Alus (SVA) is subjected to transcription-directed de novo DNA methylation during human spermatogenesis. The degree of de novo DNA methylation in SVAs varies among human individuals, which confers significant inter-individual epigenetic variation in sperm. Collectively, our results highlight potential molecular mechanisms for the regulation of retroelements in human male germ cells.DNA methylation, repressive histone modifications, and PIWI-interacting RNAs are essential for controlling retroelement silencing in mammalian germ lines. Dysregulation of retroelement silencing is associated with male sterility. Although retroelement silencing mechanisms have been extensively studied in mouse germ cells, little progress has been made in humans. Here, we show that the Krüppel-associated box domain zinc finger proteins are associated with DNA methylation of retroelements in human primordial germ cells. Further, we show that the hominoid-specific retroelement SINE-VNTR- Alus (SVA) are active in humans and can be transposed  undergo epigenetic reprogramming, characterized by DNA demethylation and global resetting of histone marks in mice and humans . A subseIn contrast to the extensive DNA hypomethylation in PGCs, the genomic DNA of sperm is highly methylated in both humans and mice . RetroelIn this study, we aimed to clarify the regulatory mechanisms of DNA methylation of retroelements in human germ cells and performed an integrative analysis using three sets of previously reported data, which included whole-genome bisulfite sequencing (WGBS) data for human PGCs (hPGCs) and sperm, the transcriptome of human male germ cells, and comprehensive human KRAB-ZFPs ChIP-exo data.To learn more about the factors that contribute to DNA demethylation resistance in hPGCs, we reanalyzed publicly available WGBS data for male hPGCs . The gloKRAB-ZFPs are important factors for retroelement silencing. Their activity is mediated by the recruitment of KAP1 and SETDB1, which induces retroelement DNA methylation . To inveIt is possible that the correlation between the DNA demethylation resistance of SVA_A and the binding potential of specific KRAB-ZNFs based on ChIP-exo mapping data in HEK293T cells could result from differences in read mappability. To determine the likelihood of this, we calculated the mappability of each transposon copy by virtually creating reads from the retroelements and mapping them onto the genome. Although highly methylated SVA_A copies showed greater mappability than those that were lowly methylated , the corZNF257 and ZNF28 in hPGCs by reanalysis of single-cell RNA-seq data from hPGCs and neighboring somatic cells (The SVA element has a region containing variable-number tandem repeats (VNTRs) in the middle segment. SVA_A contains one type of VNTR (VNTR1), whereas the other SVA classes possess two types of VNTRs (VNTR1 and VNTR2) . The ZNFic cells . HoweverWe also analyzed the correlation between KRAB-ZFP-binding motifs and the DNA methylation status of L1s and LTR12s in hPGCs. Consistent with previous reports that ZNF649 and ZNF93 bind L1s , ZNF649 Reanalysis of single-cell RNA-seq data for hPGCs and neighboring somatic cells  showed tFor the LTR12C family, we found that ZNF850 more frequently overlapped with highly methylated LTR12C/D/E copies than lowly methylated ones when we analyzed the binding peak data for 250 KRAB-ZFPs . We focuTo investigate whether lowly methylated retroelements in hPGCs acquire DNA methylation during spermatogenesis, we analyzed publicly available human sperm WGBS data from two donors . The twoAlthough the DNA methylation status of SVAs was highly variable between the sperm donors, a subset of SVA copies acquired DNA methylation or maintained a low methylation state during spermatogenesis in both sperm donors . It is pThe presence of transcription-directed retroelement silencing mechanisms, such as the PIWI/piRNA pathway , prompteInter-individual variation in DNA methylation in SVAs was also observed when another set of publicly available sperm WGBS data from three Japanese donors was analyzed . For addTo estimate the impact of SVAs on inter-individual epigenetic variations in sperm, we identified differentially methylated regions (DMRs) in two sperm donors, as shown in Finally, to investigate whether inter-individual DNA methylation variations are associated with physiological or disease conditions, we reanalyzed publicly available WGBS data from five healthy donors and six oligozoospermic patients (European Nucleotide Archive [ENA] under the accession number PRJEB34432) . The disIn this study, we showed that the binding potential of KRAB-ZFPs correlates with retroelement DNA demethylation resistance in hPGCs. Furthermore, we found that de novo DNA methylation patterns in spermatogenesis vary among the L1, LTR, and SVA retroelements. In addition, we ascertained that the SVAs located in transcription-active regions in the antisense orientation are prone to methylation during spermatogenesis, which implies that the transcription-directed DNA methylation machinery might contribute to de novo DNA methylation of SVAs in male germ cells. Notably, the extent of de novo DNA methylation of SVAs in male germ cells is variable among human individuals, with SVAs being a major source of epigenetic variation in sperms.We showed that DNA demethylation resistance in hPGCs frequently occurred in moderately young retroelements such as L1PA, SVA_A, and LTR12, but not in currently active retroelements. Because we targeted full-length transposons, our analysis was biased toward relatively young transposons. Thus, it is possible that some fragmented older transposons may also be resistant to DNA demethylation in hPGCs. A subset of LTR transposons, including LTR12, function as enhancers . It was In addition, KRAB-ZFP binding potentially contributed to the DNA demethylation resistance of L1s and SVAs. ZNF257/28, ZNF649/ZNF93, and ZNF850 were associated with the DNA demethylation resistance of SVAs, L1s, and LTR12Cs, respectively . In hPGCAdditionally, we showed that the mode of DNA methylation acquisition during spermatogenesis was very different among retroelement types. The majority of L1 copies acquired DNA methylation during spermatogenesis, whereas LTR12 maintained its DNA methylation status in hPGCs during spermatogenesis . L1HS, wSaccharomyces cerevisiae . Human semen was diluted twice with saline, layered on 5.0 mL of 20 mM HEPES buffered 90% isotonic Percoll , and centrifuged at 400 × TTATTGTAATTTTTTTGTTTGATTTTTTTGTTTTAG. SVA_1_Rv AAAAAAACTCCTCACATCCCAAAC SVA_2_Fw TTAATGTTGTTTAGGTTGGAGTGTAGTG SVA_2_Rv CAAAAAAACTCCTCACTTCCCAATA. SVA_3_Fw TTTGGGAGGTGTATTTAATAGTTTATTGAGAA SVA_3_Rv TAAACAAAAATCTCTAATTTTCCTAAACAAAAAACC. The PCR products from three sets of primers were combined, purified using a MinElute PCR Purification Kit (QIAGEN), and fragmented using Picoruptor (Diagenode) for 10 cycles of 30 s on and 30 s off. Then, the amplicon-seq library was constructed using KAPA LTP Library Preparation Kits (KAPA BIOSYSTEMS) and SeqCap Adapter Kit A (Roche). The amplicon-seq libraries were sequenced on a HiSeq X platform (Illumina).Genomic DNA was subjected to bisulfite-mediated C to U conversion using the MethylCode Bisulfite Conversion Kit (ThermoFisher Scientific), and then used as a template for PCR for 35 cycles with EpiTaq (Takara) using the following primers: SVA_1_Fw We used the hg19 version of the human genome for NGS analysis because the predicted KRAB-ZFP peaks were derived from this version. Using the newest version of the human genome (GRCh38) did not significantly affect the conclusions. The following publicly available WGBS data were used in this study: hPGCs (SRP050499), sperm , and adult tissues . For the IHEC data, we used processed data for our analysis.Quality control, read mapping, and DNA methylation calculationhttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). For WGBS data from hPGCs, the first nine bases were further trimmed. The trimmed reads were aligned to the hg19 genome using Bismark v0.14.1, with paired-end and non-directional mapping parameters  (http://genome.ucsc.edu/). For the DNA methylation analysis of retroelements, we used retroelements containing at least 10 CpG sites with a read depth of at least five reads. The methylation level of each retroelement copy was calculated by averaging the methylation levels of CpG sites within the copy.Low-quality bases and adaptor sequences were trimmed using Trim Galore version 0.3.7  . The unmClassification of retroelement copy according to DNA methylation levels.≧ 60%.Retroelement copies were classified according to their DNA methylation levels as follows: low < 20%, 20% ≦ medium < 60%, high Association of KRAB-ZFP peaks, binding motifs, and retroelements.We obtained the peak regions of 250 KRAB-ZFP, which were previously reported , from thDMR identificationDMR candidates were identified using the ‘Commet’ command in BisulFighter . To enhaThe evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model . The iniWe generated 100 bp reads from each position along the retroelement copy and aligned the simulated reads to the human genome using Bowtie with –m 1 or Bismark. Then, the mappability of each copy was calculated by dividing the number of properly mapped reads by the total number of reads derived from each copy.We reanalyzed previously reported single-cell RNA-seq data from the testes , hPGCs, We reanalyzed previously reported KRAB-ZFP ChIP-exo data , which wThe Integrative Genomics Viewer  was usedAll reads from amplicon-seq in this study have been submitted to the Gene Expression Omnibus under accession number GSE174562. Retrotransposons undergo massive reprogramming of their methylation states during germ cell development, but some elements are immune to this remodeling. This manuscript explores the contribution of binding motifs for KRAB-Zinc Finger Proteins (KZFPs) and position towards genes to explain the variable methylation dynamics of different retrotransposon families, namely L1, SVA and LTR12, as well as potential inter-individual variation during male germ cell development in humans, using an integrative analyses of available sequencing datasets. By bringing insights into the complex regulation of retrotransposons, it could be of particular interest to the epigenetics community. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Molly Przeworski as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michael Imbeault (Reviewer #1); Geoffrey Faulkner (Reviewer #2).Thank you for submitting your article "Transcriptional states of retroelement-inserted regions and specific KRAB zinc finger protein association are correlated with DNA methylation of retroelements in human male germ cells" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers agreed the study was well conducted and although was initially done on a limited number of available samples, the important observation regarding the inter-individual variability of SVA methylation in sperm was replicated in an additional dataset with more donors and further confirmed by targeted amplicon sequencing. The conclusions regarding the DNA methylation dependency towards specific KZFPs is not novel, although it was extended here to the context of primordial germ cells. The most intriguing conclusion is certainly the description of inter-individual differences in SVA methylation in the sperm of different donors. A link with being positioned inside a highly transcribed gene and in reverse orientation towards the host gene was drawn regarding the propensity of being methylated for an SVA, but the explanation for this is very uncertain. Results appear as preliminary on this matter.Please address the following points in a revised version of your manuscript, and answer the individual comments and questions raised by the reviewers in a detailed rebuttal letter, with possible corrections and additional figures to be included in the manuscript.1 – Integrate all the KZFP datasets that are available in GSE120539 (but that were not published) in your analysis2 – Is it know whether interindividual differences in SVA methylation are also found in somatic tissues, or is it a specific feature of sperm DNA? If this has not been described, please analyze available WGBS datasets focusing on one tissue of several donors, or use your amplicon sequencing strategy. This would be an important addition to the biological meaning of such differences, whether this variability also occurs during embryonic reprogramming, not only germ cell reprogramming.3 – Provide more information as to the methodology for full length retroelement analysis and ages of sperm donors4 – Please show screen shots of individual loci that follow the stated correlation of DNA methylation and KZFP binding5 – Review all sentences with over statements, as outlined by reviewer #1.Additionally, please correct the following points:– Line 304: "… subsequently, MIWI2-interacting protein SPOCD1 recruits the chromatin remodeling complex DNMT3A and DNMT3L to…". This sentence is wrong twice: (1) DNMT3A-DNMT3L does not have chromatin remodeling ability per se, it is a de novo DNA methylation complex, and (2) DNMT3C methylates piRNA-targeted retroelements in mice, not DNMT3A . Please correct as you are referring here to the process that has been described in mice.– The link between intragenic position of SVAs and their ability to undergo methylation by transcriptional readthrough/H3K36 methylation is interesting, as it means it would happen in a piRNA-independent manner. However, it does not explain why only the SVA elements with a reverse orientation to the host gene would be affected. Please report this.Reviewer #1 (Recommendations for the authors):I am a researcher active in the field of KRAB zinc finger proteins for about 12 years. In my opinion, the science in this manuscript is high quality and the findings as novel as they are interesting.I have a few small recommendations to improve some sections of the manuscript and would appreciate if they can be implemented before publication.Early in the manuscript it is stated that "we focused on full-length copies of retroelements to analyze 85 DNA methylation for at least 30 copies.". What did you consider as 'full-length', especially for LTR-containing retroviruses? Did you analyze only copies containing the internal part, discarding solo-LTRs? I could not find details describing this in the methods section. Also, this decision is probably biasing the analysis toward younger elements – not a problem in itself, but it should be stated clearly.With very young elements (L1Hs) the mappability is probably not very good with 100bp non-paired end reads. Could you provide a supplemental table with average mappability per family of transposons, or as a supplemental figure as a violinplot of mappability per family.There's more data of KZFP that is available that was not included – notably at GEO accession GSE120539 from the Trono lab – these are from the same experimental series as the ones published initially but didn't make it through analysis before publication – it would be great if you can include them.For figure 2, I would like to see statistics of enrichment  for overlaps with specific KZFPs / families of transposons and DNA methylation categories.Figure 2A – a heatmap is not the best visualization here – it could be a simple sorted barchart of hits zoomed in on the first few members with the highest scores. Same comment for figure 3H.Review all sentences that are related to conclusions to avoid overly strong wording, considering that most findings of this manuscript are purely correlative and causation has not been demonstrated. As an example (out of many): "Therefore, SVAs are methylated during spermatogenesis if these are inserted into actively transcribed genes." – this is too strong, you might want to add 'suggest, might, potentially'.Please discuss somewhere in the manuscript the potential for multiple KZFPs binding the same elements having a concerted effect on the elements.Finally, I would like to see the age distribution of the sperm donors, and some analysis to see if variability is correlating with age in any way.Reviewer #2 (Recommendations for the authors):My two major reservations are the claims around inter-individual variability being difficult to distinguish from technical variablity, which I don't have a reasonable suggestion for how to address, and the first specific point above, namely that uniquely mapped WGBS reads are unlikely to measure methylation in the core VNTR region of an SVA (or the L1HS 5'UTR CpG island). The authors could address this point by showing a composite profile of WGBS coverage and methylation levels compared to L1/SVA consensus sequences, showing the inner parts of L1 and SVA. They could also show examples of individual loci that follow the stated patterns of DNA methylation and ZFP binding that supports a correlation between the two. Another option would be to do nanopore long-read sequencing, which obviously would take time and substantial resources, but would provide a comprehensive picture of the situation. Note that this issue affects some of the more high profile mouse retrotransposons, such as IAP, to a much lesser degree because their LTRs are more accessible to WGBS.I think the figure legends for Figure 2E and Figure 2F are swapped. 1 – Integrate all the KZFP datasets that are available in GSE120539 (but that were not published) in your analysisThanks for your suggestion. We reanalyzed KZFP peak data from GSE120539 and found that ZNF850 was enriched in highly methylated LTR12C copies in hPGCs. We included the analysis of ZNF850 in Supplementary Figure S4.2 – Is it know whether interindividual differences in SVA methylation are also found in somatic tissues, or is it a specific feature of sperm DNA? If this has not been described, please analyze available WGBS datasets focusing on one tissue of several donors, or use your amplicon sequencing strategy. This would be an important addition to the biological meaning of such differences, whether this variability also occurs during embryonic reprogramming, not only germ cell reprogramming.Thanks for the suggestion. This is also important point.To address this issue, we reanalyzed IHEC DNA methylation data from adult skeletal muscle (15 samples) and CD4-positive helper T cells (18 samples). As all SVA groups showed high DNA methylation in all individuals tested, inter-individual variation of SVA DNA methylation in sperm would be cancelled during development . We stated this result in the text.“In contrast to the inter-individual epigenetic variation of SVAs in sperm, a reanalysis of WGBS data of adult skeletal muscle from 15 individuals and of helper CD4-positive T cells from 18 individuals, which was deposited in the International Human Epigenome Consortium (IHEC) portal, showed high DNA methylation of SVAs in all individuals  . Thus, inter-individual variations in DNA methylation of SVAs in sperm are essentially canceled during development.” P15. line 276-281.3 – Provide more information as to the methodology for full length retroelement analysis and ages of sperm donorsWe added following sentences in the method section for the full length retroelement analysis.“Only nearly full-length retroelements, whose length is 90% or more of the length of the consensus sequence of each subtype, were used for DNA methylation analysis of retroelements. We also included solo-LTR transposons in the DNA methylation analysis if they also possessed more than 90% of the consensus LTR sequence.” P34, line 606-609.et al., data were described in the manuscript. On the other hand, we could not access clinical information of our samples for amplicon-seq, so age data for Okae et al., was not available.The ages of sperm donors used in Hammoud 4 – Please show screen shots of individual loci that follow the stated correlation of DNA methylation and KZFP bindingI added the representative loci in Figure S1J, S4G, 3H.5 – Review all sentences with over statements, as outlined by reviewer #1.We revised following sentences to be not over described as possible as we can.Therefore, SVAs are methylated during spermatogenesis if these are inserted into actively transcribed genes.“Therefore, SVAs located in actively transcribed regions in the antisense orientation are efficiently subjected to de novo DNA methylation during spermatogenesis.” p14, line 245-246The data indicate that the effectiveness of the de novo DNA methylation varies among individuals.“These results implicate the possibility that SVAs acquire DNA methylation during DNA methylation via transcription-directed machinery, and that the effectiveness of de novo DNA methylation varies among individuals.” p15, line 255-257SVAs constitute a major source of inter-individual epigenetic variation in sperm“SVAs are a potential source of inter-individual epigenetic variation in sperm”p15, line 259In this study, we showed that KRAB-ZFPs are associated with the DNA demethylation resistance of retroelements in hPGCs.“Thus, the involvement of other KRAB-ZFPs in DNA demethylation resistance of retroelements in hPGCs is possible.” p19, line 330-331SVAs are subjected to de novo DNA methylation by the transcription-directed DNA methylation machinery.“the SVAs located in transcription-active regions in the antisense orientation are prone to methylation during spermatogenesis, which implies that the transcription-directed DNA methylation machinery might contribute to de novo DNA methylation of SVAs in male germ cells.” p18, line 302-304Our data showed that the acquisition of DNA methylation of SVAs during spermatogenesis was regulated by the inserted regions and not by the nucleotide sequence. SVAs inserted in transcriptionally active regions in the antisense direction were targeted by de novo DNA methylation during spermatogenesis.“Our data showed that the acquisition of DNA methylation of SVAs during spermatogenesis correlated with the inserted regions and not with the nucleotide sequence. SVAs inserted in transcriptionally active regions in the antisense direction are efficiently targeted for de novo DNA methylation during spermatogenesis.” P20, line 354- p21, 357Additionally, please correct the following points:– Line 304: "… subsequently, MIWI2-interacting protein SPOCD1 recruits the chromatin remodeling complex DNMT3A and DNMT3L to…". This sentence is wrong twice: (1) DNMT3A-DNMT3L does not have chromatin remodeling ability per se, it is a de novo DNA methylation complex, and (2) DNMT3C methylates piRNA-targeted retroelements in mice, not DNMT3A . Please correct as you are referring here to the process that has been described in mice.Thanks for the suggestion. We changed the sentences as bellow.“In mouse spermatogenesis, MIWI2 binds piRNAs and is recruited to the nascent transcribed regions that are complementary to piRNAs . Subsequently, MIWI2 interacting protein SPOCD1, which forms a complex with DNMT3A and DNMT3L, and potentially with a rodent-specific DNA methyltransferase DNMT3C , induces DNA methylation on transposons ..” P21, line 357- 361.– The link between intragenic position of SVAs and their ability to undergo methylation by transcriptional readthrough/H3K36 methylation is interesting, as it means it would happen in a piRNA-independent manner. However, it does not explain why only the SVA elements with a reverse orientation to the host gene would be affected. Please report this.Thank you for this comment. We described this issue as bellow.Saccharomyces cerevisiae . Thus, the machinery for gene body DNA methylation regulated by SETD2 is also a candidate for the de novo DNA methylation of SVAs during spermatogenesis.” p22, line 379-383.“Furthermore, antisense RNAs embedded within protein-coding genes are selectively silenced by H3K36 methyltransferase SET2 in Reviewer #1 (Recommendations for the authors):I am a researcher active in the field of KRAB zinc finger proteins for about 12 years. In my opinion, the science in this manuscript is high quality and the findings as novel as they are interesting.I have a few small recommendations to improve some sections of the manuscript and would appreciate if they can be implemented before publication.Early in the manuscript it is stated that "we focused on full-length copies of retroelements to analyze 85 DNA methylation for at least 30 copies.". What did you consider as 'full-length', especially for LTR-containing retroviruses? Did you analyze only copies containing the internal part, discarding solo-LTRs? I could not find details describing this in the methods section. Also, this decision is probably biasing the analysis toward younger elements – not a problem in itself, but it should be stated clearly.Thank you for your valuable and most KRAB-ZFP expert comments on our manuscript. As already pointed out, we also included solo-LTR copies in our DNA methylation analysis, if they possess more than 90% of the consensus LTR sequence. Length of consensus sequence was obtained from RepeatMasker data in UCSC genome browser. We described this in the Method section.In addition, we stated our analysis was biased toward younger elements in Discussion section as bellow.“Because we targeted full-length transposons, our analysis was biased towards relatively young transposons. Thus, it is possible that some fragmented older transposons may also be resistant to DNA demethylation in hPGCs.” P17, line 309-311.With very young elements (L1Hs) the mappability is probably not very good with 100bp non-paired end reads. Could you provide a supplemental table with average mappability per family of transposons, or as a supplemental figure as a violinplot of mappability per family.in silico and mapped the reads to human genome by bismark. As the reviewer pointed out, the younger transposon, the lower mapping efficiency. This result was included in Supplementary Figure S1A.Thanks for the suggestion. To investigate mappability of WGBS read, we generated 100-bp sequences from each full-length copy There's more data of KZFP that is available that was not included – notably at GEO accession GSE120539 from the Trono lab – these are from the same experimental series as the ones published initially but didn't make it through analysis before publication – it would be great if you can include them.Thanks for your good suggestion. We reanalyzed KZFP peak data from GSE120539 and found that ZNF850 was enriched in highly methylated LTR12C copies in hPGCs. We included the analysis of ZNF850 in Supplementary Figure S4. As we found KRAB-ZFP associated with LTR12 methylation, we removed old Figure S4 and Figure 3H.For figure 2, I would like to see statistics of enrichment  for overlaps with specific KZFPs / families of transposons and DNA methylation categories.P-value for enrichment of retroelement in KRAB-ZNF binding peaks was calculated by prop.test by R. We included these data in Supplementary Figure S1C.Expected and observed numbers of SVA_A and LTR12C that overlap with KRAB-ZNF binding peaks were calculated and Figure 2A – a heatmap is not the best visualization here – it could be a simple sorted barchart of hits zoomed in on the first few members with the highest scores. Same comment for figure 3H.Thanks for the comment.We changed the heatmap in Figure 2A and Figure 3H to scatter plot.Review all sentences that are related to conclusions to avoid overly strong wording, considering that most findings of this manuscript are purely correlative and causation has not been demonstrated. As an example (out of many): "Therefore, SVAs are methylated during spermatogenesis if these are inserted into actively transcribed genes." – this is too strong, you might want to add 'suggest, might, potentially'.Thanks for the suggestion. We revised following sentences to be not over described as possible as we can.Therefore, SVAs are methylated during spermatogenesis if these are inserted into actively transcribed genes.“Therefore, SVAs located in actively transcribed regions in the antisense orientation are efficiently subjected to de novo DNA methylation during spermatogenesis.” p14, line 245-246The data indicate that the effectiveness of the de novo DNA methylation varies among individuals.“These results implicate the possibility that SVAs acquire DNA methylation during DNA methylation via transcription-directed machinery, and that the effectiveness of de novo DNA methylation varies among individuals.” p15, line 255-257SVAs constitute a major source of inter-individual epigenetic variation in sperm“SVAs are a potential source of inter-individual epigenetic variation in sperm”p15, line 259In this study, we showed that KRAB-ZFPs are associated with the DNA demethylation resistance of retroelements in hPGCs.“Thus, the involvement of other KRAB-ZFPs in DNA demethylation resistance of retroelements in hPGCs is possible.” p19, line 330-331SVAs are subjected to de novo DNA methylation by the transcription-directed DNA methylation machinery.“the SVAs located in transcription-active regions in the antisense orientation are prone to methylation during spermatogenesis, which implies that the transcription-directed DNA methylation machinery might contribute to de novo DNA methylation of SVAs in male germ cells.” p18, line 302-304Our data showed that the acquisition of DNA methylation of SVAs during spermatogenesis was regulated by the inserted regions and not by the nucleotide sequence. SVAs inserted in transcriptionally active regions in the antisense direction were targeted by de novo DNA methylation during spermatogenesis.“Our data showed that the acquisition of DNA methylation of SVAs during spermatogenesis correlated with the inserted regions and not with the nucleotide sequence. SVAs inserted in transcriptionally active regions in the antisense direction are efficiently targeted for de novo DNA methylation during spermatogenesis.” P20, line 354- p21, 357Please discuss somewhere in the manuscript the potential for multiple KZFPs binding the same elements having a concerted effect on the elements.We added the following sentence in the Discussion section.“In hPGCs, multiple KRAB-ZNPs were correlated with DNA demethylation resistance in the same retroelements, which may contribute to more robust or cooperative retroelement suppression.” P18, line 319- p19, line 321Finally, I would like to see the age distribution of the sperm donors, and some analysis to see if variability is correlating with age in any way.et al., data were described in the manuscript . On the other hand, we could not access clinical information of our samples for amplicon-seq, so age data for Okae et al., was not available. To investigate the correlation of SVA methylation with age, we further analyzed publicly available sperm WGBS data from five healthy individuals and six oligozoospermic patients, which are deposited in the European Nucleotide Archive (ENA) under the accession number PRJEB34432  and are linked to clinical information including age. We found that both healthy and patients showed variable SVA methylation and age was not associated with SVA methylation state. We also found that testosterone level in blood was negatively correlated with SVA methylation in sperm, implicating potential link between SVA methylation in sperm and physiological conditions. These data were included in Supplementary Figure S5C, D and described in the text as follow.The ages of sperm donors used in Hammoud “Finally, to investigate whether inter-individual DNA methylation variations are associated with physiological or disease conditions, we reanalyzed publicly available WGBS data from five healthy donors and six oligozoospermic patients (European Nucleotide Archive (ENA) under the accession number PRJEB34432) . The disease condition was not associated with inter-individual DNA methylation variations in SVAs, because both healthy donors and oligozoospermic patients showed inter-individual variations of DNA methylation in “High and Low” SVA_B–F copies . On the other hand, a comparison of various physiological conditions between highly methylated individuals and lowly methylated ones (median methylation levels of “High and Low” > 50% vs < 50%) revealed that blood testosterone levels were significantly higher in lowly methylated individuals than in highly methylated ones . However, prolactin, follicle stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG) blood levels and age were not significantly different between the two groups . Although further validation of this correlation is required, DNA methylation of SVAs in sperm may be associated with physiological conditions.” P16, line 282- P17, line 296.Reviewer #2 (Recommendations for the authors):My two major reservations are the claims around inter-individual variability being difficult to distinguish from technical variablity, which I don't have a reasonable suggestion for how to address, and the first specific point above, namely that uniquely mapped WGBS reads are unlikely to measure methylation in the core VNTR region of an SVA (or the L1HS 5'UTR CpG island). The authors could address this point by showing a composite profile of WGBS coverage and methylation levels compared to L1/SVA consensus sequences, showing the inner parts of L1 and SVA. They could also show examples of individual loci that follow the stated patterns of DNA methylation and ZFP binding that supports a correlation between the two. Another option would be to do nanopore long-read sequencing, which obviously would take time and substantial resources, but would provide a comprehensive picture of the situation. Note that this issue affects some of the more high profile mouse retrotransposons, such as IAP, to a much lesser degree because their LTRs are more accessible to WGBS.Thanks for your valuable comment/suggestion. As the reviewer pointing out, it is difficult to measure DNA methylation in core VNTR regions of a younger SVA. But we were able to do it in the oldest SVA type, SVA_A. We showed the DNA methylation levels in VNTR regions of SVA_A and overlap with KRAB-ZFP peaks in new Supplementary Figure S1H. We also showed examples of individual loci showing correlation of DNA methylation in hPGCs and KRAB ZFP association in SVA_A and L1PA4 in new Supplementary Figure S1I,J and 3H. And we described about this issue as bellow.“We also confirmed that DNA methylation levels within the VNTR were correlated with ZNF257 or ZNF28 association .” p9, 149-151“We also confirmed high DNA methylation in the ZNF649 binding motifs at individual loci .” p11, 185-186I think the figure legends for Figure 2E and Figure 2F are swapped.Thank you. We fixed it.


Sympathetic nervous system (SNS) innervation into brown adipose tissue (BAT) has been viewed as an impetus for brown fat thermogenesis. However, we surprisingly discovered that BAT SNS innervation is dispensable for mice to maintain proper body temperature during a prolonged cold exposure. Here we aimed to uncover the physiological factors compensating for maintaining brown fat thermogenesis in the absence of BAT innervation. After an initial decline of body temperature during cold exposure, mice with SNS surgical denervation in interscapular BAT gradually recovered their temperature comparable to that of sham-operated mice. The surgically denervated BAT also maintained a sizable uncoupling protein 1 (UCP1) protein along with basal norepinephrine (NE) at a similar level to that of sham controls, which were associated with increased circulating NE. Furthermore, the denervated mice exhibited increased free fatty acid levels in circulation. Indeed, surgical denervation of mice with CGI-58 deletion in adipocytes, a model lacking lipolytic capacity to release fatty acids from WAT, dramatically reduced BAT UCP1 protein and rendered the mice susceptible to cold. We conclude that circulating fatty acids and NE may serve as key factors for maintaining BAT thermogenic function and body temperature in the absence of BAT sympathetic innervation. Obesity poses a serious risk for the development of a panel of metabolic diseases that greatly increase morbidity and mortality in current society . ObesityThe sympathetic nervous system (SNS) has been recognized as a key impetus for promoting BAT thermogenesis and WAT lipolysis through direct innervation into these tissues ,16,17,18Thus, in the present study, we applied a surgical denervation approach to completely abolish SNS innervation in iBAT. We found that mice with bilateral iBAT SNS denervation can tolerate a seven-day cold challenge at 5 °C. After an initial decline of body temperature in the first 24 h of cold challenge, these surgically denervated mice gradually recovered their body temperature to the level comparable to that of control mice, with only a slightly decreased UCP1 protein level in the denervated iBAT. We further explored the factors that are responsible for maintaining the cold tolerance and UCP1 protein levels in iBAT in mice in the absence of iBAT SNS innervation.All procedures conducted in the animal studies were approved by the Institutional Animal Care and Use Committee at Georgia State University (GSU) . Male C57BL/6J mice used for all experiments were purchased from Jackson Laboratory . Mice with CGI-58 knockout in adipocytes  were generated by crossing Comparative Gene Identification-58 (CGI58)-floxed mice  with Adi12-week-old male C57/BL6J mice were randomly divided into two groups: one for surgical denervation and the other for sham surgery. iBAT SNS denervation was achieved by surgically cutting 0.5cm–1.0cm intercostal nerves  bilaterally. Mice with surgical or sham denervation went on 7-day recovery and then were either housed at room temperature as control groups or challenged with cold at 5 °C for 16 h or 7 days.Mice with sham or surgical denervation were subjected to a cold challenge (5 °C) in a time course from 16 h up to seven days. At the end of the experiment, adipose tissues were dissected, weighed, snap-frozen in liquid nitrogen and stored for further analysis of protein expression and immunohistochemistry. In some cold exposure experiments, a temperature transponder  was implanted into the mouse peritoneal cavity to monitor the body temperature, as previously described . For AC5Adipose tissue was homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease inhibitor mixture and phosphatase inhibitor mixture . Tissue homogenates were separated by SDS-PAGE, which were transferred to nitrocellulose membrane , followed by blocking, washing, and incubating with primary antibodies and Alexa Fluor 680-conjugated secondary antibodies . The blots were developed with a Li-COR Imager System . The primary antibodies are UCP1 , TH , phospho-HSL (pHSL) , CD36  and α-Tubulin .Fat tissue was fixed in 10% neutral formalin, embedded in paraffin, and cut into 5 µm sections, which were further processed for hematoxylin and eosin (H&E) staining or immunostaining with the UCP1 antibody , as previously described .Norepinephrine turnover (NETO) analysis was conducted using the a-methyl-p-tyrosine (AMPT) method as previously described . BrieflySerum free fatty acids were quantified using an HR Series NEFA kit . Serum catecholamines were quantified using an ELISA kit . Serum glucocorticoids were quantified using an ELISA kit .t test or one-way ANOVA as appropriate. Statistical significance is accepted at p < 0.05.All data are expressed as mean ± SEM. Statistical differences between groups were analyzed by student To determine the importance of SNS innervation in BAT thermogenic function and whole body thermoregulation, we conducted a bilateral surgical SNS denervation in interscapular BAT (iBAT). After a seven-day recovery from surgery at room temperature, the denervated mice were overall healthy and weighed similarly to sham-operated control mice . While tIn contrast, iBAT SNS denervation markedly induced UCP1 protein contents in iWAT, with a tendency seen for increased TH protein but no change in phospho-HSL A. ImmunoWe next challenged the denervated and sham control mice with a 5 °C cold in a seven-day time course. As expected, body temperature of denervated mice dropped to a significantly lower level than that of sham controls during the first 24 h of cold exposure A. Body tTo determine what drove body temperature lower in the denervated mice in the first 16 h of cold exposure, we characterized the iBAT and iWAT of these animals. There were no differences in body weight between the denervated mice and sham controls after a 16-h cold challenge , nor werTo determine what drove the body temperature recovery in denervated mice during prolonged cold exposure, we next examined iBAT and iWAT of the denervated mice after a seven-day cold exposure. There was no difference in body weight between denervated mice and sham controls after the seven-day cold challenge . There wThe catecholamines released from the sympathetic nerve terminals in adipose tissue during the cold exposure are the driving force for activation of brown/beige thermogenesis ,24. To gAfter a seven-day cold challenge, there was no difference in basal NE content between denervated and sham-operated iBAT C. HoweveWe also measured circulating catecholamine and glucocorticoids levels in denervated and sham-operated mice during the course of seven-day cold exposure. We found that cold exposure significantly increased circulating catecholamines, including NE and epinephrine E,F. InteFatty acids promote brown fat thermogenesis by serving as a fuel and a UCP1 activator ,25. We tTo test the importance of fatty acids in maintaining body temperature and UCP1 expression in mice without sympathetic innervation in iBAT, we characterized cold-induced thermogenesis of a genetic mouse model (AC58KO) deficient in adipocyte Comparative Gene Identification-58 (CGI-58), a coactivator of Adipose Triglyceride Lipase (ATGL) required for intracellular lipid droplet lipolysis ,26. We fThe premise of this study was derived from our prior observations on the hamsters with iBAT SNS denervation . As muchWe want to first highlight two observations that link the dynamic changes of iBAT UCP1 protein and NE contents to the fluctuated body temperature of denervated mice during the cold exposure. For one, the pattern of more reduction of UCP1 protein (40%) in denervated iBAT of 16 h vs. lesser reduction of UCP1 (12%) in denervated iBAT of seven-day cold mirrors the dynamic change of body temperature: the body temperature of denervated mice falls at the early stage of cold exposure and gradually recovers along the course of cold exposure, which eventually reaches to a level comparable to that of sham control mice on day six. This observation highlights the importance of brown fat UCP1 in maintaining body temperature. On the other hand, the change of denervated iBAT UCP1 protein from 16 h to seven-day also parallels the pattern of NE contents in denervated iBAT along the course of cold exposure: lower iBAT NE content in 16 h cold exposure when body temperature declines and higher iBAT NE content in seven-day cold when body temperature recovers. This also underscores the importance of brown fat NE in maintaining brown fat UCP1 protein and body temperature.Severing SNS surgically indeed achieved a complete denervation in iBAT, evident by complete loss of TH protein, barely detectable basal NE content and NETO in iBAT. However, in the absence of SNS-derived NE, the denervated iBAT still partially recovered basal NE, especially in mice with the 16-h and seven-day cold challenge, which may be replenished by circulating NE that displays a significant increase during the cold challenge on day 1 and a tendency of increase on day seven. Although the circulating NE only exhibits a trend of increase on day seven, iBAT NE contents are similar between denervated mice and the sham controls, suggesting that a dynamic replenishment of NE into denervated iBAT may drain circulating NE to a lower level. With this in mind, future studies are warranted to examine the pathways underlying NE metabolism in fat tissues such as recently identified NE transporters in sympathetic neuron-associated macrophages and adipocytes ,28. On tThe circulating NE may be the major source for the increased basal NE in iWAT of the denervated mice with overnight cold exposure because the SNS innervation into iWAT of the denervated mice remains at the same level as that of the sham controls evident by unchanged NETO and TH protein. The increased NE in circulation is presumably derived from adrenal medulla in response to the stress such as cold and loss of iBAT innervation. However, future studies involving adrenalectomy would be required to confirm whether circulating NE compensates for the loss of SNS-derived NE in sustaining thermogenesis during cold exposure. Although alternatively activated macrophages (M2) also have been shown to produce catecholamines that promote BAT thermogenesis and lipolysis , the stuChemical denervation of iBAT in hamsters indeed impaired BAT thermogenesis evident by lower BAT temperature . HoweverWe believe that fatty acids play a significant role in maintaining iBAT UCP1 protein and thermogenesis during cold exposure as we observed a significant increase in circulating fatty acid levels on day seven of cold challenge when the denervated mice recovered their body temperature. In support of this, we observed a strong correlation between circulating fatty acid levels and iBAT UCP1 protein in denervated mice, suggesting the importance of fatty acids for the mice to maintain the sizable brown fat UCP1 in the absence of SNS innervation. Further, the increased expression of CD36 protein in iBAT of denervated mice indicates that circulating fatty acids are actively transported into brown adipocytes and may serve as a driving force to retain the UCP1 protein in the denervated brown fat. To further confirm the vital role of fatty acids in this process, we examined the phenotype of AC58KO mice lacking lipolytic capacity, which are extremely cold sensitive with little presence of UCP1 protein in iBAT. Fatty acids serve as not only a fuel but also as a UCP1 activator for BAT thermogenesis ,25. CircIn sum, we show that mice with surgical denervation of iBAT SNS are able to tolerate a seven-day cold challenge, with proper maintenance of UCP1 protein in iBAT and body temperature during the cold challenge. The denervated mice have a significant increase in circulating NE, which may account for the basal NE levels in the denervated iBAT, maintaining UCP1 protein. Furthermore, the denervated mice also display increased free fatty acid levels in circulation, which may be important for maintaining UCP1 expression and activity in iBAT in the absence of sympathetic innervation. Indeed, surgical denervation of mice deficient in WAT lipolysis dramatically reduces UCP1 protein and causes susceptibility to cold. We conclude that circulating fatty acids and NE are the key factors for maintaining a proper cold-induced thermogenesis in the absence of BAT sympathetic innervation.


Coagulopathy and changes in coagulation biomarkers after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is recognized but few details have been studied;The study revealed that the 6% incidence rate of symptomatic VTE within 6 months of CRS and HIPEC was similar to comparable abdominal cancer surgeries. Furthermore, the study results revealed that residual tumor at the completion of surgery, as well as elevated D-dimer on day 2, were independent risk factors for postoperative VTE.D-dimer on day 2 and incomplete cytoreduction of CC2–3 may be useful clinical markers to identify patients at higher risk for VTE after surgery; this could identify patients in need of prolonged prophylaxis. Further research on thromboprophylaxis and on changes in post-HIPEC coagulation biomarkers is needed, particularly research investigating whether clinically relevant cut-off values for D-dimer may exist.


Whilst feration .ex vivo studies showed similar effects in AML patient blasts and in vitro AML cell lines models [et al., 2010 [Our previous studies linked increased NOX2 derived ROS production with cellular proliferation in human hematopoietic stem and progenitor cells (HSPC) . Follow s models . Furthers models . Clinicas models , whilst s models . Buildinl., 2010 , our grol., 2010 . Data prl., 2010 . OverexpHigh and ROSLow producing blasts. Analysis of this data showed ROSHigh blasts had higher intracellular glucose levels than ROSLow blasts and additionally AML blasts had higher levels of intracellular glucose and lactate than normal hematopoietic (control) cells [Mass spectrometry was employed to determine the impact of inhibition of ROS production on metabolism in AML cell lines . These dl) cells . Metaboll) cells , 13 alsol) cells . The genl) cells ), has bel) cells .HIF-1α expression [HIF-1α mRNA correlated with increased ROS levels and given that PFKFB3 is known to be a target of HIF-1α it was reasoned that changes in expression may be driving PFKFB3 expression. However, HIF-1α is mainly post-translationally regulated and immunoblotting showed that HIF-1α was not expressed at detectable levels and furthermore, knock-down of the mRNA for this protein did not result in any changes in glucose uptake or proliferation. We also investigated the potential role of the stress response kinase p38, which is known to be activated in response to ROS [PFKFB3 gene contains a serum response element which has been shown to be activated by the p38MAPK pathway [MAPK by ROS has been shown to occur in our studies (unpublished data), inhibition of p38MAPK did not lead to changes in PFKFB3 expression.ROS has previously been linked to metabolism, having been shown in endothelial cells to increase HIF-1α expression which in turn upregulates numerous glycolytic enzymes . To try pression . The date to ROS . Further pathway . Whilst ROS is also known to activate both AKT and AMPK. We showed that inhibition of mitochondrial proteins (UCP2) led to decreased p-AMPK and PFKFB3 levels . AMPK isSCL16) family of genes. The main transporter of lactate out of the cell is MCT4 (SLC16A3). While MCT4 showed no ROS dependent changes in expression, expression of MCT1 (SLC16A1) the main transporter of lactate into the cell was not examined (data not shown). Overexpression of MCT1 has been observed in several cancers and imported lactate can be used for both energy production and biosynthesis [+, which can then be used to support an increased glycolytic rate.Whilst our studies clearly established a link between ROS, PFKFB3 expression, cellular glucose uptake and proliferation, several areas warrant further investigation. It is often stated as a key tenet of the Warburg effect, that transformed cells show an increase in aerobic glycolysis, accompanied by increased cellular glucose uptake and lactate secretion. Whilst increases in ROS were shown to increase cellular glucose uptake, corresponding changes in lactate levels were not observed when stratified according to ROS level and the reasons for this have not been delineated. One plausible explanation is that ROS effects changes in monocarboxylate transporter (MCT) or LDH expression, resulting in decreased lactate excretion, or increased lactate uptake and increased conversion of lactate into pyruvate. Given the hydrophilic nature of lactic acid, transport across membranes necessitates transporters that belong to the MCT family encoded by the solute carrier 16 (ynthesis , 25. In ynthesis . Furtherin vitro [et al., is consistent with the hypothesis that elevated ROS results in increased flux through the PPP.Lactate may also be retained in the cell to promote the antioxidant response as lactate is known to inhibit PFK activity  and thusin vitro . AnalysiCurrent treatments in AML appear to have reached their therapeutic limit and the need for either novel stand-alone therapies or adjuncts to traditional chemotherapeutic treatments is clear. There is a growing body of evidence implicating aberrant ROS production and redox signalling in the modification of cellular metabolic pathways, whilst PFKFB3 is known to be dysregulated in numerous cancers. Work by our group has established a causal link between increased ROS production and changes in PFKFB3 expression levels in AML models. However, further work is urgently needed to delineate the full impact of dysregulated ROS production on cellular metabolic function in AML.


Cine Fingerprint Extractor and Risk Predictor. The former extracts cardiac structure and function features from cine CMR in a form of cine fingerprint in a fully unsupervised fashion, and the latter takes in the cine fingerprint and outputs disease outcomes as a cine risk score. Patients with VA (n = 96) had a significantly higher cine risk score than those without VA. Multivariate analysis showed that the cine risk score was significantly associated with VA after adjusting for clinical characteristics, cardiac structure and function including CMR-derived scar extent. These findings indicate that non-contrast, cine CMR inherently contains features to improve VA risk prediction in primary prevention ICD candidates. We solicit participation from multiple centers for external validation.Better models to identify individuals at low risk of ventricular arrhythmia (VA) are needed for implantable cardioverter-defibrillator (ICD) candidates to mitigate the risk of ICD-related complications. We designed the CERTAINTY study (CinE caRdiac magneTic resonAnce to predIct veNTricular arrhYthmia) with deep learning for VA risk prediction from cine cardiac magnetic resonance (CMR). Using a training cohort of primary prevention ICD recipients  who underwent CMR immediately prior to ICD implantation, we developed two neural networks:  Although the survival benefit of primary prevention ICD is incontrovertible, the rate of appropriate ICD therapies due to ventricular arrhythmia (VA) is relatively low at 1.1–5.1% per year2. In contrast, the adverse event rate exceeds the rate of appropriate therapies in individuals at low risk for SCD. For example, device infection rates are 1.4–2.0% per year3, and rates of inappropriate shocks approach 5–20% per year4, which are associated with increased mortality5 and decreased quality of life6. In addition, LVEF improvement occurs in up to 25–50% of patients which correlates with diminished SCD risk due to VA7. Thus, LVEF is far from being a comprehensive imaging feature to predict VA. Recently, other imaging features of cardiac structure and function were found to be independent predictors of VA, including the extent of heterogeneous myocardial tissue (‘gray zone’) on late gadolinium enhancement (LGE) cardiac magnetic resonance (CMR)8, and right ventricular (RV)9 and left atrial (LA) function10.Implantable cardioverter-defibrillator (ICD) therapy has become the cornerstone for the primary prevention of sudden cardiac death (SCD) in patients with systolic heart failure (HF) and reduced left ventricular ejection fraction (LVEF)11. Recently, AI has been shown to predict personalized prognosis, such as individual responses to lung cancer therapy12 and survival for patients with pulmonary hypertension13. While traditional machine learning approaches rely on handcrafted, previously recognized features extracted from medical images, AI can also automatically generate a patient-specific fingerprint containing inherent features of cardiac structure and function from cine CMR14 in an unsupervised fashion15.Artificial intelligence (AI) algorithms based on deep learning consist of learning complex models directly from data sets. Initial success of AI applications in medical imaging was demonstrated by confirming expert-level diagnosesThe CERTAINTY study (CinE caRdiac magneTic resonAnce to predIct veNTricular arrhythmia) utilizes deep learning for VA risk prediction for individual patients from non-contrast cine CMR images in primary prevention ICD candidates. The findings from the CERTAINTY study are expected to improve our understanding of the mechanisms that predispose to VA, with the hope to develop a new paradigm to identify high- and low-risk individuals by extracting features associated with increased VA risk from cine CMR images in an unsupervised fashion. This article presents an overview of the CERTAINTY design, a descriptive analysis of the demographics of the study cohort, deep learning network architecture, results within the training cohort, and solicitation of participation to contribute external validation data sets.1. Baseline characteristics of the training cohort (n = 350) by VA occurrence are summarized in Table The inclusion and exclusion criteria of the CERTAINTY study population are described in Table 1hort n = 50 by VA First, we assessed the value of cine fingerprint to predict outcomes within the training cohort by applying a univariate Cox hazards model to cine risk scores calculated by the risk predictor autoencoder network Fig. . For allNext, we assessed the covariates together within multivariate Cox Proportional Hazards Regression models Fig. . We perfWe also used unadjusted and adjusted Cox proportional regression analyses to assess the performance of cine risk score to predict the primary endpoint Table . Univari16. Its rationale is based on a electrophysiological assumption that the gray zone represents transitional tissues between scar and normal myocardium, and that slow conduction within the gray zone serves as a substrate for reentrant arrhythmia17. More recent studies identified LA function19, quantified by echocardiography or cine CMR, as independent predictors of SCD. However, quantification of chamber dysfunction in prior studies relied on pre-specified feature extraction with chamber segmentation in multiple imaging views. In contrast, our algorithm automatically extracts features from only cine CMR in an unsupervised fashion. In addition, our findings showed that the predictive value of cine risk score is independent of LA function (Supplementary Table Our main findings are summarized as follows: 1) cine CMR inherently contains features of cardiac structure and function that improve VA risk prediction in primary prevention ICD candidates; (2) deep learning can automatically extract those features in the form of a cine risk score; and (3) the cine risk score is an independent biomarker of risk associated with VA and all-cause death in primary prevention ICD candidates. To our knowledge, this is the first study to demonstrate the incremental prognostic value of cardiac structure and function assessed from cine CMR in primary prevention ICD candidates. Earlier studies on predictive biomarkers for VA mainly focused on the presence, extent, and characteristics of myocardial scar detected by LGE CMR cine CMR20 and risk stratification12, it suffers from a lack of transparency and explainability21. In our study, the algorithm does not identify specific 4cv cine CMR function features associated with increased risk for VA. One possibility is imaging features associated with structural and functional interactions among the four chambers. Neural network-based algorithms can handle high-dimensional vector space simultaneously. This ability enables assessment of feature interactions as emergent phenomena that cannot be evaluated by studying each feature in isolation. Another possibility is imaging features associated with cardiac hemodynamics such as increased LV filling pressures, decreased LV compliance, increased LV wall stress associated with clinical heart failure. Because low RV22 and LA function19 are independent predictors of SCD, it is possible that the algorithm identified imaging features of impending biventricular failure associated with VA. Importantly, VA risk prediction approaches to date have not comprehensively incorporated metrics from these two possibilities, which are not mutually exclusive. Future studies are needed to address the knowledge gap as to the potential clinical significance of these metrics in VA prediction.Although deep learning is emerging as a powerful tool for diagnosis23. Notably, the algorithm is applied to 4cv cine CMR, which does not require intravenous contrast agents. This is particularly important for individuals with severe HF and cardiorenal syndrome who are considered for primary prevention ICD. The key innovation of the developed algorithm is that it is does not require manual segmentation of the heart chambers, which allows quicker risk assessment without cognitively biased human intervention.The proposed algorithm has a potential clinical impact to help primary prevention ICD candidates and their physicians make an informed decision regarding ICD implantation during the shared decision making processOur findings clearly indicate that non-contrast, cine CMR inherently contains features that improve VA risk prediction in primary prevention ICD candidates. However, the training cohort is of relatively small sample size and derives from a single institution. To assess the generalizability of the algorithm, it needs to be tested in an external validation cohort. The unique value of the CERTAINTY study is the long follow-up duration 10 years), because, unlike pharmacologic or ablation interventions, ICD is usually a lifetime commitment. The ICD candidates need to be informed of long-term implications at the time of shared decision making on ICD implantation. This unique value of long-term follow-up unfortunately limits the data availability of the CERTAINTY study. Even with the advent of remote ICD monitoring, most institutions have very few data sets with this duration of follow-up. Therefore, we encourage participation of multiple institutions in the CERTAINTY study by contributing to the external validation cohort. The baseline characteristics of the CERTAINTY population is described in Table 0 years, There are two limitations associated with the study. First, we used only 4cv cine CMR, which was included in a routine image acquisition protocol. Therefore, it is possible that the algorithm underestimated the degree of abnormal structure and function by missing regions that were not covered by the 4cv view. However, we believe that the advantage of our approach outweighs the disadvantage of including multiple views to assess 3-D structure and function, which would increase the scan time and post-processing burden including a higher training complexity for the algorithm. Second, the proposed method focuses on the extraction of cardiac function features while additional imaging features capturing anatomic abnormalities could further improve the risk prediction.Non-contrast, cine CMR inherently contains imaging features that can improve VA risk prediction in primary prevention ICD candidates without the need for manual contouring or contrast-enhancement. The deep learning algorithm could be easily implemented in routine clinical practice and provide valuable information during the shared decision-making process.Left Ventricular Structural Predictors of SCD, ClinicalTrials.gov Identifier: NCT01076660). The device type included a single-, dual-chamber ICD, and CRT-D based on current guidelines1. Patients were evaluated every 6 months and after any ICD shock. Patients who were not seen in-person underwent a telephone interview to update history. Out of this cohort, we have previously reported the association between LGE gray zone extent and VA inducibility at electrophysiology study (n = 47 with ICM)24, or appropriate ICD firing (n = 235 with ICM and NICM)8, the association between LGE-based arrhythmia simulation and appropriate ICD firing (n = 41 with ICM)25, the association between LGE scar characteristics and LVEF improvement (n = 202 with ICM and NICM)26, the association between LA function and inappropriate ICD firing (n = 162 with ICM and NICM)27, the association between LGE scar complexity and VA (n = 122 with ICM)28, and the association between time-varying risk covariates and appropriate ICD firing (n = 382 with ICM and NICM)29. In this study, 350 patients out of this cohort were included where both LGE and 4cv cine CMR images were available to test whether a deep learning algorithm can extract features associated with VA from 4cv cine CMR images in an unsupervised fashion. The primary outcome was defined as adjudicated appropriate ICD shock or appropriate anti-tachycardia pacing (ATP) for VA, including ventricular tachycardia and fibrillation. The secondary endpoint was death due to HF and all-cause death. Deaths were classified according to the most proximate cause after review of ICD interrogations, medical records, death certificates, autopsy reports, and eyewitness accounts.The protocol was approved by the institutional review board of the Johns Hopkins Medical Institutions, and all the patients provided written informed consent. All methods were carried out in accordance with relevant guidelines and regulations. We retrospectively analyzed a training cohort with ICM and NICM who underwent CMR at Johns Hopkins Medical Institutions  using 1.5-Tesla whole body scanners prior to primary prevention ICD implantation (median 3 days) between 2003 and 2015 with a standard imaging protocol  was used to obtain phasic LA volumes, strain, and strain rate from four-chamber cine CMR images27.The cohort (n = 350) were studies in one of two different types of scanners . Details of CMR imaging and analysis are described in Supplementary Appendix 14 was trained to extract cardiac structure and function features from 4cv cine CMR in a form of cine fingerprint in a fully unsupervised fashion  with Tensorflow  backend.In this work, the above-mentioned cine CMR dataset was used to train two independent neural networks. First, a probabilistic encoder-decoder neural networkχ2 test for categorical variables and the Student t-test or Mann–Whitney U test for parametric or nonparametric continuous variables, respectively. For risk prediction, we report the mean concordance (C-) index32 between predicted risk score and actual event time. We applied bootstrapping31 with resampling of 100 times for computing the C-index and CI to confirm the cross-validated results. We computed the hazard ratio (HR) including CI and p-value by fitting a linear Cox regression model on the predicted risk scores. To this end, the median risk value was used to divide the cohort in a low- and a high-risk group. Kaplan–Meier estimates for cumulative survival rates for the low- and high-risk groups were determined and statistically evaluated with the log-rank test. Cox proportional hazards models were used to estimate the association between variables and endpoints. Univariable analyses of all baseline variables were performed. Multivariable analyses were performed separately for each CMR feature by adjusting for clinical variables significantly associated with outcomes in univariable analysis (p < 0.05) and/or clinically deemed important (Model 1). We also performed competing risk analysis for the cumulative incidence of VA with death as a competing event, using the method by Fine and Gray33 and net reclassification improvement (NRI) analysis34. We used STATA  and lifelines  for statistical analysis. A two-sided p-value of < 0.05 was considered statistically significant.We used Pearson's Supplementary Information.


Interferon λ (IFN-λ) is critical for host viral defense at mucosal surfaces and stimulates immunomodulatory signals, acting on epithelial cells and few other cell types due to restricted IFN-λ receptor expression. Epithelial cells of the intestine play a critical role in the pathogenesis of Inflammatory Bowel Disease (IBD), and the related type II interferons (IFN-γ) have been extensively studied in the context of IBD. However, a role for IFN-λ in IBD onset and progression remains unclear. Recent investigations of IFN-λ in IBD are beginning to uncover complex and sometimes opposing actions, including pro-healing roles in colonic epithelial tissues and potentiation of epithelial cell death in the small intestine. Additionally, IFN-λ has been shown to act through non-epithelial cell types, such as neutrophils, to protect against excessive inflammation. In most cases IFN-λ demonstrates an ability to coordinate the host antiviral response without inducing collateral hyperinflammation, suggesting that IFN-λ signaling pathways could be a therapeutic target in IBD. This mini review discusses existing data on the role of IFN-λ in the pathogenesis of inflammatory bowel disease, current gaps in the research, and therapeutic potential of modulating the IFN-λ-stimulated response. Inflammatory bowel disease (IBD) is the collective term for Ulcerative Colitis (UC) and Crohn’s Disease (CD). IBD has historically been more prevalent in North America, Europe and Oceania, but worldwide incidence is rising, especially in developing nations . The CenThe intestinal epithelium plays a critical role in the pathogenesis of IBD , so fundIFNs are named for their important role in interfering with viral replication , 11, butThree human IFN-λs  were discovered in 2003  and a foThe IFN-λ receptor (IFNLR) is a heterodimeric complex composed of IL10RB and IFNLR1 subunits. IFNLR1 provides specificity for IFN-λ while IL10RB is shared by other IL-10-family cytokines with anti-inflammatory and wound healing functions  , 21, 27.In the context of complex inflammatory disease such as IBD, multiple cytokine pathways and transcription factors may act in concert , with syvia sequestration of STAT1 monomers  pathways . In contollagens . Howeverollagens , and it Biopsies from IBD patients show increased expression of both IFN-λ and IFNLR in the intestinal mucosa, with no discernable differences between CD and UC . HoweverUSP18, which has been shown to desensitize cells to the IFN-α/β response (IFNLR knockout mice exhibited increased tissue inflammation following dextran sulfate sodium (DSS)-induced colitis relative to WT controls, suggesting that induction of IFN-λ could be tissue protective in this acute inflammation model . The autresponse , 54. Addresponse , 56. In response . Increasresponse . Howeverresponse . Other rresponse . Taken tThere are also indications of a protective role for IFN pathway factors. IRF3 and IRF7 are important for induction of IFN-λ, IFN-α/β, and ISGs; experiments in IRF3/IRF7 double knockout mice with DSS colitis showed greater tissue inflammation relative to controls , suggestvia defense against pathogenic microbes that could trigger IBD symptom onset or exacerbate flares (Atg16L1 (autophagy- and CD-associated gene) to increase risk of DSS-triggered colitis -conjugated IFN-λ therapy has been examined for viral infections, including Hepatitis C, Hepatitis B, and recently SARS-CoV-2. PEG-IFN-λ has not been tested as a therapy for IBD. However, IFN-α/β has been tested in multiple trials, but there was no improvement compared to placebo and generally low tolerability with increased withdrawal due to adverse events . These rThe JAK/STAT pathway is an attractive therapeutic option based on the potential synergistic effects across IFNs and other cytokine networks, and has currently approved inhibitors and several investigational products. JAK inhibitors may have differential efficacy in IBD subsets due to evidence for differential regulation in CD vs UC . TofacitIn addition to therapeutic modulation of IFN signaling, IFN-λ or ISGs may be useful biomarkers to predict responsiveness to anti-TNFα treatments. The heterogeneity in therapeutic responsiveness and the significant systemic adverse effects associated with anti-TNFα therapy have compelled an extensive search for predictive biomarkers . Althougvia ISG expression in IECs themselves as well as via regulation of other inflammatory cell types such as neutrophils. However, a maladaptive role might be assumed in the setting of chronic inflammation. In particular, studies of Paneth cells in human subjects and mouse models suggest that IFN-λ takes a detrimental rather than protective role in the small intestine in CD. Finally, IFN-λ as a biomarker for prediction of response to anti-TNFα is an intriguing area for investigation, and continued consideration of JAK inhibitors that act across IFN types will be important.IFN-λ is critical for immunological function and barrier integrity at mucosal surfaces. Control of pathogenic microbes at the intestinal epithelium without inducing overly robust inflammatory responses is a well-established normal function of IFN-λ. Additionally, there is evidence that IFN-λ promotes healing in colonic epithelial tissues and JW wrote the initial draft and all authors revised, edited, and approved the final manuscript. All authors contributed to the article and approved the submitted version.DC was supported by NIH grant T32-AI007472 and the Medical Research Foundation of Oregon (OHSU). TN was supported by NIH grant R01-AI130055. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, the correlation between LRs cholesterol and the clinical profile showed a significant association with autistic traits  and adaptative behavior . These results support the clinical significance of LR cholesterol alterations in FXS. Further studies are warranted to investigate the implication of LRs in FXS pathophysiology and ASD.Fragile X syndrome (FXS) is the most prevalent monogenic cause of intellectual disability and autism spectrum disorder (ASD). Affected individuals have a high prevalence of hypocholesterolemia, however, the underlying mechanisms and the clinical significance remains unknown. We hypothesized that decrease in the plasma cholesterol levels is associated with an alteration of cholesterol content within the lipid rafts (LRs) which ultimately affects the clinical profile of FXS individuals. The platelets LRs were isolated by ultracentrifugation on sucrose gradient from 27 FXS and 25 healthy controls, followed by measurements of proteins, cholesterol, and gangliosides content. Autistic and adaptive behaviour of affected individuals were respectively assessed by the Social Communication Questionnaire and Adaptive Behavior Assessment System. Our results suggest a decrease in the cholesterol content of LRs in FXS individuals as compared to controls. As opposed to controls, LR cholesterol was significantly associated with plasma total cholesterol (r = 0.47;  In most cases, the mutation results from a large expansion of CGG repetitions in the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene leading to a deficit of the Fragile X Mental Retardation Protein (FMRP), an ubiquitary RNA-binding protein highly expressed in the brain3. FMRP controls the translation of many mRNAs encoding the proteins critical for synaptic structure and functions4. Studies in fmr1 knockout (KO) mice have shown that FMRP deficiency triggers: an upregulation of signaling pathways related to the group 1 metabotropic glutamate receptor (mGluR1 and mGluR5) activation6; an excessive internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR); and a reduction of N-methyl-D-aspartate receptors (NMDAR)8. All the mentioned factors contributes to exaggerated long-term depression (LTD), a hallmark of FXS10.Fragile X Syndrome (FXS), an X-linked neurodevelopmental disorder, is the most prevalent monogenic cause of inherited intellectual disability (ID) and autism spectrum disorder (ASD)11. These platforms recruit important synaptic receptors including mGluRs, NMDAR, and AMPAR12. Indeed, LRs facilitate protein–protein and protein-lipid interactions13. Moreover, LRs play crucial roles in the compartmentalization of signaling molecules on the cell membrane and intracellular trafficking17. Studies in rat brain tissues have shown that cholesterol depletion alters the morphology of dendrites (reduce the number and increase the size of dendritic spines) and modulates the synaptic activity mediated by AMPAR and NMDAR19. To the best of our knowledge, no previous study has investigated the implication and correlation of the LRs cholesterol content with the cognitive dysfunction and autistic traits of the FXS affected humans.Lipid rafts (LRs) are dynamic structures enriched in cholesterol, proteins, and gangliosides that transiently move within the plasma membrane in order to facilitate a cellular response20. The clinical profile of the FXS affected individuals is frequently associated with hypocholesterolemia, a condition characterized by plasma cholesterol lower than the 5th centile of a normalized population. Specifically, 90% of FXS individuals have plasma cholesterol below the 50th centile of the normalized population and up to 30% fulfill the criteria of hypocholesterolemia21. Our recent report suggested a decrease of proprotein convertase subtilisin/kexin type 9 (PCSK9) activity linked to an alteration of serine-phosphorylation as an underlying mechanism of FXS associated hypocholesterolemia22. Indeed, PCSK9 is implicated in the degradation of low-density lipoprotein (LDL) receptors, and therefore a decrease in PCSK9 activity is associated with low plasma cholesterol. Moreover, our results suggest a negative correlation between plasma cholesterol and aberrant behavior as evaluated by the Aberrant Behavior Checklist-Community (ABC-C) total score21. Since cholesterol is essential for cell membrane integrity, synaptic development and neurotransmitter release, a decrease of plasma cholesterol could alter the content in cholesterol of LRs and potentially contribute to the FXS physiopathology24.Each individual with FXS is unique and displays variable degrees of ID, adaptive and aberrant behaviors25. Platelets express many receptors and markers that are also found in neurons and thus may recapitulate the defects observed in FXS neurons, such as glutamate signaling pathways28. Moreover, FMRP levels quantified in the platelets of the FXS affected individuals are lower than those observed in the healthy controls and significantly correlate with cognitive functions29.In humans, brain tissue is barely accessible. Platelets have been described as a plausible surrogate to study neurons integrity in living animals and humans31, Smith-Lemli-Opitz-Syndrome32, Parkinson33, and Huntington34. Moreover, perturbation of the cholesterol content of LRs in platelets has been revealed in neurodevelopmental and neurocognitive diseases37. However, the implication of LRs in the FXS human pathophysiology remains unclear. This study thus aims to explore the lipid content of LRs in platelets of FXS individuals as compared to healthy individuals. We also investigated the relation of LRs cholesterol with lipid profile and cognitive functions in FXS.Perturbations of plasma cholesterol have been reported in several cognitive disorders such as Alzheimer21. Characteristics of FXS and control participants are shown in Supplementary Table at Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke (CHUS), between January and June 2015. Individuals with acute conditions, disorders affecting lipid metabolism, or treated with lipid-lowering drugs were excluded. The LipidX study was approved by the Scientific and Ethics Board of the local research center. All participants provided informed consent prior to participation in the study or by the tutor/caregiver on behalf of the mentally disabled minors and adults with FXS. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Centre de Recherche du Centre Hospitalier de l’Université de Sherbrooke .The design and population of the LipidX study have been described previouslyCIUSSS de l’Estrie-CHUS: plasma total cholesterol (TC), triglycerides (TG), C-HDL were measured by enzymatic methods (Modular Roche P800); C-LDL was calculated using the Friedewald formula; apolipoprotein B (ApoB) and apolipoprotein A (ApoA) were determined by immunoturbidimetric assays . FMRP quantitation was performed on platelets by Western Blot as previously described29.Medical history and anthropometric measures including weight, height, body mass index (BMI) were obtained for each participant. The clinical profile of each FXS participant was assessed by the following questionnaires and completed by their caregiver: (1) age-adjusted Adaptive Behavior Assessment System – Second Edition (ABAS-II); (2) Aberrant Behavior Checklist-Community (ABC-C); (3) Social Communication Questionnaire (SCQ) and (4) Anxiety, Depression, and Mood Scale (ADAMS). Fasting blood samples were collected in acid citrate dextrose (ACD) (BD vacutainer) from all participants to perform plasma lipid analyses and platelet isolation. Specifically, the following tests were performed at the clinical biochemistry laboratory of the 29. Briefly, the platelet-rich plasma (PRP) was separated from whole blood after a 10 min centrifugation at 300 xg at room temperature and collected without disturbing the buffy coat. The platelet count was then performed on a flow cytometer analyzer . After a 15 min centrifugation at 2,400 xg, the platelet pellet was isolated and then washed twice in phosphate-buffered saline (PBS) containing EDTA 5 mM (Invitrogen by Thermo Fisher Scientific).Platelets were isolated from the whole blood as previously described39. Briefly, the platelet pellet was disrupted in lysis buffer  for 30 min and homogenized using a syringe and needle 27G (Terumo). The platelet lysate was mixed with 2 mL of 80% sucrose followed by two 4 mL layers containing respectively 30% and 5% sucrose to allow a gradient formation during the centrifugation. The lysate was centrifuged at 37,000 RPM for 18 h at 4 °C . After the centrifugation, 12 fractions of 1 mL were gently collected from the top (F1) through to the bottom layer (F12). The enriched LR fractions were identified by monitoring the elution of flotillin-1 by Western Blot. Briefly, 10 µL of each fraction were separated on a 4–10% gradient SDS polyacrylamide gel and proteins were transferred on nitrocellulose. Membranes were blocked in 5% non-fat dry milk, incubated with anti-flotillin-1  followed by anti-rabbit IgG coupled to Alexa FluorVR 680 . Fluorescence was revealed using an Odyssey Infrared Imaging System (LI-COR Biosciences). Semi-quantification of flotillin-1 was performed with Image-J software .Cholesterol measurement was performed using the Amplex® Red Cholesterol Assay Kit (Life Technologies). LRs fractions were diluted 4 times in the reaction buffer and 50 µL were used for the assay. Fifty microliters of working solution  was added to the samples. After an incubation of 30 min, at 37 °C in the dark, the fluorescence was measured at 560 nm in a microplate reader . Dot-blot was used to measure gangliosides: 200 µL of the standards Ganglioside GM1, Asialo (Santa Cruz)  and samples (diluted 5 times) were loaded on nitrocellulose. Each dot was washed three times with PBS, blocked with 5% non-fat dry milk in PBS for 1 h at room temperature, washed twice with PBST (PBS with 0.05% Tween 20) and incubated overnight at 4 °C on a shaker with 1 µg/mL cholera toxin B/horseradish peroxidase conjugate (HRP-CTB)  in 5% non-fat milk. The following day, membranes were washed twice with PBST and once with PBS then revealed using an enhanced chemiluminescence (ECL) detection kit on a ChemiDoc system (Bio-Rad) .Descriptive analyses were performed on demographic variables. Depending on the normality distribution assessed by the Shapiro–Wilk test, the Mann–Whitney U test was used for the comparison between the two groups. The Pearson correlation coefficient (r), univariate linear, and multiple regressions were respectively performed to investigate the association and to predict clinical profile. Statistical significance was established at an alpha level of rejection of 0.05. Multiple testing correction was performed using FDR (Benjamini and Hochberg). Outliers were removed from the analyses according to the ROUT method (Q = 1%) in Graphpad Prism software version 9 The average distribution of LRs components including cholesterol, proteins, and gangliosides GM1, all adjusted to obtain concentrations expressed in one million platelets are shown in Fig. vs 0.007 (µg/mL)/million platelets, and 0.013 vs 0.019 (ng/mL)/million platelets, respectively, although the differences are not statistically significant  , social  and practical . Furthermore, LRs cholesterol significantly correlates with SCQ score   Fig. A as wellp = 0.042), LDL-C , ApoB , and ApoA1 . Meanwhile, no significant correlations between these parameters are observed in the control cohort. In addition, no significant correlation is obtained between total cell cholesterol  and plasma total cholesterol, in neither FXS affected individuals, nor in control healthy individuals.Associations between LRs cholesterol and lipid profile are shown in Fig. 43. Indeed, platelets have been used as a surrogate to study neuron function of human brain in several neurocognitive and neurodevelopmental disorders including Alzheimer disease or ASD44. The present study reports the first investigation of LRs from platelets of FXS participants as compared to healthy controls.Lipid rafts are plasma membrane microdomains rich in cholesterol that are important for signal transduction. In neurons, LRs serve as ordered platforms that coordinate signaling molecules through membrane receptors and are involved in synaptic transmission. LRs from human platelets have been investigated in cognitive disorders such Alzheimer diseases = 0.49)21, while the present study reports a stronger association between LRs cholesterol and all adaptive domains. In addition, lower LRs cholesterol was associated with a higher score of autistic traits as evaluated by the SCQ questionnaire. These data agree with our previous report in ASD population that showed an increase of cognitive impairment risk when the plasma cholesterol is below the 10th centile of normalized population45. Taken together, these findings suggest a potential mechanistic implication of low cholesterol in cognitive dysfunction of FXS individuals probably through disruption of cholesterol content in LRs.Our data show a slight reduction of LRs cholesterol in FXS as compared to healthy controls. Although the difference was not statistically significant, the direct correlation of LRs cholesterol with adaptative functions suggests clinical importance. Specifically, LRs cholesterol correlated positively with adaptative behavior as evaluated by ABAS: FXS participants with lower LRs cholesterol displayed a lower degree of three adaptive domains including practical (r = 0.62), social (r = 0.66), and conceptual skills (r = 0.67). In a previous study, we reported a positive link between plasma cholesterol and ABAS practical domain  after cholesterol depletion of LRs, followed by a higher expression of mGluR1 in hippocampus neurons46. Another study showed that cholesterol depletion of LRs resulted in a significant reduction of NMDA receptors in cultured wild rat neurons47. Importantly, the excessive signaling of mGluR1 is a hallmark of FXS and a recent report suggests an association with NMDA receptor hypofunction7. Similarly, in vitro research suggests an alteration of platelet function following cholesterol depletion of LRs48. We thus hypothesize that LRs from platelets are a study model of LRs from neurons. Indeed, a reduction of cholesterol content in LRs might contribute to FXS pathophysiology and ultimately to clinical profile.In neurons, LRs are implicated in synapse morphology and functions45. Our results show a significant direct association between the plasma total cholesterol (including ApoB and ApoA1 containing lipoproteins) and LRs cholesterol only in the FXS group. In contrast, no significant association is obtained between plasma total cholesterol and total cell cholesterol in neither FXS nor control group. We believe thus that abnormal distribution of membrane cholesterol into lipid rafts might be the underlying cause of low cholesterol content in LR, observed in FXS. Indeed, cell cholesterol is determined not only by in situ synthesis but also by cellular influx and efflux mediated by LDL and HDL respectively49. ApoB represents lipoproteins (mainly LDL) delivering hepatic cholesterol to the peripheral tissues, while ApoA1 (mainly HDL) mediates the reverse transport of cholesterol from peripheral cells to the liver52. The cholesterol distribution into lipid rafts is regulated by Apolipoprotein A-I binding protein (AIBP)53. The association of AIBP with ApoA1 or HDL-C allows a rapid and effective efflux of cholesterol through an interaction with ATP-binding cassette transporter type A1 (ABCA1) or through cytoskeleton changes54. In vitro and in vivo animal studies suggest that high HDL levels are associated with low LRs cholesterol content. However, AIBP does not promote cholesterol efflux in the absence of ApoA1 or HDL53. The finding that FXS had significantly lower HDL-C compared to healthy controls might explain the direct link observed between ApoA1 and LR cholesterol in FXS group, whereas correlation results in our control group did not corroborate with previous in vitro findings55. The limited number of participants or the cellular model used in the present study (platelets) might account for this discrepancy. However, this different interaction of cholesterol pools in FXS as compared as controls highlight the possibility of an impairment of cholesterol regulation in LRs. Further studies with labeling technics are warranted in FXS to validate the hypothesis that an alteration of control mechanisms of cholesterol influx and efflux alters the distribution of cholesterol into LRs56.Considering the high prevalence of hypocholesterolemia in FXS and ASD as well as the reported link of plasma cholesterol with clinical profile, we explored whether plasma level of cholesterol is associated with cholesterol content in LRs57. Although two pools are separated by hematoencephalic barrier, the same genes are implicated in cholesterol metabolism and thus similar lipid alterations might be present in both, brain and peripheral circulation. Several studies in neurodevelopmental and neurodegenerative conditions reported associations between plasma cholesterol and cognitive or behavioral functions60. We hypothesize that brain cholesterol levels are also decreased in FXS and might trigger to some extend the clinical profile. Since FMRP regulates the expression of many cytoplasmic proteins; it might be involved in lipid synthesis and transport and its absence might dysregulate lipid rafts properties. However, further studies are needed to fully understand the interplay between FMRP and cholesterol metabolism, particularly if it regulates sterol regulatory element binding proteins.It is noteworthy to mention brain cholesterol is synthesized in situ and is independent from peripheral cholesterol metabolism62. It has been shown that lovastatin inhibits the ERK pathway in FXS resulting in a normalization of protein synthesis63. On the other hand, lovastatin is a cholesterol-lowering agent that inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme of cell cholesterol biosynthesis64. It has been shown that lovastatin decreases cholesterol content in LR of dendrites of cultured hippocampal neurons and cholesterol depletion following statin treatment leads to a gradual loss of synapses18. In FXS, there is an abnormally high density of dendritic spines that are usually long and immature66. In the light of our results, we can hypothesize that lovastatin might lead to a decrease in the number of these abnormal spines through depletion of cholesterol content in LR. This might justify short-term treatments with lovastatin in FXS. However, we should be careful when considering long-term treatment periods with statins in these individuals, especially those with hypocholesterolemia.Few study trials of lovastatin have been performed on FXS individuals, specifically short-term treatment periods showing discrepancy results of phenotype improvementfmr1 KO mice neurons. Second, the small sample size of each group limited us to conclude significant differences between LRs components from FXS and controls. Moreover, the small number of FXS females, limited our conclusion about the reflection of sex effect in our results. The sample size was primarily calculated to detect a difference in plasma cholesterol of the FXS population (LipidX study)21. A larger study of FXS individuals is warranted to validate the correlations of LRs components with lipid and clinical profiles. Third, since lipid rafts are enriched in other lipids, such as, sphingolipids or saturated fatty acids, a deeper lipidomic analysis would have improved the interpretation of our findings67. Fourth, since we did not have controls affected by hypocholesterolemia, it remains unclear whether our results are caused by the pathogenesis of FXS or is a consequence of hypocholesterolemia in FXS individuals for instance, demyelination due to the lack of cholesterol and leading to clinical manifestations.Some limitations should be considered in the present study. First, although, we used platelet as a model to investigate neurons LRs, we agree that biopsies of neuronal tissue such as motor endplates or peripheral nerves might strengthen the conclusions. However, considering the invasive procedure, it would be difficult to justify of human biopsies beyond clinical necessity. As an alternative, the implication of cholesterol content in LRs in FXS pathogenesis might be further investigated in fmr1 and genes involved in the lipid metabolism might further elucidate the implication of FMRP in hypocholesterolemia pathogenesis.In summary, this study was a first attempt to investigate composition of LRs from platelets of FXS participants, specifically the association of LRs cholesterol with cognitive functions of FXS such as adaptative behavior and autistic profile. Our results support the use of platelets as a surrogate model for the neuron to study the implication of LRs alteration in FXS. However, larger studies including lipidomic analysis of LRs are warranted to validate our results not only on FXS, but also in other neurodevelopmental disorders such as ASD. Moreover, bioinformatic studies investigating the link between Supplementary Information.