the offspring are exposed to additional insult after weaning, such as obesity or diabetes. Such hypothesis requires validation in future studies.Oxidative stress is often linked to inflammatory responses and fibrotic changes, which were also observed in the SE offspring with increased levels of inflammatory  and fibrotic markers (fibronectin and collagen IV). The AGEs-RAGE interaction has also been shown to associate with enhanced production of intracellular ROS, which can mediate further inflammatory response44. Therefore, therapeutic application of mitochondrial-targeted therapies may offer potential alternatives for the prevention and treatment of such conditions, instead of the generic antioxidants which are normally poorly taken up by the mitochondria. The most widely investigated mitochondria-specific antioxidant to date is MitoQ45. The beneficial effects of MitoQ have been reported in various disorders, such as metabolic disease22, neurodegenerative diseases23, kidney damage related to diabetes46, Parkinson’s disease47, and liver inflammation in hepatitis C virus infection48. Importantly, our previous study using the same cohort of mice demonstrated that maternal MitoQ supplementation during pregnancy and lactation is beneficial in reducing lung inflammatory and oxidative stress responses in the adult offspring caused by maternal SE24. As we have demonstrated that oxidative stress plays an important role in maternal SE related renal disorders in the male offspring15, this study extended the investigation of the impact of maternal MitoQ supplementation on renal disorders.Mitochondrial dysfunction occurs in several human disorders, which is considered as the major driver for cellular and organ failure. Adverse effects of cigarette smoke have been attributed to increased oxidative stress together with mitochondrial dysregulation, which play a key role in the progression of renal injury and development of CKD46. Mukhopadhyay and colleagues also found that MitoQ treatment prevented renal dysfunction caused by cisplatin nephrotoxicity49. Such improvement in the offspring is closely related to reduced renal ROS level and normalised mitochondrial density in both mothers and offspring. Interestingly, renal MnSOD level was not increased in the offspring as a consequence of maternal administration of MitoQ. This was different to that observed in the lungs24, suggesting that ROS was suppressed by other antioxidative mechanisms or due to reduced mitochondrial ATP production. The impact of maternal SE on TOM-20, the mitochondrial outer membrane receptor for the translocation of cytosolically synthesized mitochondrial preproteins, was partially reversed in SE offspring, suggesting some improvement in mitochondrial function. However, mitochondrial DNA copy number was not reversed in the SEMQ offspring compared with the SEMQ mothers, suggesting oxidative stress may not be the only factor to damage mitochondrial DNA in the offspring. Foetal kidneys are likely to be more vulnerable to the damage from the chemicals in the cigarette smoke, since nicotine level is 15% higher in the foetal blood than the maternal blood50. This may also affect the fibronectin level in the offspring’s kidney51, which was also unaffected by maternal MitoQ supplementation although the inflammatory markers MCP-1, CD68, F4/80 and ROS level were reduced. As the aim of the study was to determine whether MitoQ protects the offspring from maternal smoking we did not include a sham group treated with MitoQ. Hence we are unable to be definitive about whether MitoQ may cause changes in the parameters studied in “normal’ animals. However, Rodriguez-Cuenca et al., have previously examined the long-term consequences of MitoQ on wild-type mice in the absence of injury and demonstrated that MitoQ has no effect on mitochondrial function, mitochondrial DNA, food consumption or whole body metabolism25.In the current study, our results showed that MitoQ supplementation during pregnancy can significantly mitigate small body weight due to in-utero SE. Moreover, we demonstrated that MitoQ treatment can restore smaller kidney size and glomerular numbers with nearly normalised renal function in adult offspring from the SE mother. These results suggested that MitoQ exert beneficial effects on offspring’s health, despite continuing maternal SE during gestation and lactation. Our results are consistent with earlier reports that showed that MitoQ treatment prevented renal disorders in a mouse model of type 1 diabetesIn summary, our study demonstrates the beneficial effects of maternal MitoQ supplementation during gestation and lactation on renal under development and pathology by maternal SE. It also reduced renal ROS accumulation and mitochondrial density in both mothers’ and offspring’s kidneys. Although MitoQ was unable to reverse the increase in fibrotic markers, it may still protect the offspring against maternal SE induced renal pathology and potentially future CKD through reduction of inflammation and oxidative stress. This is yet to be confirmed in future human clinical trials.Supplementary information


Omega-3 polyunsaturated fatty acids (PUFAs) have a highly anti-angiogenic effect in animal models. However, the clinical relevance of omega-3PUFAs in human retinal pathologies remains unclear. The ARED 2 study found no effect of omega-3 PUFA supplementation on progression of age related macular degeneration (AMD). The aim of this study was to compare serum levels of omega-3- and omega-6 PUFAs between patients with diabetic retinopathy (DR), AMD and retinal vein occlusion (RVO), and to identify potential confounders of serum level measurements.Venous blood samples were collected from 44 patients with DR, 25 with AMD, 12 with RVO and 27 controls. The lipid phase was extracted and analyzed using mass spectrometry. Retinal disease staging was done by indirect funduscopy and FAG where appropriate. Patient demographics and medical history including current medication and fasting state were acquired. Tukey contrasts for multiple comparisons of the mean and linear regression analysis were used for statistical analysis.p > 0.858). Uncorrected omega-3 PUFA levels were significantly higher in patients with AMD compared to DR but not compared to controls (p = 0.004). However, after correcting for possible confounders such as body mass index (BMI), age, sex, fasting and use of statins, no statistically significant difference remained for serum omega-3 PUFA levels. Fasting was identified as an independent confounder of total omega-6 PUFAs, three individual omega-6 PUFAs and one omega-3 PUFA(p < 0.0427). Statin use was identified as an independent confounder of α-linolenic acid .Our data revealed no significant differences in omega-6 PUFA serum levels between patients with AMD, DR, RVO and controls  contains supplementary material, which is available to authorized users. Angioproliferative retinal diseases such as diabetic retinopathy or the wet type of age related macular degeneration are responsible for visual impairment among many working age individuals and the elderly , 2 and lOmega-3-polyunsatureted fatty acids (PUFAs) are major structural lipids of retinal photoreceptor outer segment membranes and have important structural and protective functions in the retina . The antIn the mouse model of oxygen-induced retinopathy (OIR), omega-3PUFAs were found to have potent anti-angiogenic effects on retinal neovascularization . For exaDifferent from AREDS2, however, Merle et al.found serum EPA to be associated with a lower risk for neovascular AMD in 290 patients from the Nutritional AMD Treatment 2 Study (NAT2) who had neovascular AMD in one eye and early AMD in the other eye . Merle eThese contradicting results of various AMD studies demonstrate the need for characterization of confounding parameters which might affect lipid measurements in serum samples. This is not only important for AMD research but also for research in other areas of retinal disease where lipid imbalance may play a role such as diabetic retinopathy (DR) and retinal vein occlusion (RVO). To date, significantly less scientific data exist for DR and RVO compared to AMD with regard to the potential effect of lipids on disease development or progression.The aim of our study was therefore to identify potential confounders which may affect a perceived correlation of omega3 PUFAs with the degree of AMD, DR or RVO. In addition to omega-3 PUFAs, we also investigated omega-6 PUFAs as well as monounsaturated and saturated fatty acids.Venous blood samples were obtained from 81 patients with DR, AMD or RVO. In addition, samples from 27 control patients without any of the above diseases were collected. Patients who had more than one of the mentioned pathologies were excluded. Diabetic patients were sub-classified in five groups: diabetes without DR (5 patients), mild non-proliferative DR (6 patients), moderate non-proliferative DR (14 patients), severe non-proliferative DR (5 patients) and proliferative DR (15 patients). The grading was done according to the EDTRS–guidelines . PatientAdditional clinical data were acquired by indirect fundoscopy and FAG where appropriate. For all patients additional information was gathered including medical history, current and past medication (including vitamin supplementation), state of fasting and body mass index (BMI).The study was registered with the Ethics Committee at the University Hospital Freiburg. After obtaining written informed consent, one serum sample per patient was acquired by venous blood draw and stored at −80 °C until being used for lipid extraction. Lipid extraction was performed by the Department of Clinical Chemistry at the University of Freiburg as previously described at a temperature of 4 ° C in a two-step procedure . 100 μl p = 0.00405; Table p > 0,858; Table Our initial analysis of native sample readings without correction for potential confounders suggested significantly higher omega-3PUFA levels in patients with AMD compared to patients with DR , the use of oral statin medication and the state of fasting (defined as at least six hours fasting prior to blood sampling). The results shown in Table p < 0.0427). Statin medication was identified as an independent confounder of α-linolenic acid . Interestingly, patient age and BMI were no independent confounders of serum lipid measurements. The small patient number especially in the RVO subgroup has to be considered when interpreting the results of this pilot study.Next, we investigated whether individual fatty acids were significantly affected by individual patient variables. Table Numerous clinical studies have investigated a possible protective effect of omega-3PUFA supplementation on the progression of AMD. These studies yielded variable and contradicting results , 16, 17.Despite the small sample size of this pilot study, our data clearly suggest that serum lipid levels cannot be used for clinical ophthalmological studies without vigorous correction for potential confounders. The most important confounder is patient fasting state, which affects various unsaturated and saturated fatty acid levels. Most of the existing AMD studies have corrected for potential confounders and report fasting status in their publications. For example, the study by Merle et al. which identified EPA as a risk variable for neovascular AMD did correct for a list of potential confounders including fasting state of the patient . It has The second important finding of our study is that, in our cohort, total omega-3 or omega-6 PUFA levels were not independently associated with the presence of AMD. Different from our study, Merle et al. found associations between AMD and EPA, an omega-3 PUFA . While MWe would like to point out that larger patient numbers would be necessary in order to more reliably investigate clinically relevant differences between patient groups. The main purpose of this manuscript, however, was to identify and flag potent confounders of lipid serum measurement that have to be accounted for when performing these studies. Even with our low patient numbers we were able to identify such confounders – an indication of their potency in affecting results from serum lipid measurements. In this respect, our current manuscript has to be viewed as preparatory work for larger studies by providing important information on methodological aspects of serum lipid measurements that have to be accounted for in larger clinical trials.In summary, our data together with the discussed literature emphasize the need for (i) correction for identified confounders of serum lipid profile measurements and (ii) interpretation of lipid profiles in light not only of the disease entity studied but also the underlying patient population. One limitation of our study is the relatively low sample number. Larger patient numbers are thus needed to obtain a full understanding of all factors influencing serum lipid measurements.


P<10−6), and 8 SNPs exert cis-regulation effects on 11 eQTL target genes. Among the 8 SNPs, 2 SNPs (RPL31 rs2278729 and LRP5 rs3736228) were confirmed to impact the expression of 3 genes  that were differentially expressed between human subjects of high BMD group and low BMD group. All of the functional evidence suggested the important functional mechanisms underlying the associations of the 2 SNPs (rs2278729 and rs3736228) and 3 genes  with osteoporosis. This study may provide novel insights into the functional mechanisms underlying the osteoporosis associated genetic variants, which will help us to comprehend the potential mechanisms underlying the genetic association for osteoporosis.Osteoporosis is characterized by increased bone loss and deterioration of bone microarchitecture, which will lead to reduced bone strength and increased risk of fragility fractures. Previous studies have identified many genetic loci associated with osteoporosis, but functional mechanisms underlying the associations have rarely been explored. In order to explore the potential molecular functional mechanisms underlying the associations for osteoporosis, we performed integrative analyses by using the publically available datasets and resources. We searched 128 identified osteoporosis associated SNPs ( Osteoporosis is a skeletal disease characterized by low bone mineral density (BMD) and micro-architectural deterioration of bone tissue, leading to decreased bone strength and increased the risk of fracture. OsteopoGenome-wide association studies (GWAS) have been successful at identifying a number of promising genetic variants that are associated with osteoporosis and related traits. However, the statistical association between OP and genetic variants were only established in the DNA level at the present stage, functional relevance has rarely been explored. Such established associations do not provide direct understanding into functions of significant candidate genes or regulation of genes expressions that functionally establish a connection between OP phenotype and gene code information.Transcriptional regulation plays an important role in functional mechanisms underlying genetic association. The change of binding affinity between variants and regulatory factors altered by genetic variants may influence the transcription and/or translation of target proteins. Numerous public gene expression datasets are available, which are invaluable and may provide the functional data support for a better understanding of the association between variants and phenotype.We combined the integrative analyses–8 (gene www.ncbi.nlm.nih.gov/gap/PheGenI/) is a bioinformatics online tool that can provide robust view and download analystic data  for published studies[P value <10−6 by searching PheGenI[Phenotype-Genotype Integrator (PheGenI) (d studies. Using pg PheGenI.http://www.broadinstitute.org/mpg/grail/), which is an online statistical method that examining relationships automatically between seed regions and candidate genes were selected through PubMed abstracts to prioritizes the best candidate gene[Under normal circumstances, one disease-associated SNP has one or more predisposing genes or locus in the region near this SNP. In order to carry out the further functional studies, selecting genes located at the two sides of the associated SNP as potential candidate genes is a conventional method to identify the candidate genes. With thdate gene. The seedate gene.http://eqtl.uchicago.edu/cgi-bin/gbrowse/eqtl/). We used this database to detect the eQTL effects of the identified OP-associated SNPs in monocytes and lymphoblastoid cell lines (LCLs). In humans vivo, the favorable working cell model for studying gene/protein expression patterns and their regulation mechanisms with reference to osteoporosis risk is monocyte[Variation occurs at the level of DNA may lead to the changes of genes expression, which subsequently account for a significant proportion of the phenotypic variance  of for the identified eQTL genes between low and high BMD were detected by using t-tests.Three previous in vivo genome wide gene expression studies in our group using Human Genome U133 Plus 2.0 or U133A Arrays were performed to identify genes differentially expressed in monocytes or B cells between low and high BMD women., and we uploaded those data to GEO (gene expression omnibus) Datasets. The experimental procedures and data analysis were detailed in the original studies, and the GSE numbers are: GSE7158, GSE13850, GSE2208 –34. The http://compbio.cs.queensu.ca/F-SNP/). RegulomeDB[http://regulome.stanford.edu/). Different regulomeDB score (1–6) is represent the different degree for functional variant as transcription factor binding site. Score 1 indicates the strongest evidence for a SNP being located in a functional region.For the identified eQTL SNPs, we used two online tools to predict the potential functions. F-SNP is a database to provide the latest information about already confirmed and presumed potential functional effect of SNPs at the multiple level, such as splicing, transcriptional, translational, and post-translational(egulomeDB is an onhttp://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) is an online tool to predict and analyze the secondary and tertiary structure and function of proteins[Phyre2  showed significantly differential expressions in GSE7158 and GSE13850 cell samples, with p value<0.05. The genes carnitine palmitoyltransferase 1A (CPT1A) and metallothionein-like 5 (MTL5) showed significantly differential expressions in GSE13850 cell sample  . The thrRPL31 rs2278729, CREB3L1 rs1007738 and LRP5 rs3736228), scored 1f, were annotated as the most credible functional variants with potential regulation effects and likely accounted for allelic-specific expressions of the 5 eQTL target genes .We used the Regulome DB to investigate whether the 8 eQTL SNPs are functional variants with putative regulation effects. As shown in LRP5 protein with amino acid residue alteration from Alanine (A) to Valine (V).We used 101 amino acids (50 amino acids before and 50 after the missense mutation of rs3736228) to predict the secondary structure of protein. We found that the amino acid residue alteration from Alanine (A) to Valine (V) caused loss of alpha helix and gain of beta strand before the missense mutation  were identified. Among these SNPs, two eQTL SNPs (RPL31 rs2278729 and LRP5 rs3736228) likely accounted for allelic-specific expressions of the 3 eQTL target genes  which showed significantly differential expression in monocytes and LCLs.In this study, we performed integrative analyses to explore functional mechanisms underlying the associations for OP by using publically available datasets. A total of 8 SNPs  acting as cis-effect regulators on the 11 corresponding eQTL genes  and OP.Integrative analyses by utilizing the public data resources can provide new understandings into the molecular genetic mechanisms of human diseases. In genetics, the genetic information carried on DNA is transferred to RNA through transcription and then translated into protein molecules. The funLRP5 gene was strongly associated with the BMD[CPT1A and MTL5[CPT1A is a key regulator to facilitate the transfer of long-chain fatty acids across the mitochondrial membrane for β-oxidation in mammals[MTL5 may play a central role in the regulation of cell growth and differentiation[CPT1A and MTL5[CPT1A located in BIOCARTA_LEPTIN_PATHWAY which is important in the biology and etiology of osteoporosis[CPT1A and MTL5 in B cells were significantly differential expressed between high and low BMD group (P<0.05). SNP rs3736228 acts as potential eQTL cis-effect on CPT1A and and MTL5. Therefore, we speculate that rs3736228 plays important roles in the pathological mechanism of osteoporosis by regulating differential mRNA transcriptions of CPT1A and MTL5. On the other hand, since rs3736228 caused a missense mutation for LRP5 protein, LRP5 rs3736228 may influence and even induce osteoporosis through changing the amino acid sequence and secondary structure of LRP5 protein. Our finding strongly suggested that rs3736228 was a causal variant functioning either by regulating expression of CPT1A and MTL5 and/or by influencing function of LRP5 protein. Further functional study on the molecular mechanism of CPT1A, MTL5 gene and LRP5 rs3736228 may help us comprehend the association between osteoporosis and candidate gene, understand the pathogenesis of osteoporosis and the genetic mechanism.The previous study showed that rs3736228 in the h the BMD. Through and MTL5. CPT1A in mammals. MTL5 maentiation. Previou and MTL5genes wereoporosis. In thisRPL31, belongs to the L31E family of ribosomal proteins. The encoded protein is one of the members of the ribosomal 60S subunit. Ribosomal proteins have a lot of functions, such as participation in regulation of gene transcription and translation, involved in DNA repair, regulation of cell proliferation, differentiation, apoptosis and so on. In addition, ribosomal proteins may play an important role in tumor occurrence, development, metastasis and tumor suppression[RPL31 expression in lymphoc-ytes and osteoblasts. In this integrative study, we confirmed that SNP rs2278729 acts as cis-effect regulators for RPL31. We speculate that rs2278729 can regulate differential expression of protein by regulating differential mRNA transcriptions of gene RPL31, therefore, rs2278729 plays important roles in the variation of susceptibility to OP.ppression,47. AlleWe identified two SNPs having combined OP-associated functional evidence among the searched 128 OP-associated SNPs. Failure to find functional evidence for some promising functional SNPs in BMD hot loci, like RANKL-OPG, ESR and LRP5 in this study does not exclude the importance of the associations of the BMD hot loci with OP. Instead, those associated SNPs may be involved in pathogenesis of OP by other mechanisms (such as epigenetic regulation or protein translation) instead of directly regulating target gene expression. Small effect size of the SNPs and small sample size will lead to limited statistical power, which is a reason for the lack of association between OP-associated SNPs and mRNA expression. Further functional studies are required to understand the functional association between genetic variants and OP, which may provide new insights into mechanisms underlying the association detected at DNA level.There are two limitations to this study. First, although we identified some functional evidence for the associations between 2 SNPs and OP, it is still needed to further study the causal variants with potential functional effects. Second, although we used two methods to select the candidate genes, there is still another possibilities that other genes near the associated-SNPs are also directly or indirectly involved in the association around the SNPs. In shorRPL31,CPT1A and MTL5), at the same time, these 3 genes were differentially expressed between high BMD group and low BMD group. The above evidence provides some important clues for us to perform further study on the functional molecular mechanism between OP and OP-associated genetic variants. Performing the integrative analyses by utilizing the public data resources may provide a new perspective into the functional molecular genetic mechanisms underlying human complex diseases.In summary, all of the functional analysis results we found in this study demonstrated the significance of the 2 candidate OP-associated SNPs (rs2278729 and rs3736228). We confirmed that the 2 SNPs have cis-regulation effects on the expression of 3 genes  and the “Gene 1” and “Gene 2” represent the nearest genes physically located at two sides of the SNP. "Implicated gene" are the results from Gene Relationships Across Implicated Loci (GRAIL) analysis. PMID: Pubmed ID for association results."RegulomeDB Score" is a scoring scheme of RegulomeDB and represents the source of supporting evidence. Among those, "1f" refers to the evidence from "eQTL + TF binding/DNase peak", “2b” refers to TF binding + any motif + DNase Footprint + DNase peak”, “3a” refers to TF binding + any motif + DNase peak, "4" refers to TF binding + DNase peak, "5" refers to TF binding or DNase peak, "6" refers to the evidence from "other"LCLs: lymphoblastoid cell lines.(DOCX)Click here for additional data file.


Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating  Dengue virus, belonging to the Flaviviridae family. It affects nearly 390 million people every year worldwide345Dengue is a mosquito-borne disease caused by a group of viruses collectively known as Dengue viruses (DV) are enveloped, ssRNA viruses with a single open reading frame encoding three structural and seven non-structural proteins810121314151618192021250–270) was visualised using available structures of DV E proteins in pre- and post-fusion forms , 120 of Tick-borne encephalitis virus (TBEV), 120 of West Nile virus (WNV), were selected randomly from GenBank as well as the 70 sequences of Yellow fever virus (YFV) and 63 of Zika virus (ZKV), available, aligned with MUSCLE and visualized all together with WebLogo. Sixteen out of 21 amino acids in this peptide were 100% conserved in every DV sequence evaluated, being DV4 the most divergent among the four serotypes. When compared to other Flaviviruses at least seven amino acids from E250–270 are highly conserved  were analysed along with the same number of genomes for each other DV serotype, that were randomly selected through their NCBI sorting numbers . This unbiased dataset, comprising 480 sequences , supplemented with m7G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs). RNA was purified , and transfected into Huh7.5 cells with Lipofectin (Invitrogen). Cells and supernatant were recovered at indicated hours post transfection (h.p.t.) and the RNA extracted using RNeasy Mini Kit or QIAamp Viral RNA Mini Kit, respectively (both Qiagen). Using RT-qPCR for non-structural protein 5 (NS5) mRNA , only decreasing quantities of CSMut1 NS5 transcript were observed in both cell associated RNA or culture supernatant, suggesting an impaired ability of this mutant virus to replicate , using Alexa Fluor 488 rabbit anti-mouse IgG  as a secondary antibody, and DAPI counter stain (Molecular Probes). In agreement with RNA detection , suggesting that the defect caused by the mutations were not due to a host specific factor.The impact of CSMut1 on DV1 was also assessed by immunofluorescence for the viral envelope antigen in Huh7.5 cellsetection , E proteetection . To unde250–270 was assessed in patient sera. Serum samples were obtained from an outbreak in Paraná state, Brazil (2013) according to the approved guidelines of Fiocruz , within Instituto Carlos Chagas . Experiments involving human subjects were approved by the committee of ethics and research (Comitê de Ética e Pesquisa – CEP) of Fiocruz-RJ under protocol 617/11. Informed consent was obtained from all donors. 70 serum samples were evaluated for DV infection . Samples corresponded to 54 DV1 sera designated positive by at least one assay and from patients in the acute phase of disease (up to the seventh day after the onset of symptoms) and 16 control sera from non-infected individuals. An indirect ELISA protocol was used to identify anti-E250–270 IgG antibodies253–270 peptide (it was not possible to solubilise a full length E250–270 peptide), blocked and incubated with serum samples. An HRP-linked, goat anti-human IgG  was used as secondary antibody, and the plates were treated with o-phenylenediamine in citrate phosphate buffer containing 30% hydrogen peroxide. These ELISA data demonstrated this peptide to be antigenic in human natural infections, despite its predicted buried nature in the dimeric E form. One explanation for the development of antibodies to this site would be the dynamic movement of the virus particle, described as ‘breathing’37et al.250–270250–270 might have a functional role in the DV life cycle. In agreement with our data other groups suggest that amino acids within this region are key to the E protein biology. Accordingly, a single substitution at V251A moderately restricts DV replication and viral particle production in C6/36 and Vero cells, and reduces viral E protein detection by immunofluorescence in C6/36 and BHK-21 transfected cellset al. using replicons with random point mutations in E protein did not verify significant differences in E expression, budding and infectivity when mutating residues of the peptide´s N-terminus , or residues 266 and 27029250–270 to viral infectivity and further investigations should be done to elucidate the exact function this conserved peptide. Moreover the detection of antibodies anti E250–270 in natural infected patients point the possibility to the use of these peptide as immunological target, as recently shownThese data show that, despite the great diversity among the serotypes of dengue viruses there are at least two polypeptides within its main antigenic determinant that are highly conserved at both the sequence and structural levels. These two regions were also reported during analysis of pan-DV sequences as potential immune-relevant T cell determinants250–270 peptide, a systematic analysis of other highly conserved regions could suggest potential immunological/pharmacologic target for dengue treatment and control. Moreover, these knowledge can be extrapolated for other ssRNA genome viruses, and contribute to understand the evolution of their cryptic conserved peptides.Our strategy enables identification of the most conserved regions in DV genomes, other flavivirus such as ZKV, and also the rational designing of mutant viruses to investigate the importance of these regions in viral fitness and infectivity. As carried out for EHow to cite this article: Fleith, R. C. et al. Genome-wide analyses reveal a highly conserved Dengue virus envelope peptide which is critical for virus viability and antigenic in humans. Sci. Rep.6, 36339; doi: 10.1038/srep36339 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.