In one case (lnc-phox2bb), absence of putative DNA regulatory-elements, but not of the lncRNA transcript itself, resulted in abnormal development. LncRNAs might have redundant, subtle, or context-dependent roles, but extrapolation from our results suggests that the majority of individual zebrafish lncRNAs have no overt roles in embryogenesis, viability and fertility.Hundreds of long non-coding RNAs (lncRNAs) have been identified as potential regulators of gene expression, but their functions remain largely unknown. To study the role of lncRNAs during vertebrate development, we selected 25 zebrafish lncRNAs based on their conservation, expression profile or proximity to developmental regulators, and used CRISPR-Cas9 to generate 32 deletion alleles. We observed altered transcription of neighboring genes in some mutants, but none of the lncRNAs were required for embryogenesis, viability or fertility. Even RNAs with previously proposed non-coding functions ( LncRNAs have been proposed to affect the expression of neighboring or distant genes by acting as signaling, guiding, sequestering or scaffolding molecules . The funxist , tsix  and imppx  are welinc-pint , and upppperhand  are esse  and cyr defects . However defects . These c defects , (Han et defects . We thersquint).Transcriptomic studies of early embryonic development  and fiveThe genomic location of selected lncRNAs are depicted in Using CRISPR-Cas9  we generPrevious observations in mammalian cell culture systems suggested that lncRNA promoters can affect the expression of nearby genes . To testTo determine the developmental roles of our selected lncRNAs, we generated maternal-zygotic mutant embryos  and analyzed morphology from gastrulation to larval stages, when all major organs have formed. Previous large-scale screens  have shocyrano is evolutionarily conserved lncRNA and based on morpholino studies, has been suggested to have essential functions during zebrafish embryogenesis  for HuR during neuronal proliferation (miR-7 mediated embryonic stem cell differentiation (miR-7 in the adult mouse brain (cyranoa171) or the gene (cyranoa172), including the highly conserved miR-7 binding-site (oip5) was not affected in either of these mutants  into wild type and homozygous deletion mutants. The TSS-mutant allele lacked the e1i1 morpholino-binding site and the gene deletion allele lacked the CMiBS morpholino-binding site    by sequestering specific microRNAs (gas5 transcript can mimic Glucocorticoid Response Element and act as a decoy factor (riborepressor) for the Glucocorticoid Receptor (nr3c1)-mediated transcription (a173 embryos (at 1-dpf) and found significant upregulation for ptena in MZgas5a173 mutants (a173gas5 mutants were indistinguishable from wild type (cific 5)  that is cific 5)  and hostcific 5) . Knockdocific 5)  have indppressor  and exerppressor . Howeverto +127)  and resuto +127) . Expressd by 50% . PreviouicroRNAs . Additiocription . We anal mutants . Despiteild type , reachedLnc-setd1ba is the zebrafish orthologue of human LIMT  (lnc-setd1baa174). In situ hybridization and qRT-PCR revealed absence of lncRNA expression (setd1ba (setd1ba and the other neighboring gene rhoF at one-day post fertilization (1-dpf) (a174lnc-setd1ba mutants were indistinguishable from wild type (man LIMT  (LncRNA irection . Evolutielopment  promptedpression  and stro(setd1ba  during c (1-dpf) . Despiteild type , reachedSquint encodes a Nodal ligand involved in mesendoderm specification squint lead to delayed mesendoderm specification and partially penetrant cyclopia  that lacked most of the protein coding region and the 3’UTR, including the Dorsal Localization Element (DLE) implicated in maternal squint RNA localization (squint transcript was greatly reduced (~90%). MZsquinta175 embryos displayed partially penetrant cyclopia, similar to existing protein-disrupting squint alleles lnc-phox2bb (lnc-phox2bb (a176lnc-phox2bb) lacked lnc-phox2bb RNA   led to j2bba177) , and no x2bb RNA , but in n allele . This fi were used as wild-type fish in this study. Fish were maintained on daily 14 hr (light): 10 hr (dark) cycle at 28°C. All animal works were performed at the facilities of Harvard University, Faculty of Arts and Sciences (HU/FAS). This study was approved by the Harvard University/Faculty of Arts and Sciences Standing Committee on the Use of Animals in Research and Teaching Guide RNAs (gRNAs) were designed using CHOPCHOP  and syntVisual assessment of live embryos and larvae performed  to identAt day 5, formation of swim bladder and overall appearance of the embryos were checked again (at any stage 60–100 embryos were scored). Sixty to hundred fish from heterozygous mutant crosses were grown to adulthood and genotyped to identify the viability of adult homozygous fish. Validated homozygous mutant fish were further crossed together to test for potential fertility phenotypes or putative maternal functions of candidate lncRNAs.Antisense probes for in situ hybridization were transcribed using the DIG RNA labeling kit (Roche). All RNAs were purified using EZNA Total RNA kits (Omega Biotek). Embryos were fixed in 4% formaldehyde overnight at 4°C (embryos younger than 50% epiboly fixed for 2 days). In situ hybridizations were performed according to standard protocols . NBT/BCIef1a. Three technical replicates were used per condition. The qPCR primer sequences are listed in Total RNA was isolated from three individuals or sets of 10–20 embryos per condition using EZNA Total RNA kits (Omega Biotek). cDNA was generated using iScript cDNA Synthesis kit (Bio-Rad). qPCR was conducted using iTaq Universal SYBR Green Supermix (Bio-Rad) on a CFX96 (Bio-Rad). Gene expression levels were calculated relative to a reference gene, Embryos were anesthetized in Tricaine (Sigma) and mounted in 1% low melting temperature agarose (Sigma) with Tricaine, then imaged using a Zeiss SteREO Discovery.V12 microscope or Zeiss Axio Imager.Z1 microscope. Images were processed in FIJI/ImageJ . Brightnsquint mRNA, non-protein coding squint transcript  and the a175squint transcript were synthesized as gBlocks (IDT) containing 5’ XhoI cut site and 3’ NotI site. Fragments were digested and inserted the pCS2 plasmid. Positive colonies were selected, and sanger sequenced to assure the accuracy of the gene synthesis process. Sequences of the constructs are provided in h2b-gfp was used as control mRNA. Each injection mix contained 30 ng/ul of squint or control mRNA). 1 nl of mRNA mix was injected into the yolk of one-cell stage embryos.The sequences for the wild-type Morpholinos were ordered from Gene Tools and injected based on  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Elisabeth M Busch-Nentwich as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Shawn Burgess (Reviewer #3).Thank you for submitting your article "Long non-coding RNAs are largely dispensable for zebrafish embryogenesis, viability and fertility" 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:This study describes the generation and morphological characterisation of whole gene or transcription start site deletion mutants in 25 zebrafish lncRNAs. The authors find that the mutants display neither overt developmental phenotypes nor viability or fertility defects. The phenotype of one of the whole gene deletion mutants is due to loss of regulatory elements of neighbouring protein-coding genes which reside within the deleted lncRNA. The authors conclude that, based on these 25 examples, most zebrafish lncRNAs are dispensable for development, viability and fertility.The experiments are well designed and carefully controlled. The results challenge previous findings using knockdown strategies and will be of high interest.Essential revisions:1) The title is not fully supported by the data. "Largely dispensable" implies that some phenotypes have been observed. Also, the extrapolation to all lncRNAs suggested in the title is overreaching based on 25 genes. A less general title, e.g. "Zebrafish embryogenesis, viability and fertility are not overtly affected by loss of embryonically expressed long non-coding RNAs", would be more appropriate. Similarly, in the abstract, the authors state that the lncRNAs are "dispensable for embryogenesis." We suggest softening that statement. There has been a high profile paper that has walked back similar assertions about ultraconserved non-coding sequences . There could easily be (as the authors acknowledge in the conclusions) subtle differences in embryogenesis not detected by eye but that are important to fish in the wild. Saying the fish can survive without the lncRNAs is as bold a statement as you can make with the data presented.The authors state "Previous large-scale screens have shown that the visual assessment of live embryos and larvae is a powerful and efficient approach to identify mutant phenotypes, ranging from gastrulation movements […] to the formation of brain, […] and gut." This is certainly true, but it is also true that many phenotypes are missed at this level of scrutiny and a sentence admitting that should be included.2) More detail needs to be provided on the chosen lncRNAs. The selection criteria need to be described more thoroughly. How was evolutionary conservation determined? Conservation of synteny or sequence conservation? What are the thresholds for these criteria ? Also, please provide Figure 1 as a table rather than an image and provide the used lncRNA gene models as files that can be uploaded and viewed in a genome browser.Does expression of the lncRNAs correlate with their neighbours? The authors show expression dynamics across development based on RNA-seq data from their 2012 publication. In the meantime more detailed gene expression datasets have been published, not least single cell data from the same lab. It would be interesting to see whether some of the early expressed lncRNAs can be detected in the single cell data and whether any lineage commitment can be identified.How do previously described targets (e.g. trans targets for gas5) behave in the mutants?3) There has been controversy around the squint locus for some time, and while the presented data are fairly convincing, they are not definitive in ruling out squint as a cncRNA family member, as there are still pieces of the gene left both 5' and 3' that could maintain residual function. To maintain the assertion, a complete deletion would be required. Alternatively, it would be acceptable to acknowledge that there may still be an  chance that the remaining gene sequences could have residual non-coding functions.setdb1a in the lnc-setdb1a mutant is very interesting, but not investigated any further. The authors state that lnc-setdb1a mutants are viable and fertile, but it is possible that maternal-zygotic mutants show impaired fertilisation or develop a gametogenesis defect over time. Have the authors looked at fertilisation rates in successive clutches from MZ mutant lnc-setdb1a adults? Is there an effect on setdb1a target gene expression in oocytes?4) The 500-fold increase of 5) Data on relative expression: Statistical tests should be used to determine whether the difference between wild type and mutant is significant. Further, relative expression levels are based on three biological replicates with three technical replicates each. This means a maximum of nine points underlie each bar in the bar charts. Similarly, N=4 in panel 5E. It would therefore increase data transparency to show the individual data points (and mean and SD) instead of bar charts.In Figure 3D, Figure 4E, Figure 5C and Figure 6E it is not clear what the expression is relative to. Only Figure legend 5C states "qRT-PCR […] on wild-type and MZ squint embryos", which suggests all measurements are normalised to ef1a levels  and then the wild-type level is set to 1 for each measured gene, but this needs to be clarified in the figure legends. Essential revisions:1) The title is not fully supported by the data. "Largely dispensable" implies that some phenotypes have been observed. Also, the extrapolation to all lncRNAs suggested in the title is overreaching based on 25 genes. A less general title, e.g. "Zebrafish embryogenesis, viability and fertility are not overtly affected by loss of embryonically expressed long non-coding RNAs", would be more appropriate. Similarly, in the abstract, the authors state that the lncRNAs are "dispensable for embryogenesis." We suggest softening that statement. There has been a high profile paper that has walked back similar assertions about ultraconserved non-coding sequences . There could easily be (as the authors acknowledge in the conclusions) subtle differences in embryogenesis not detected by eye but that are important to fish in the wild. Saying the fish can survive without the lncRNAs is as bold a statement as you can make with the data presented.We have changed the Title to:“Individual long non-coding RNAs have no overt functions in zebrafish embryogenesis, viability and fertility”We also modified the last sentence of the Abstract to: “LncRNAs might have redundant, subtle, or context-dependent roles, but extrapolation from our results suggests that the majority of individual zebrafish lncRNAs have no overt roles in embryogenesis, viability and fertility”.The authors state "Previous large-scale screens have shown that the visual assessment of live embryos and larvae is a powerful and efficient approach to identify mutant phenotypes, ranging from gastrulation movements […] to the formation of brain, […] and gut." This is certainly true, but it is also true that many phenotypes are missed at this level of scrutiny and a sentence admitting that should be included.We added to the Discussion section: “Our phenotypic screen does not exclude more subtle phenotypes; e.g. in behavior or brain activity”.2) More detail needs to be provided on the chosen lncRNAs. The selection criteria need to be described more thoroughly. How was evolutionary conservation determined? Conservation of synteny or sequence conservation? What are the thresholds for these criteria ?In the current manuscript we have state that:“For our knockout study we selected 24 bona fide lncRNAs based on syntenic and sequence conservation, expression dynamics and proximity to developmental regulatory genes (see Table 1). These criteria were chosen to increase the likelihood of functional requirement.”We have revised the manuscript as follows:“For our mutant analysis we selected 24 bona fide lncRNAs based on synteny (conserved relative position on at least one other vertebrate genome), sequence conservation, expression dynamics  and proximity to developmental regulatory genes (see Table 1). These criteria were chosen to increase the likelihood of potential functional requirements of the selected lncRNAs.”We have also extended the section “selection criteria” in Table 1 to better represent the main selection criteria for each lncRNA.Also, please provide Figure 1 as a table rather than an image and provide the used lncRNA gene models as files that can be uploaded and viewed in a genome browser.We replaced Figure 1 with Table 1 and provided a genome-browser-compatible file for the coordinates of the studied lncRNAs (Supplementary file 2).Does expression of the lncRNAs correlate with their neighbours?We did not systematically investigate this question, but we have provided the expression dynamics of investigated lncRNAs and their immediate neighbors in a 200kb window. In our selection, we did not find unifying themes for co-expression patterns of lncRNAs and their immediate neighboring genes.The authors show expression dynamics across development based on RNA-seq data from their 2012 publication. In the meantime more detailed gene expression datasets have been published, not least single cell data from the same lab. It would be interesting to see whether some of the early expressed lncRNAs can be detected in the single cell data and whether any lineage commitment can be identified.Based on this suggestion, we tested all of our lncRNAs with assigned transcript ID, in the single cell data from Farrell et al., 2018. We observe clear trajectory restriction only for squint and a potential partial enrichment might exist for lnc-3852. Expression distribution and trajectory trees for these lncRNAs are provided in Author response images 1-10. No data could be retrieved for four lncRNAs . Although not very informative, we can add this analysis to the paper if the reviewers wish.How do previously described targets (e.g. trans targets for gas5) behave in the mutants?a173 embryos (at 1-dpf) relative to WT by qRT-PCR using previously published primer pairs for these genes in zebrafish. T-test analysis revealed significant upregulation for ptena in MZgas5a173 mutants.Previous studies have shown that gas5 lncRNA can act in trans to affect pten expression (ptena and ptenb in zebrafish) by sequestering specific microRNAs including miR-103, miR-222 and miR-21. Additionally, gas5 transcript can mimic Glucocorticoid Response Element and act as a decoy factor for the Glucocorticoid Receptor (nr3c1) mediated transcription. We analyzed the expression level changes of these genes in MZgas53) There has been controversy around the squint locus for some time, and while the presented data are fairly convincing, they are not definitive in ruling out squint as a cncRNA family member, as there are still pieces of the gene left both 5' and 3' that could maintain residual function. To maintain the assertion, a complete deletion would be required. Alternatively, it would be acceptable to acknowledge that there may still be an  chance that the remaining gene sequences could have residual non-coding functions.The previously published claims were related to the function of specific conserved element in the 3’UTR of squint. Our deletion mutant is sufficient to rule out the proposed dorsalizing function for the 3’UTR of maternal squint. In our mutant allele, 525 basepairs comprising of 5’UTR (178bp), first exon (280bp) and part of the second exon (67bp) remain in the genome. The above-mentioned sequences encode part of the squint prodomain and might still provide some unclaimed non-coding function.We revised text as follows:a175) that lacked most of the protein coding region and the 3’UTR, including the Dorsal Localization Element (DLE) implicated in maternal squint RNA localization52 . In this allele 525bp  out of the 1592bp-long mature transcript remain in the genome .”“To investigate the non-coding roles of squint mRNA we generated a deletion allele (squint“These results indicate that squint 3’UTR does not have the previously proposed noncoding functions and that the squint transcript may not be a member of the cncRNA family.”4) The 500-fold increase of setdb1a in the lnc-setdb1a mutant is very interesting, but not investigated any further. The authors state that lnc-setdb1a mutants are viable and fertile, but it is possible that maternal-zygotic mutants show impaired fertilisation or develop a gametogenesis defect over time. Have the authors looked at fertilisation rates in successive clutches from MZ mutant lnc-setdb1a adults? Is there an effect on setdb1a target gene expression in oocytes?a176 fish (17-month old), that resulted in clutches which did not show significant differences by multiple t-test analysis in any of the assayed categories .We did not systematically quantify the clutch size and fertilization rates. We scored four independent crosses of Wild type -AB (14-month old) and MZlnc-setd1ba5) Data on relative expression: Statistical tests should be used to determine whether the difference between wild type and mutant is significant. Further, relative expression levels are based on three biological replicates with three technical replicates each. This means a maximum of nine points underlie each bar in the bar charts. Similarly, N=4 in panel 5E. It would therefore increase data transparency to show the individual data points (and mean and SD) instead of bar charts. In Figure 3D, Figure 4E, Figure 5C and Figure 6E it is not clear what the expression is relative to. Only Figure legend 5C states "qRT-PCR […]) on wild-type and MZ squint embryos", which suggests all measurements are normalised to ef1a levels  and then the wild-type level is set to 1 for each measured gene, but this needs to be clarified in the figure legends.All the bar-graphs in the main figures are changed to represent individual data points, and appropriate statistical analysis is performed to define the significance of observed differences.


Our findings suggest that normal non-cycling differentiated cells may serve as a reservoir of iatrogenically induced mutations. These results provide more insight into the possible molecular mechanisms that underlie late-life morbidities in cancer survivors exposed to chemotherapy.Many anticancer drugs are genotoxic agents inducing DNA breaks in actively proliferating cancer cells. However, these same drugs also induce mutations, mostly genome structural variations (GSVs). The detection of GSVs in normal cells and tissues is a major challenge due to the very low abundance of these mutations, which are essentially only detectable in clonal outgrowths, such as tumors. Previously we developed Structural Variant Search (SVS) – an NGS-based assay for the quantitative detection of somatic GSVs in normal cells. Using an improved version of SVS we now demonstrate that the same dose of the anti-cancer drug bleomycin induces about 5 times more somatic GSVs in quiescent primary human fibroblasts than in proliferating cells. GVS induction in non-dividing, normal cells was subsequently confirmed  However, successful treatment is often associated with compromised quality of life due to adverse effects of the applied therapy on normal cells and tissues. Short-term complications, such as hair loss2, disorders of excretory3 and gastrointestinal4 systems, are considered to be a consequence of cytotoxic or cytostatic effects of anticancer drugs on actively dividing host cells and show up shortly after treatment start. Long-term complications of cancer treatment, such as symptoms of premature aging5 and secondary neoplasms6, are developing years after treatment and their exact mechanism is understudied.Advances in early cancer diagnosis and treatment strategies have resulted in over 14 million cancer survivors in the United States7. Notably, virtually all genotoxic anticancer drugs are potent clastogens exerting their cytotoxicity through induction of DNA breaks in affected cells8. However, DNA break repair mechanisms (non-homologous end joining and homologous recombination being most prominent10) are able to recover the DNA chemical structure and rescue the cell, but often at the cost of compromised genome integrity, namely formation of genome structural variants (GSVs)11. Hence, it is conceivable that chemotherapy will accelerate the normal age-associated accumulation of GSVs in tissues13, offering one possible explanation for the multiple late-life morbidities, including secondary malignancies and early onset of symptoms typically associated with aging, in cancer survivors treated with chemotherapy16.Many anticancer drugs are genotoxic agents and induce apoptosis in actively dividing cancer cells as a response to DNA damage17. Recently we developed Structural Variant Search (SVS) – a novel next-generation sequencing (NGS)-based analytical tool for the quantitative detection of somatic GSVs18. This assay utilizes ultra-low coverage sequencing data and allows accurate genome-wide assessment of frequency and spectra of low-abundant GSVs induced by bleomycin, a cancer therapeutic agent. Using an improved version of SVS we now show that quiescent cells are significantly more susceptible to the mutagenic effects of chemotherapeutic drug bleomycin than proliferating cells and further demonstrate that GSVs are induced in vivo in postmitotic tissues of mice treated with the drug. These results shed light on a possible mechanism of late-life effects of cancer chemotherapy.Chemotherapy-induced GSVs are independent events unique for each affected cell. Thus, while their overall frequency may be high, chemotherapy-induced GSVs involve ultra-low abundant mutations dispersed among affected cells and considered undetectable in normal cells. Thus far, accurate evaluation of these chemotherapy-associated GSVs in normal tissues of cancer patients was not feasible because currently available approaches are not capable of detecting somatic GSVs due to the low abundance of each particular GSV19. Cells were maintained in 10% CO2 and 3% O2 atmosphere at 37 °C in DMEM  supplemented with 10% FBS (Gibco). The quiescent state of the cells was achieved by culturing in the medium supplemented with 0.1% of FBS for 24 hours prior to the experiment start. For the treatment serum-free medium with bleomycin  was prepared at the time of application from stock solution of the drug (10 mM in DMSO) and applied for 1 hour at the atmospheric O2 level. At the end of the treatment, cells were washed twice with PBS and cultured further in the medium supplemented either with 10% FBS (proliferating cells) or 0.1% FBS (quiescent cells). Proliferating cells were trypsinized 24 hours and 72 hours after the treatment, half of the cells was collected for DNA extraction and half was left in culture until the next time point. Quiescent cells were maintained without passaging and collected for DNA extraction at the time points designated in the text.Human dermal fibroblasts (HDFs) from a 6-year-old male human were provided by H. Choi . The human fibroblasts were collected and protocols were approved as described inTo determine viable cell numbers, trypsinized cells were stained using Guava ViaCount Reagent  and analyzed using Guava EasyCyte Plus flow cytometer (MilliporeSigma) as recommended by the manufacturer. The cell doubling time was calculated using the formula:2) was delivered by IP injection; control mice received an injection of saline. Mice were euthanized 24 hours after injections and liver, lung and heart tissues were collected aseptically and kept at −80 °C until use.All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Albert Einstein College of Medicine and performed in accordance with relevant guidelines and regulations. Six three-month-old female C57BL/6 mice were obtained from the National Institute on Aging and maintained in the animal husbandry facility of the Albert Einstein College of Medicine until treatment. A single dose of bleomycin in saline  following the manufacturer’s instructions. DNA concentration was determined using the Qubit 2.0 fluorimeter .Total DNA was isolated from tissue and cultured cells using the Quick-gDNA20.Long-range PCR was performed with the Takara LA PCR kit  according to the manufacturer's protocol. Five DNA fragments were amplified from the mouse genomic DNA using previously designed and validated primers targeting distant regions of the mouse genome Table 20.Table 21 were filtered out during analysis of the potential variants.Obtained amplicons were mixed together in equimolar quantities and used as a template for sequencing library preparation. The resulting “artificial genome” is guaranteed free from “interchromosomal” rearrangements between these five fragments simulating different chromosomes because each of them was amplified individually and chances that potential PCR-mediated errors will produce rearrangements between these particular regions of genome are negligibly small. Possible PCR-mediated recombination between target DNA fragments and the rest of the genome occurring via template-switching mechanism18, but some modifications were made to improve its accuracy further. The improvements mainly reside in sequencing libraries preparation protocol. Ligation-based sequencing libraries were prepared using NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent , following the manufacturer’s instructions. Transposon-based libraries were prepared as we described previously22. Ligation-mediated chimera-free (LCF) libraries were prepared as follows:Fragmentation using NEBNext dsDNA Fragmentase (NEB);End-repair I using S1 nuclease ;A-tailing using Terminal Transferase (TdT) polymerase (NEB) and dATP (NEB);NNNNNNNNNNTTTTTTTT-3′ ;Ligation of single-stranded sequencing adaptors using Taq DNA Ligase (NEB). The sequencing adaptor oligos were: 5′-CCTCTCTATGGGCAGTCGGTGATTTTTTTT-3′  and 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGEnd-repair II using T4 DNA polymerase (NEB) and dNTP mix (NEB).The SVS method used has been described previously18.All completed libraries were size selected using the PippinHT System  and quantified using KAPA Library Quantification Kit for Ion Torrent Platform . Sequencing was performed on Ion Proton System (Life Technologies Corporation) using the Ion PI™ Hi-Q Sequencing 200 Kit and Ion PI™ Chip Kit v3, following the manufacturer’s instructions. Sequencing data analysis was performed using our proprietary software as described previouslyThe descriptive statistics and the P values were calculated using a two-sample t-test (Microsoft Excel software package).18. To improve accuracy of GSV calling in SVS we have designed a Ligation-mediated Chimera-Free (LCF) sequencing library preparation protocol . Utilization of the PCR-free LCF protocol resulted in further suppression frequency of artifacts to 0.08 rearrangements per 1 million sequencing reads  and human lung fibroblasts (IMR90) at different time points after treatment. As expected, bleomycin treatment led to a statistically significant increase of somatic GSV frequency in both cell strains with the maximum observed value at 24 hours after treatment Fig. .Figure 2Next we analyzed GSV frequency induced by bleomycin treatment in quiescent HDFs and IMR90 cells. The quiescence state was generated by culturing cells at low serum concentration (0.1% FBS). Similar to its effect on proliferating cells, bleomycin caused a statistically significant increase of GSVs in quiescent cells of both strains at 24 hours after treatment Fig. . By contNot unexpectedly, bleomycin treatment of actively dividing human cells led to a significant decrease in cell growth rate during the first 24 hours after the intervention; population doubling time was ~75 and ~124 hours compared to ~22 and 18 hours for control HDFs and IMR90 cells, respectively Fig. . HoweverTreatment with bleomycin caused substantial changes in the type of somatic GSVs in proliferating HDFs and IMR90 cells Fig. . While fin vivo. For this purpose we used C57BL/6 mice treated with sub-lethal doses of bleomycin. The experimental group received an intraperitoneal (IP) injection with bleomycin (40 mg/m2) in saline, while control animals were injected with the same (0.5 ml) volume of saline only. Mice were sacrificed for analysis 24 hours after the treatment. We found that a single IP injection of bleomycin induced a statistically significant elevation of somatic GSV frequency in the mouse liver and heart tissues  is a challenging task due to their low abundance, which makes them indistinguishable from NGS-associated errors. Several approaches have been introduced allowing assessment of point mutations and small indels, but not genome structural variants (GSVs)23. One plausible explanation of the observed more than 5-fold difference in mutational response is that dividing and quiescent cells cope with DNA DSBs in a different way. While bleomycin-induced DNA DSBs in proliferating cells are predominantly repaired by relatively error-free homologous recombination mechanism, the repair of DNA DSBs in quiescent cells is mediated by error-prone non-homologous end joining, resulting in significantly higher amounts of somatic GSVs25.Using bleomycin-treated quiescent cells as a surrogate for non-dividing cells in cancer patients treated with chemotherapeutic drugs we found that cells withdrawn from cycling are susceptible to bleomycin-induced mutagenesis even more than actively proliferating cells. Notably, this phenomenon was observed with two different normal primary human cells, confirming that the observed effect does not depend on genetic features of the cells, but rather has general character. Of note, these results are in line with previously reported increased accumulation of genome rearrangements in quiescent cells upon treatment with hydrogen peroxideDNA mutations are stable irreversible changes in DNA sequence and the observed decline of somatic GSV load in actively proliferating cells was not expected because cells can repair DNA damage such as breaks, but lack mechanisms capable of restoring sequence integrity. Hence, once mutations are generated, as a consequence or erroneous repair or replication, they should be irreversible and remain in the affected cell population unless cell death removes a mutated cell. Thus, the most likely explanation of the decrease of GSVs in proliferating cells is a growth advantage of cells with low levels of GSVs. Heavily mutated cells would either die or display a significantly lower growth rate. In our experiment with proliferating cells treated with bleomycin, the cells with the lowest mutation load, continue to proliferate and dilute the cells with higher numbers of mutations. Indeed, unlike proliferating cell populations where both HDFs and IMR90 cells were rapidly losing bleomycin-induced GSVs after 24 hours, quiescent HDFs, least susceptible to the toxic effects of the drug of the two cell strains used Fig. , showed in vivo experiments confirm our observations with quiescent cells in culture. They demonstrated that a single dose of bleomycin leads to a statistically significant increase of somatic GSV frequency in the mouse liver and heart, two postmitotic tissues.Our subsequent 26. SVS provides a practical approach for further systematic studies addressing an urgent unmet need to understand and predict how chemotherapy affects normal tissue and leads to adverse events.In summary, using our new, NGS-based method for direct GSV detection in normal, non-clonally developed tissue we provide clear evidence indicating that non-cycling differentiated cells can serve as a reservoir of iatrogenically induced somatic mutations. Our present findings provide an immediate approach for studying the molecular mechanisms of late morbidities in cancer survivors exposed to chemotherapy. This is particularly important for childhood cancer survivors whose successful treatment is often associated with compromised quality of consecutive life, which averages more than 60 yearsSupplementary tables


Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization.RPGR gene, 353-bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified.Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing. Inherited retinal diseases (IRDs) are observed in highly variable phenotypes in 1 in 2000 people –5. The gIn contrast to dideoxy sequencing, next generation sequencing (NGS) has reduced the time it takes to sequence massive amounts of DNA from decades to months. Whole exome sequencing (WES) selectively targets the 20,000 coding genes that constitute approximately 2% of the human genome, as they are predicted to be responsible for 85% of rare and common inherited diseases . HoweverIn this study, we present individuals and their family members in whom no disease-causing variants had been identified by clinical exome sequencing. Pathogenic or likely pathogenic variants were subsequently identified by targeted single-gene sequencing, gene panels, whole genome sequencing (WGS), or array CGH analysis, which provided genetic diagnoses of retinitis pigmentosa (X-linked RP) (MIM 300455), (RP62) MIM 614181), (RP25) (MIM 602772), (RP11) (MIM 600138), Stargardt disease 1 (STGD1) (MIM 248200), and Leber congenital amaurosis 1 (LCA1) (MIM 204000). Through our investigation, we propose possible molecular mechanisms underlying the missed variant calls and emphasize the need for continued search for the causative variant in such cases. Furthermore, we suggest increased utilization of WGS, a more comprehensive type of NGS that has recently shown a significant reduction in cost [181, , indirect ophthalmoscopy, and retinal imaging performed following pupillary dilation. Color fundus photography, infrared reflectance imaging, spectral-domain optical coherence tomography (SD-OCT), and short-wavelength fundus autofluorescence , were obtained using Spectralis HRA + OCT device . Wide-angle color fundus photography was performed using Daytona Optos device .RPGR gene was evaluated using long range PCR followed by DNA fragmentation and long read (250 bp-paired end) high-depth Illumina sequencing.DNA was isolated from peripheral whole blood of each participant for WES at the Personalized Genomic Medicine Laboratory at Columbia University Irving Medical Center. WES was performed as first-tier genetic testing for the unbiased search for the genetic cause of disease. WES was performed with Agilent SureSelectXT Human All Exon V5 + UTRs capture  and Illumina HiSeq2500 sequencing technology . The WES output reads were mapped against the reference genome (GRCh 37/hg19) using NextGENe software  and our own proprietary analytical pipeline to sequence alignment for variant calling. Due to the technical limitations of sequence capture employed in this test, intronic variants were not predicted to be identified. Targeted sequencing of the The following molecular diagnostic tests were ordered based on the patient’s family history and the clinical features: targeted gene sequencing and inherited retinal dystrophy panels due to the 100% exon coverage and 99% sensitivity for nucleotide base alterations as well as small deletions and insertions, WGS for the detection of noncoding variants, and array CGH of IRD genes for the detection of structural variants such as CNVs with 99% sensitivity for the detection of nucleotide base changes. Gene sequencing was conducted at the Personalized Genomic Medicine Laboratory at Columbia University . Targeted gene sequencing was conducted at Molecular Vision Laboratory , or University of Utah Genome Center . Retinal dystrophy panels were conducted at Blueprint Genetics , Casey Eye Institute Diagnostic Laboratory at Oregon Health & Science University , Prevention Genetics , or GeneDx . WGS was performed at New York Genome Center . Array CGH was analyzed at Molecular Vision Laboratory . Technical information for each gene testing is found in Table The molecular test report of each patient was reviewed for genes known to cause IRDs. We used a joint consensus recommendation of the ACMG and the Association for Molecular Pathology  for the RPGR, MAK, EYS, PRPF31, ABCA4, and GUCY2D (Table RPGR), autosomal recessive RP (MAK and EYS), autosomal dominant RP (PRPF31), Stargardt disease (ABCA4), and Leber congenital amaurosis (GUCY2D). Molecular genetic testing predicted the variants were genetically deleterious according to the ACMG guidelines. There were seven previously undescribed variants: two protein-truncating variants of RPGR open reading frame of exon 15 (ORF15) c.2752G > T (p.Glu918*) and RPGR ORF15 c.2501_2502del (p.Glu834Glyfs*244), two large EYS exonic deletions from exon 15 to 18 and 20 to 22, one large PRPF31 exonic deletion from exon 1 to 9, two deep intronic variants of ABCA4 c.4539 + 2085G > A, and GUCY2D c.1378 + 151C > G.Of 250 patients and family members that received WES between 2013 and 2018, 108 received results that reported no pathogenic variants and therefore offered no genetic explanation for their clinical diagnosis. Of these, a total of 26 cases (21 patients and 5 family members) received additional genetic testing. The remaining 82 cases did not receive subsequent genetic sequencing. WES did not identify 26 variants in the following genes: RPGR variants found in ORF15, including 12 frameshift deletions and three nonsense mutations. These variants were identified by targeted gene sequencing. The homozygous 353-bp Alu insertion variant in exon 9 of the MAK gene was also missed by WES, which was identified by a gene panel . In the EYS gene, WES did not detect two large exonic deletion variants spanning exons 15 to 18 and 20 to 22 out of a total of 43 exons, each over 54 kb and 49 kb in length, respectively. These were subsequently identified with array CGH of IRD genes. The exonic deletion variant of over 52 kb in length in the PRPF31 gene that spanned exons 1 to 9 out of a total of 14 exons was identified by a gene panel . In the ABCA4 gene, WES did not identify two deep intronic variants, c.4539 + 2085G > A and c.2160 + 584A > G, which were discovered by targeted gene sequencing of the ABCA4 gene. The deep intronic variant c.1378 + 151C > G in the GUCY2D gene that was not identified by multiple tests, including WES, array CGH analysis, and single-gene analysis for deletion and duplication, was subsequently detected by WGS. Clinical descriptions of selected cases representative of each gene are provided below. The case images of RP are shown in Fig. Overall, WES did not detect 15 RPGR carriers  variant in the proband and his daughter.Case 13 is a 44-year old man who was diagnosed with RP at the age of 8 .Case 21 is a 51-year-old woman who was diagnosed with RP 20 years ago  variant in the WES has contributed to a significant advancement in our understanding of the genetic causes of inherited diseases through the discovery of novel variants, enhancement of important genotype-phenotype associations, and progression of gene-directed therapy. Approximately 2600 gene therapy clinical trials in 38 countries have been or are being conducted .WES as first-tier genetic testing enabled an unbiased search for the genetic causes of disease. This “WES-first” approach has been associated with a lower incremental cost per additional diagnosis than the traditional WES-later approach –24. The RPGR ORF15, which constitutes a large 3′ terminal region of the RPGR gene, is a mutational hotspot associated with up to 60% of pathogenic mutations of X-linked RP [RPGR ORF15 variants were the most common, as observed in Cases 1 to 15. Compared to the constitutive RPGR isoform that spans exons 1 to 19, the ORF15 isoform terminates in intron 15, a GA-rich region that encodes Glu-Gly acidic domains [RPGR gene following the negative WES analysis. Additionally, the tapetal-like reflex observed in the daughter strongly suggested a carrier status for an RPGR variant  [MAK gene  . They prEYS gene and one in the PRPF31 gene. The WES pipeline is prone to miss these variant types because it is constructed to detect SNVs or short indels [Three exonic deletion variants were not detected by WES: two in the t indels . In a stt indels .The second class of limitations of WES involves the remaining 98% of the genome beyond its sequencing scope. By design, WES does not cover intronic variants, as exons have been perceived as the primary regions of the genome that when disrupted are responsible for causing disease. However, genome sequencing has revealed the clinical significance of structural and regulatory variants of the noncoding genome. Deep intronic mutations can be pathogenic by activating non-canonical splice sites, changing splicing regulatory elements, or disrupting transcription regulatory motifs .ABCA4, and one in the GUCY2D gene. The genetic variants of deep intronic nature in the ABCA4 gene have been previously reported as the cause for the missing variant of STGD1 [Three intronic missense variants were not identified by WES: two in of STGD1 ; 67% of of STGD1 , 37. Theof STGD1 . ConversGUCY2D gene harbored in Case 25, which is predicted to activate a new splicing donor site. Like STGD, LCA shows autosomal recessive inheritance that manifests with the presence of bi-allelic variants. Therefore, when WES identifies only one variant in a gene known to cause LCA, it justifies for the subsequent search for the second variant, most likely one of a deep intronic nature, as this type is commonly associated with LCA. Previous studies have consistently established the association of a deep intronic c.2991 + 1655A > G variant in the CEP290 gene with LCA, occurring in more than half of CEP290-associated cases [CEP290 c.2991 + 1655A > G. If the search warrants an unbiased approach, WGS would be recommended.WGS detected the heterozygous deep intronic variant in the ed cases , 40. Thied cases . TherefoOur study illustrates that following a negative WES report, further genetic testing, such as targeted gene panels that cover deep intronic and highly repetitive regions or WGS, is needed to account for these limitations. These alternative tests are particularly important when the patient’s clinical phenotype is compelling. However, the interpretive limitation of these sequencing platforms should also be noted. The clinical significance of the identified variant is predicted based on previously reported findings, which constitute a body of medical knowledge that is continuously expanding.RPGR ORF 15 region still represent a technical challenge for WGS because of the highly repetitive regions that lead to poor coverage. Further analysis, including targeted long-range PCR following DNA fragmentation and long read high depth sequencing, are therefore required in addition to WES, or WGS are required for these types of cases.Further investigation of gene variants in a larger cohort will strengthen the need to re-evaluate negative WES results with additional genetic testing. Although it functions with a lower overall coverage depth of 30x compared to WES (100x), WGS performs at a higher hybridization efficiency because it has a more consistent read depth and covers the non-targeted regions of WES. Compared to using WES alone, supplementing unresolved WES cases with WGS identified 14 out of 45 additional pathogenic variants, which translates to a detection rate of 31% [RPGR ORF15, MAK 353-bp Alu insertion, large exonic deletions in EYS and PRPF31, and intronic variants in ABCA4 and GUCY2D. While the current cost per diagnosis is higher for WGS compared to that of WES, it continues to fall [Despite the high diagnostic yield of WES, there are inherent technical limitations that lead to missed variant callings. As achieving genetic diagnosis is imperative for clinicians and patients to move forward with potential treatments such as gene replacement therapy, a negative WES analysis should be re-evaluated when compelling clinical findings support the presentation of a distinct genetic etiology. We used 14 targeted gene sequencing, 10 gene panels, one WGS, and one array CGH to identify the undetected gene variants of high GA-repeat regions of


Placental inflammation, often presenting as acute chorioamnionitis (aCA), is commonly associated with preterm birth. Preterm birth can have both immediate and long-term adverse effects on the health of the baby. Developing biomarkers of inflammation in the placenta can help to understand its effects and potentially lead to new approaches for rapid prenatal diagnosis of aCA. We aimed to characterize epigenetic variation associated with aCA in placenta (chorionic villi) and fetal membranes (chorion and amnion) to better understand how aCA may impact processes that lead to preterm birth. This study lays the groundwork for development of novel biomarkers for aCA.Samples from 44 preterm placentas (chorionic villi) as well as matched chorion and amnion for 16 of these cases were collected for this study. These samples were profiled using the Illumina Infinium HumanMethylation850 BeadChip to measure DNA methylation (DNAm) at 866,895 CpGs across the genome. An additional 78 placental samples were utilized to independently validate the array findings by pyrosequencing.Using a false discovery rate of < 0.15 and average group difference in DNAm of > 0.05, 66 differentially methylated (DM) CpG sites were identified between aCA cases and non-aCA cases in chorionic villi. For the majority of these 66 DM CpGs, the DNAm profile of the aCA cases as compared to the non-aCA cases trended in the direction of the blood cell DNAm. Interestingly, neutrophil-specific DNAm signatures, but not those associated with other immune cell types, were capable of separating aCA cases from the non-aCA cases.Our results suggest that aCA-associated placentas showed altered DNAm signatures that were not observed in the absence of aCA. This DNAm profile is consistent with the activation of the innate immune response in the placenta and/or reflect increase in neutrophils as a response to inflammation.The online version of this article (10.1186/s13072-018-0234-9) contains supplementary material, which is available to authorized users. Approximately 15 million children globally  are born before 37 weeks of gestation and thus classified as preterm births (PTB) , 3. PretAcute chorioamnionitis may be associated with microbial invasion of the amniotic space; however, it can also occur in the absence of detectable microorganisms and may be triggered by non-microbial “danger signals” including cellular stress and cell death , 10. TheEpigenetics may be a useful tool to characterize some of the molecular and cellular processes involved in placental inflammation, yet its role in the context of aCA is substantially understudied. Epigenetic influences refer to mitotically heritable chemical modifications to DNA or the proteins around which DNA is bound that occur in the absence of a change to the nucleotide sequence. DNA methylation (DNAm), the addition of a methyl group to the 5′carbon of cytosine, most typically at cytosine–phosphate–guanosine (CpG) dinucleotides, is a commonly interrogated epigenetic mark in human population studies. The relationship between DNAm and gene expression regulation is complex and tends to be dependent on genomic context . In the While DNAm may reflect changes to gene expression in specific cell types, it may also reflect altered cell composition. DNAm signatures differ strikingly between different cell lineages, including those in extra-embryonic tissue. Chorionic villi have a unique DNAm landscape compared with maternal decidua, fetal membranes (chorion and amnion), and embryonic tissues  . In wholN = 42 aCA cases, 36 non-aCA cases) by a site-specific technique for measurement of DNAm (pyrosequencing). Finally, we used unsupervised hierarchical clustering with neutrophil-specific CpG sites to segregate chorionic villus samples based on aCA status. Collectively, our genome-wide array analysis of aCA placentas was able to capture some immunity-related changes, which may be in part attributable to changes to immune cell-type ratios and gene expression during placental inflammation in aCA.An overview of the study design is presented in Fig. This study was approved by the research ethics committees of University of British Columbia and the Children’s and Women’s Health Centre of British Columbia (H04-70488). Samples used in this study were de-identified, and only non-identifiable clinical information was available for this study. A small piece of the fetal membranes (amnion and chorion) was removed and washed thoroughly to minimize maternal blood contamination. Independent samples of chorionic villi were taken from 2 to 3 distinct locations from the fetal side of the placenta to avoid maternal decidua. Extraction of DNA was performed by the standard salting out method , and DNADiscovery cohort: Placentas from 44 PTBs, 22 with aCA and 22 without aCA were collected from the deliveries at the BC Women’s Hospital. A subset of these samples has been previously described in Wilson et al. . DiagnosSince ancestry was largely unknown in the discovery cohort, a panel of 57 ancestry informative marker (AIM) single nucleotide polymorphisms (SNPs) –23 was uValidation cohort: Fresh frozen placental chorionic villi was obtained from the Anatomical Pathology Laboratory at BC Children’s & Women’s Health Centre. Clinical characteristics of the validation cohort are presented in Table n = 44), amnion (n = 16), and chorion (n = 16) from the discovery cohort samples were run on the Illumina Infinium HumanMethylationEPIC BeadChip (850K array), which quantifies DNAm at 866,895 CpG sites across the genome  and was verified in the discovery cohort (p < 0.003). Furthermore, the differences at these three CpG sites were also confirmed in the validation cohort (p < 0.01)  in chorionic villi in the discovery cohort  and in an additional set of 42 aCA and 36 non-aCA cases . One CpG site, cg01276475 (IRX2) and cg01276475 (RIMS1)  and identified more DM sites when comparing chorionic villi to amnion than when comparing chorionic villi to chorion , no sites were DM in amnion, while ~ 20% (13/66) of chorionic villi sites were also DM in chorion, higher than expected by chance (p = 0.0001). This suggests that chorion may show a somewhat overlapping response to infection as compared to chorionic villi; this may have been missed in the 850K array analysis because we were underpowered with the smaller sample size in amnion and chorion.As the aCA changes seemed limited to chorionic villi, despite that diagnosis of aCA is based on inflammation of the fetal membranes, we wanted to further understand the relationship between these tissue types. DNAm patterns vary widely by tissue type , 58. BecThe majority of the aCA samples in our study were also diagnosed with acute villitis, i.e., inflammation with histological evidence of the presence of neutrophils within the chorionic villi. We therefore hypothesized that some of the aCA-associated DNAm changes captured by the 850K array could be attributable to an increase in immune cell number, especially neutrophils during placental inflammation. Therefore, we expected the 66 DM sites associated with aCA in chorionic villi may trend in the same direction as DNAm associated with blood-derived immune cell types. To test whether the aCA-associated DNAm changes were driven by changes in immune cells ratios in the placenta, we first sought to compare whether the DNAm profile of the aCA chorionic villi cases shifted in the same direction as the blood immune cell types. Specifically, when aCA chorionic villi cases were hypermethylated or hypomethylated compared to the non-aCA chorionic villi cases, we anticipated the blood immune cell types will show similar DNAm patterns compared to the non-aCA chorionic villi cases.β value distributions of 36/66 DM sites available in GSE35069 for each immune cell type to our data. Taking neutrophils as an example, 24/36 (66.6%) DM sites showed a similar trend in DNAm to the aCA cases versus non-aCA cases, which is higher than what we would expect by chance (p = 0.00001). This pattern was also observed across all the other immune cell types including whole blood, suggesting that the DNAm profile of the aCA chorionic villi cases may be more shifted in the direction of blood immune cell type than a non-aCA-associated placenta . As these data were run on a previous version of the Illumina array, the 450K array, only data for 36 of our 66 DM sites were available. We compared the p < 0.005) which separated most aCA cases from the non-aCA cases , suggestive of a “placental inflammation-mediated” phenotype in this group. Neutrophil-specific cluster 2 consisted predominantly of non-aCA cases. Although two sub-clusters were observed in this latter group, the sub-clusters were not stable as determined by multiscale bootstrap resampling (1000 permutations).As we only expected neutrophils and not other blood cells to be increased in the placental samples, we next sought to detect whether there was a specific immune cell type more likely to be linked to aCA-associated placentas. We identified immune cell-type-specific CpG sites using this same data by a differential methylation analysis (see Methods). Unsupervised hierarchical clustering was performed in the discovery cohort of chorionic villus samples using these immune cell-type-specific CpGs. Neutrophil-specific sites (2069 CpGs) produced two stable and significantly different clusters  associated with aCA in chorionic villi. Among the 66 were a number of CpGs located in inflammation-related genes including HLA-E, CXCL14, RAB27A, IRX2, and HSD11B2. Expression of HLA-E molecules in extravillous trophoblast cells of the placenta inhibits the cytotoxic activity of maternal natural killer (NK) cells, resulting in immune tolerance to the fetus during pregnancy [CXCL14 is highly expressed in the placental trophoblast cells and shown to regulate trophoblast invasion during pregnancy [CXCL14 was reported in spontaneous PTB placentas [RAB27A plays a vital role in the regulation of multiple neutrophil functions such as chemotaxis, adhesion to activated surfaces, and exocytosis of granules with anti-microbial properties [IRX2 has been shown to control chemokine expression in breast cancer cells [HSD11B2, on the other hand, is regulated by proinflammatory cytokines and may influence the inflammatory microenvironment [HSD11B2 in chorioamnionitis-associated placentas has been reported [To our knowledge, this is the first study to investigate genome-wide aCA-associated DNAm alterations in the placenta and fetal membranes. Our EWAS identified 66 DM CpG sites  cannot distinguish the canonical 5-methylcytosine mark from its oxidized derivative 5-hydroxymethylcytosine (5-hmc), though 5-hmc values tend to be low in placenta . AlthougThis work addresses the gaps in our understanding of the epigenetic dynamics associated with aCA, which is linked to an inflammatory response in the newborn. We found aberrant array-wide DNAm associated with aCA placentas and confirmed some of these DNAm changes in an independent set of samples. Although our candidate list of aCA-associated CpG sites may contribute toward identification of diagnostic biomarkers for aCA, further studies with larger sample sizes are required to confirm these placental disease-specific DNAm signatures in maternal blood.Additional file 1. Table S1: is a table listing the demographic, clinical and technical variables for the individual tissue samples in the discovery cohort. Table S2: is a table listing the demographic and clinical variables for the independent set of chorionic villus samples in the validation cohort. Table S3: is a table listing the probe filtering characteristics for chorionic villi, amnion, and chorion. Table S4: is a table listing probes/CpG sites investigated for the biologically relevant candidate CpG site analysis. Table S5: is a table listing the probe filtering characteristics for the matched tissue dataset. Table S6: is a table listing probes/CpG sites identified as differentially methylated in the discovery cohort (chorionic villi), along with their relevant gene information.Additional file 2. Figure S1: shows the distribution of samples across the ten chips for the discovery cohort. Figure S2a and S2b: shows unsupervised hierarchical clustering and PCA on all probes in the 850K array . Figure S3: shows the distribution of M values plotted for each pair of technical replicate samples. Figure S4: summarizes the primer sequences and reaction conditions used for each pyrosequencing assay. Figure S5: shows hierarchical clustering on the 66 differentially methylated CpG sites in chorionic villi. Figure S6: shows sex-specific array-wide volcano plots in chorionic villi. Figure S7: shows differential methylation at the three candidate CpG sites in males and females. Figure S8: shows variation in DNAm between fetal sexes over gestational age. Figure S9: shows the distributions of correlation coefficients (R) between DNAm in chorionic villi and fetal membranes compared against the null distribution. Figure S10: shows the array-wide volcano plots of differential methylation analysis between the tissue pairs .Additional file 3. Methods S1: provides detailed description of data preprocessing of matched tissue dataset. Methods S2: provide detailed description on differential methylation analysis on matched tissue comparison dataset.


This pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes.Radial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating.These results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations.Trial registration NCT02311075. Registered December 8, 2014.The online version of this article (10.1186/s12933-019-0843-z) contains supplementary material, which is available to authorized users. Despite standard treatments, the endothelial dysfunction of conduit arteries persists in type 2 diabetic patients and remains a main contributor of increased cardiovascular morbidity and mortality in this population, representing thus a critical therapeutic target , 2. The The aim of the present study was to assess the mechanistic involvement of the EET/sEH pathway in the endothelial dysfunction of conduit arteries in type 2 diabetes in particular using biological approaches based on the quantification of endothelium-derived factors at baseline and during sustained blood flow stimulation induced by hand skin heating. Because essential hypertension is associated with an altered EETs bioavailability in conduit arteries , 10, we 2) were excluded from the study. Patients with type 2 diabetes must not have a HbA1c > 9.4 mmol/L (7.5%), obtained with lifestyle management and standard hypoglycaemic agents except insulin.This study was performed in a total of 74 subjects. Because the presence of hypertension may affect the evaluation of EETs pathway , this coRadial artery endothelium-dependent flow-mediated dilatation was assessed using the previously validated hand skin heating technique, from 34 to 44 °C, allowing to induce a progressive and sustained increase in blood flow , 10, 11.3H]-trans-1,3-diphenylpropene oxide-based radioactivity assay respectively  per min for 2 min each, and then 80 mU/m2 of BSA per min) to achieve and maintain either a target steady-state capillary glucose concentration of 11 mmol/L (200 mg/dL) for the hyperglycemic clamp or 5.5 mmol/L (100 mg/dL) for the hyperinsulinemic clamp, without change in glucose infusion rate for at least 30 min before assessing radial artery endothelium-dependent and -independent dilatation. A control procedure was also performed with saline infusion. Blood sampling were performed before beginning the clamp, at glycemic steady-state before hand skin heating and at the end of hand skin heating to determine the evolution of nitrite, EETs and DHETs bioavailability.Hyperglycemic and hyperinsulinemic euglycemic clamps were performed in 8 subjects selected from the healthy group  and explored on two separate occasions after a 12 h overnight fasting. Briefly, a venous catheter was inserted in a large vein at the antecubital fossa of the dominant arm, allowing a variable infusion rate of a 20% glucose solution alone or combined with continuous regular insulin administration  and R software (version 3.5.1). The Shapiro–Wilk test was used to assess normality of data.Differences between healthy, hypertensive, type 2 diabetic and hypertensive type 2 diabetic groups were assessed by Fisher’s exact test for categorical variables and by one-way ANOVA or nonparametric Kruskall–Wallis test for continuous variables, followed in case of significance, by Tukey’s test or Dunn’s test for multiple comparisons.Significant predictors for radial artery flow-mediated dilatation  were assessed using both backward and forward stepwise regression based upon Akaike Information Criterion (AIC) using a null model without predictors and a full model including diabetes, hypertension, sex, age, smoking status, mean arterial blood pressure, BMI, LDL cholesterol level, triglyceride-glucose (TyG) index (log [fasting triglycerides (mg/dL) × fasting glucose (mg/dL)/2]), creatininemia, baseline radial diameter, variation in shear stress and GTN-induced dilatation as predictors. Similar analyses were performed for shear stress variation during heating and GTN-induced dilatation.Pearson correlation analyses were performed to determine linear relationships between biological and functional variables of interest.P ≤ 0.05 was considered statistically significant.Differences between the impact of saline infusion, hyperglycemic clamp and hyperinsulinemic clamp on the variations in radial artery parameters at baseline and during heating and on the variations in the increase in diameter following GTN administration were assessed by ANOVA using a generalized linear model with group and subject as factors followed, in case of significance, by a Tukey’s test for posthoc pairwise comparisons. The impact of the hyperglycemic and the hyperinsulinemic euglycemic clamps on biological parameters at baseline and during heating were assessed by paired t-test or Wilcoxon matched pairs test. A value of Baseline characteristics of the subjects included are summarized in Table There was no significant difference between groups for baseline radial artery diameter and mean wall shear stress assessed at 34 °C before heating  in type 2 diabetic patients and this was statistically significant in hypertensive diabetic patients compared to healthy subjects. Plasma nitrite increased during heating in healthy and hypertensive subjects but not in the diabetic groups (Fig. Regarding the EETs pathway, no difference was found between groups in the baseline plasma levels of EET Fig. a or DHETThe hyperglycemic clamp increased plasma glucose and insulin concentrations while the hyperinsulinemic clamp increased insulin levels to a higher level, without significantly affecting glucose level (Additional file Compared to saline infusion, the basal radial artery diameter was increased by both clamps at steady-state before hand skin heating but statistical significance was only reached with the hyperinsulinemic clamp, without change in mean wall shear stress and in systemic hemodynamics (Additional file The plasma nitrite levels were not significantly modified by both clamps at steady-state before hand skin heating Fig. d. NitritThe major finding of the present work is that the alteration of conduit artery endothelial function in type 2 diabetic patients was partly due to an alteration of the EETs pathway, in parallel to the expected contribution of the decreased NO availability.In this study, we carefully selected type 2 diabetic patients treated according to recent recommendations for optimization of glycemic control together with management of associated risk factors, mainly LDL cholesterol and blood pressure. To evaluate the proper impact of type 2 diabetes independently from the rise in blood pressure, we compared the functional and biological data of healthy, essential hypertensive, type 2 diabetic and hypertensive type 2 diabetic subjects.In this context, we demonstrated that type 2 diabetic patients either in absence or in presence of hypertension display a decreased sustained radial artery flow-mediated in response to heating, taking into consideration cardiovascular risk factors, the flow-dependent stimulus and endothelium-independent dilatation to GTN. These results demonstrate that well-controlled type 2 diabetic patients still exhibit an endothelial dysfunction of peripheral conduit arteries, as previously shown using more transient increase in blood flow induced by post-ischemic hyperemia –16. In aRegarding the NO pathway, the basal nitrite level was elevated but did not further increase during the sustained endothelial stimulation in the two groups of diabetic patients. This may be related to the increase in oxidative stress in diabetic patients illustrated by the elevation in the whole blood levels of ROS. Indeed, previous work suggested that hyperglycemia may lead to an increased expression of endothelial NO-synthase to compensate for the enhanced inactivation of NO by superoxide anions and promoting increased peroxynitrite formation , 20. A pFurthermore, our results demonstrate for the first time in humans that the endothelial dysfunction of conduit arteries in type 2 diabetes is associated with an alteration in the EETs pathway. In fact, according with previous clinical studies , 23, 24,In contrast, no change in the plasma levels of other epoxy fatty acids and diols derived from linoleic acid or omega-3 were observed, suggesting that their altered metabolism does not contribute to the endothelial dysfunction in our population of well-controlled type 2 diabetic patients. These results extent recent works showing that that the levels of omega-3 and linoleic acid appear no altered when glycemic control is achieved and that omega-3 supplementation is ineffective to improve the metabolic and inflammatory status of patients with well-controlled type 2 diabetes , 33. FinGoing further, we assessed the role of hyperglycemia and hyperinsulinemia in the development of endothelial dysfunction using clamp studies in healthy subjects. Both clamps slightly induced an increase in radial artery diameter, without significant change in mean wall shear stress, but this increase appears stronger with the euglycemic hyperinsulinemic clamp reaching statistical significance compared to basal conditions. This arterial vasodilating effect was probably related to the potentiation of endothelial factor release by insulin, although the increase in nitrite levels was not significant. Previous results showed that acute hyperglycemia or hyperinsulinemia dilates peripheral resistance arteries by a NO-dependent mechanism involving endothelial NO-synthase phosphorylation , 36, andDespite this basal vasodilator effects and as generally observed using post-ischemic hyperemia –44, hypeAltogether, our results demonstrate that a decreased EETs production and increased EETs degradation by sEH together with altered NO bioavailability are both associated with conduit artery endothelial dysfunction in type 2 diabetic patients on top of standard treatments. In contrast, treated essential hypertensive patients appeared to only display a decreased EETs production but maintained NO levels (Additional file Additional file 1. Supplementary Methods and Results.


BP and heart rate (HR) were measured using continuous finger-pulse photoplethysmography. SAS signals were recorded using an SAS monitor. Oxyhaemoglobin saturation (SaO2) and end-tidal CO2 (EtCO2) were measured using a medical monitoring system. Procedure 1 consisted of breathing spontaneously and at controlled rates of 6 breaths/minute and 6 breaths/minute with inspiratory resistance for 10 minutes. Procedure 2 consisted of breathing spontaneously and at controlled rates of 6, 12 and 18 breaths/minute for 5 minutes. Wavelet analysis with the Morlet mother wavelet was applied for delineation of BP and SAS signals cardiac and respiratory components. Slow breathing diminishes amplitude of cardiac BP and SAS oscillations. The overall increase in BP and SAS oscillations during slow breathing is driven by the respiratory component. Drop in cardiac component of BP amplitude evoked by slow-breathing may be perceived as a cardiovascular protective mechanism to avoid target organ damage. Further studies are warranted to assess long-term effects of slow breathing.The aim of the study was to assess cardiac and respiratory blood pressure (BP) and subarachnoid space (SAS) width oscillations during the resting state for slow and fast breathing and breathing against inspiratory resistance. Experiments were performed on a group of 20 healthy volunteers (8 males and 12 females; age 25.3 ± 7.9 years; BMI = 22.1 ± 3.2 kg/m Yogic breathing (pranayama) represents an example of such ancient practice of controlled breathing, often performed in conjunction with meditation or yoga3. Significantly, a number of studies based on modern experimental and computational approaches have confirmed that ancient techniques with controlled respiration may exert a beneficial impact on overall well-being. For instance, resonant breathing (6 breaths per minute) results in improvements in cardiovascular functions such as blood flow to internal organs and sensitivity of the sympathetic component of the baroreflex and ventricular elastance6. Various yoga techniques based on slow breathing were also reported to improve cognitive functions among their practitioners7. Several studies have demonstrated a reduction in acute mean arterial pressure (MAP) during controlled slow respiration9.Slow breathing practices have been practiced for thousands of years amongst Eastern cultures due to their perceived health benefits10. However, it remains unclear how controlled slow breathing affects BP oscillations of cardiac origin. Delineation of the cardiac and respiratory components may help to better understand those BP-related mechanisms that are implicated in cardiovascular disease. Importantly, the calculating wavelet amplitude ensures the identification of cardiac and respiratory components during provocative tests.Controlled slow breathing, particularly at 6 breaths per minute, is associated with augmented BP fluctuations of respiratory origin, as compared to the BP oscillations observed during spontaneous breathing11; however, the regulatory mechanisms underlying CSF motion have yet to be unveiled. Evidently, back-and-forth periodic CSF motion is secondary to cardiac and respiratory cycles (reviewed recently by12). Nevertheless, the precise description of these two physiological processes is difficult due to the technical shortcomings of magnetic resonance imaging (MRI). Apart from inadequate resolution, the imaging of CSF motion during the cardiac and respiratory cycles requires different MRI sequences, a fact which precludes simultaneous measurements of these two components13.Several studies have suggested that one of the fundamental roles of cerebrospinal fluid (CSF) is buffering detrimental cerebral blood flow pulsatility12. Periodic oscillations of SAS width can be measured non-invasively in humans with a near-infrared transillumination/backscattering sounding (NIR-T/BSS) method. In short, the main assumption for NIR-T/BSS technique is that translucent CSF in SAS acts as a propagation duct for infrared radiation 15. This allows for measurement of the SAS width to estimate changes in CSF volume18. NIR-T/BSS has been validated against MRI, showing comparable SAS width alterations induced by shifts in body position. Regression analysis of data derived from both methods shows that SAS width alterations observed in supine vs. abdominal-lying positions yield high interdependence between both methods 19.Recently, we proposed using subarachnoid space (SAS) width pulsatility as a surrogate for CSF pulsatile flow20. Recent analyses have shown that the power spectrum density levels of SAS oscillations are characterized by detectable peaks at both cardiac and respiratory frequencies23. Taken together, NIR-T/BSS is capable of synchronized assessment of instant cardiac and respiratory influences on SAS width, an exclusive feature of this technique23.Importantly, in accordance with the Nyquist theorem, the NIR-T/BSS high sampling frequency allows for signal analysis up to 35 Hz. Thus, as no other available technique does, the NIR-T/BSS accurately identifies rapid SAS width changes resulting from systole-diastole fluctuations in the cerebral blood volume1). In particular, more negative intrathoracic pressures lead to substantial increase in respiratory CSF shifts between cranial compartment and the cervical spinal canal (reviewed by24). Thus, increased respiratory resistance should exacerbate the effects of slow breathing on the investigated variables. The aim of the present study was to delineate cardiac- and respiratory-generated BP and SAS oscillations during the resting state, slow and fast breathing and breathing against respiratory resistance. We hypothesized that induced slow breathing will augment amplitudes of BP and SAS respiratory oscillations while at the same time it may diminish amplitudes of BP and SAS cardiac oscillations.BP and SAS fluctuations are further augmented by inspiratory resistance due to more negative intrathoracic pressures (reviewed by2) during all stages of procedure 1. We also showed (Table Non-parametric Friedman test showed that there was a significant differences (p < 0.05) for all measured signals  for four variables: HR, DBP, MAP and SaOLEFT) signal. Wavelet analysis with the Morlet mother wavelet can detect oscillations with logarithmic frequency resolution and follow the variations of their frequencies and amplitude in time26. The minimal frequency is not the same for procedures 1 and 2 due to the different time of the signals. It is clearly visible that collected signals manifest over a wide frequency range, but in our study we were interested only in two frequency bands which correspond to cardiac and respiration activity. Previous studies revealed that intervals (0.6 – 2 Hz) and (0.145 – 0.6 Hz) are related to the cardiac and respiration functions25. We extended the bottom limit of the respiration interval to 0.1 Hz because in both procedures we asked volunteers to take a breath 6 times per minute, which gives us a frequency of 0.1 Hz. For all signals, we observed a similar cardiac component with a frequency of about 1 Hz. Additionally, there are clearly visible respiration components 0.1 Hz (6 breaths per minute), 0.2 Hz (12 breaths per minute) and 0.3 Hz (18 breaths per minute) for the second procedure  and procedure 2 (panel b). Phase coherence at each frequency was considered significant if its value was above the 95th percentile of 380 intersubject surrogates. All statistically significant WPCO peaks were subjected to statistical analysis to establish more quantitatively the difference between subjects for different stages of the procedures.Wavelet phase coherence (WPCO) were calculated between BP and both (left and right) SAS signals, and directly between the SAS signals. Figure LEFT and SASLEFT- SASRIGHT).The results of our statistical analysis illustrate Fig. Our study has demonstrated that the overall increases in BP and SAS oscillations observed during slow breathing are strictly dependent on the respiratory component, and this phenomenon is particularly evident when inspiratory resistance is applied Fig. . Second,1). We now report an additional mechanism evoked by controlled slow breathing: a lessening of cardiac-derived BP pulsatility. Physiologically, there are two main mechanisms influencing blood flow velocities. One is dependent on vessels’ performance, where arteries’ buffering capacity protects from pressure-related damage to peripheral organs. As documented in human studies, increased arterial stiffness (e.g. commonly seen in long-lasting hypertension or obstructive sleep apnoea) confers cardiovascular risk and translates into higher rates of target organ damage28. The other regulatory component implicated in blood flow pulsatility is dependent on the heart’s performance, where interaction between HR and SV plays an essential role. The respiration synchronous variation in SV is driven by changes in intrathoracic pressure and direct mechanical interactions between the right and the left ventricles32.The beneficial cardiovascular effects of slow breathing are usually explained by the increase in both baroreceptors’ sensitivity and heart rate variability  motion has been recently linked to impairments in white matter structure and function in ageing38, hypertension39 and multiple sclerosis40. Thus, controlled slow breathing techniques may offer a non-pharmacological aid to decrease heart-driven CSF oscillations. Importantly, our study suggests that duration of slow breathing exercise matters; a lessening of cardiac-derived BP pulsatility was statistically significant only in procedure lasting 10 minutes. 5 minutes period of slow breathing is too short to evoke described changes. In future studies the optimal time to achieve maximal benefit for the patients should be established.Exposure to highly pulsatile pressure is a known predictor of cerebral vascular damage and increased risk of cerebrovascular events, even in the absence of elevated MAP2O elevates both BP and cerebral blood flow velocity oscillations within frequency range (0.04 to 0.4 Hz) during simulated hypovolemia. Interestingly, mean cerebral blood flow velocity is not affected41. In this study presyncopal symptoms were delayed by ∼4 min (245 s) when subjects were exposed to enhanced BP and cerebral blood flow velocity oscillations. Consequently, regular respiratory driven CBF fluctuations into higher maximum velocities may represent a protective mechanism for maintaining adequate cerebral perfusion that delays the onset of presyncopal symptoms and prolongs tolerance to hypovolemia41. Similarly, observed in our study augmented BP and CSF oscillations at respiratory frequency can potentially reflect similar phenomena (i.e. support brain perfusion). In addition we may speculate that increased CSF motion at respiratory and slower frequencies may support interstitial fluid flow in the brain42.Increased respiratory resistance set at −7 cm H22. This mechanism is apparently weakened by slow breathing. As both BP and SAS oscillations are heart driven36 BP-SAS lower coherence during slow breathing might be related to augmented heart rate variability43.To the best of our knowledge we demonstrated for the first time that slow breathing results in lower coherence of cardiac components of BP and SAS oscillations. We analysed both amplitude  is challenging even to healthy subjects. Therefore, we limited the number of measured signals, for instance we did not record a real-time sympathetic firing with microneurography. The volunteers exercised symmetrical breathing which is closer to yoga ujjayi breathing than to physiological breathing (1/3 for inspiration and 2/3 for expiration). However, symmetrical breathing was reported to be the best technique for improving baroreflex sensitivity in yoga-naive subjects while differences between symmetrical and non-symmetrical breathing are very limited51 while is not present in healthy subjects52. Our experiments were performed in non-trained yoga-naïve subjects. We observed the effects of controlled (timed) breathing imposed by visual signals. Based on available data we are not able to say whether BP and SAS oscillations alter chronically and consequently, whether observed phenomena provide long-term BP-SAS regulation and organ protection. Future studies with pre-trained or yoga-breathing practitioners are warranted to assess long-term effects of slow breathing on BP-SAS oscillations.Recent studies suggest the long-term beneficial effect of slow breathing might be visible in heart-failure patients9, HR accelerations when inspiratory resistance applied42, increase blood oxygenation regardless the application of inspiratory resistance1. Additionally, the study participants were able to avoid hyperventilation with subsequent changes in EtCO2, which allowed to precisely delineate regulatory mechanisms. All the aforementioned aspects increase reliability of our results.The results of our study are highly consistent with previous reports, which definitely stand for reliability of the applied techniques and findings . None of them were smokers or received any chronic pharmacotherapy (including diet supplements), nor were any of the participants involved in advanced yoga breathing or meditation techniques such as resistance breathing. This study was carried out in accordance with the recommendations of Helsinki. The experimental protocol and the study were approved by the Ethics Committee of Medical University of Gdansk (NKBBN/265/2016). All subjects were informed in detail about the study’s objectives and any potential hazard to their health. All volunteers gave written informed consent to participate in the study. Participants were asked to refrain from coffee, tea, cocoa and any food and beverages containing methylxanthine for at least 8 hours before the tests. All procedures were preceded by 10 minutes of rest in the sitting position in a comfortable and quiet room.Experiments were performed with a group of 20 healthy volunteers . Following the mask fixation and dry-run practicing with the equipment, subjects were asked to rest before the baseline recordings were initiated. Inspiration and expiration pace was imposed by visual signals displayed on the computer screen.2O. The target pressure of the generated inspiratory resistance equalled −20 cm H2O. Procedure 2 consisted of breathing spontaneously and at controlled rates of 6, 12 and 18 breaths/minute for 5 minutes , both inspiration and expiration lasted 1.67 seconds.LEFT – left hemisphere, SASRIGHT – right hemisphere) signals were recorded using SAS Monitor . Detailed description of SAS Monitor was provided previously22. Oxyhaemoglobin saturation (SaO2) was measured using a medical monitoring system . Gas samples from the mouthpiece were constantly analysed using the side-stream technique for end-tidal CO2 (EtCO2) with the metabolic module of the same medical monitoring system. All parameters were recorded continuously for further analysis, and BP and SAS signals were synchronized on a beat-to-beat basis22.BP and heart rate (HR) were measured using continuous finger-pulse photoplethysmography . Finger BP was calibrated against brachial arterial pressure. SAS  is the wavelet coefficient, g(u) is the time series and φ is the Morlet mother wavelet, scaled by factor s and translated in time by t. The Morlet mother wavelet is defined by the equation:53. The wavelet coefficients are complex numbers in the time-frequency plane when the Morlet wavelet is used:To detect physiological processes that are responsible for generating oscillations in the cardiovascular system, we need an appropriate mathematical tool. One such tool is the wavelet analysis with the Morlet mother wavelet, which can detect these oscillations with logarithmic frequency resolution and follow the variation of their frequencies and amplitudes in timeThey define the instantaneous relative phase,54:tn and frequency fk56:During the measurement, external factors such as respiration or heartbeat may create an amplitude and phase modulations. Mathematical tool to find relationship between an amplitude of two signals is the cross wavelet transform defined asCθ(fk) is between 0 and 1. When two oscillations are unrelated, their phase difference continuously changes with time, thus their Cθ(fk) approaches zero. If the Cθ(fk) is around 1, the two oscillations are related and the phase difference between the two signals at a particular frequency remain constant.The value of the WPCO function 58.Surrogate data testing is a method which is used to test whether the estimated values of phase coherence are statistically significant or not. The method has been successfully used in the analysis of signals from living systems54. This assumes that the signals collected from different subjects must be independent, while having similar characteristic properties. We therefore calculated surrogate values of, for example, the BP phase coherence using a signal from one subject and the SAS from another. In our studies, we analysed data from 20 subjects. The significance level was estimated as the 95th percentile of 380 (2-permutations of 20 subjects) intersubject surrogates. The actual value of phase coherence obtained at each frequency can then be compared with the surrogate threshold. When the phase coherence is located above the threshold it is considered to be statistically significant.As we know for oscillations with lower frequencies, there are fewer cycles within a given time interval. This causes artificially increased phases coherence, even in cases where there is none. We must find a significance level above which the phase coherence may be regarded as physically meaningful. To estimate significance levels for the wavelet phase coherence, we used intersubject surrogatesNonparametric statistical tests were used for all comparisons, to avoid the assumption of normality in the results. A one way, between stages of both procedures, non-parametric ANOVA (Friedman test) was conducted to find the differences among stages in the procedures. Post hoc comparisons, using Tukey test, was used to find differences between stages of procedure when Friedman test was statistically significant (p < 0.05). The results of our calculations we placed in Figs