Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology.Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects.Comparison of the newly characterized Monarch butterfly  contains supplementary material, which is available to authorized users. Despite experimental efforts in a number of taxa, a robust determination of parasperm function has yet to be attained.Spermatozoa exhibit an exceptional amount of diversity at both the ultrastructure and molecular levels despite their central role in reproduction . One of Bombyx mori) . In. In47]. lization . Consist09) Fig. . Aside fongation .p = 7.06E-19), as well as more moderate enrichments in several categories relating to peptidase activity or regulation of peptidase activity (data not shown). The broad representation of proteins involved in proteolytic activity is worthy of discussion, not solely because these classes of proteins are abundant in other sperm proteomes, but also because proteases are involved in the breakdown of the fibrous sheath surrounding Lepidoptera eupyrene sperm upon transfer to the female [B. mori) [Manduca [Manduca , are excellent candidates for a sperm activating factor(s) in Lepidoptera.Molecular Function GO analysis revealed an enrichment of oxidoreductase proteins acting on NAD(P)H p = .06E-19, B. mori)  and a si[Manduca . Blast2GManduca was the first foray into the molecular biology of Lepidopteran sperm and was motivated by our interest in the intriguing heteromorphic sperm system that is found in nearly all species in this order [Manduca sperm proteome [Drosophila identified 203 (35%) Monarch proteins with orthology relationships, of which 166 (82%) were within “one-to-one” orthology groups , as would be expected given the taxonomic relationship of these species. Additionally, gene duplication does not appear to be a widespread contributor to divergence relating to sperm form or function between Lepidoptera and Drosophila. It is also noteworthy that 27 orthologous proteins between Monarch and Drosophila were identified that differed substantially in length (>35%). Additional comparative analyses with gene models in other available Lepidoptera and Drosophila genomes indicated that 17 of these cases represent bona fide divergence in gene length, while the remainder are likely to represent gene model annotation errors in the Monarch genome. These issues were most commonly the result of inclusion/exclusion of individual exons with adjacent gene models and full gene model fusions , categories associated with energy metabolism, including ATP metabolic process (p = 1.64e-15), generation of precursor metabolites and energy (p = 9.77e-21), and multiple nucleoside and ribonucleoside metabolic processes. Analysis of cellular component GO terms revealed a significant enrichment for mitochondrion related proteins (p = 3.72e-22), respiratory chain complexes (p = 8.25e-12), dynein complexes (p = 1.37e-5), and axoneme (p = 3.31e-6). These GO category enrichments are consistent with a core set of metabolic, energetic, and structural proteins required for general sperm function. Similar sets of core sperm proteins have been identified in previous sperm proteome comparisons [Drosophila. This includes proteins associated with sperm individualization, including cullin3 (Cul3) and SKP1-related A (SkpA), which acts in cullin-dependent E3 ubiquitin ligase complex required for caspase activity in sperm individualization [Recent comparative analyses of sperm composition across mammalian orders successfully identified a conserved “core” sperm proteome comprised of more slowly evolving proteins, including a variety of essential structural and metabolic components. To characterize the “core” proteome in insects, we conducted a GO analysis using parisons , 52, 53.lization , gudu, alization , and porlization . Two prolization  and an alization .Manduca and Drosophila sperm, although this analysis was limited by the extent of orthology between these taxa [Manduca revealed a significant correlation   were identified. S-LAPs are members of a gene family first characterized in Drosophila that has recently undergone a dramatic expansion, is testis-specific in expression and encodes the most abundant proteins in the D. melanogaster sperm proteome [Despite the more proximate link between proteome composition and molecular phenotypes, transcriptomic analyses far outnumber similar research using proteomic approaches. Nonetheless, recent work confirms the utility of comparative evolutionary proteomic studies in identifying both conserved  and diveese taxa . To furt15) Fig. . We noteproteome . As woulManduca have distinct mating systems; female Monarch butterflies remate considerably more frequently than Manduca females, increasing the potential for sperm competition [P < 0.05; Fig. p value = 0.2757). Several of these proteins are worthy of further discussion given their role in sperm development, function or competitive ability. Proteins identified as more abundant in the Monarch sperm proteome were heavily dominated by mitochondrial NADH dehydrogenase subunits  and other mitochondria-related proteins, including ubiquinol-cytochrome c reductase core protein 2 (UQCR-C2), cytochrome C1 (Cyt-C1), and glutamate oxaloacetate transaminase 2 (Got2). Additionally, two proteins with established sperm phenotypes were identified as more abundant in Manduca. These included dynein light chain 90F (Dlc90F), which is required for proper nuclear localization and attachment during sperm differentiation [We next sought to identify proteins exhibiting differential abundance between the two species. As discussed earlier, Monarch and petition . These dntiation , and cutntiation . Serine ntiation , 51. AltManduca identified 111 acetylated peptides within 63 proteins. We found evidence for conserved PTMs within Lepidoptera in 19 proteins (36% of those identified in Monarch), including Lys40 of alphaTub84B.During spermatogenesis, the genome is repackaged and condensed on protamines and the cellular machinery required for protein synthesis are expelled. Consequently, mature sperm cells are considered primarily quiescent . NonetheManduca (71%). This included the ortholog of the Y-linked Drosophila gene WDY. Although a specific function for WDY in spermatogenesis has yet to be determined, WDY is expressed in a testis-specific manner and under positive selection in the D. melanogaster group [Drosophila sperm samples purified in a similar manner . Lastly, identical acetylation and phosphorylation PTM patterns were identified for Monarch and Manduca HACP012 (DPOGS213379), a putative seminal fluid protein of unknown function previously identified in the Postman butterfly (Heliconius melpomene) [In contrast to acetylation, only 75 Monarch sperm proteins showed evidence of phosphorylation, 53 of which were also modified in er group . The reler group . Howeverer group . We notelpomene) , 70. Thep < 0.05; Fig. p < 0.01). Orthology relationships in OrthoDB are established by a multi-step procedure involving reciprocal best match relationships between species and identity within species to account for gene duplication events since the last common ancestor. As such, the underrepresentation of orthology relationships is unlikely to be accounted for by lineage-specific gene duplication. Therefore, rapid evolution of sperm genes appears to be the most reasonable explanation for the breakdown of reciprocal relationships (see below). This conclusion is consistent with a diverse body of evidence that supports the influence of positive selection on male reproductive genes [Rapid gene evolution  and geneve genes , 74, incve genes , 75–78. n = 171) were considered “unresolved”. Although the number of Monarch-specific proteins is considerably higher than the eight Manduca-specific proteins found in our previous study, the number of Lepidoptera specific is comparable to our previous estimate in Manduca (n = 126). These observations support the hypothesis that a substantial subset of lepidopteran sperm proteins are likely rapidly evolving and thus exhibit little detectable similarity. To pursue this possibility, we calculated nonsynonymous divergence (dN) for 10,212 genes across four species of butterfly and compared dN between Lepidoptera specific sperm proteins, sperm proteins with homology outside of Lepidoptera and the remainder of the genome  and the remainder of the genome . Interestingly, sperm proteins with homology outside of Lepidoptera also evolve significantly slower than the genome as whole (D = 0.30. p = 3.14 × 10−6). Consistent with these trends, 17.7% of Lepidoptera specific sperm proteins where amongst the fastest evolving in the genome (top 5%), compared to only 2.6% of sperm proteins with homology outside of Lepidoptera. In light of the rapid divergence of Lepidoptera specific proteins we next sought to assess their potential contribution to sperm function using protein abundance as a general proxy in the absence of functional annotation for nearly all of these proteins. As was observed in Whittington et al. [p = 0.0009, Fig. The second analysis aimed to characterize the distribution of taxonomically restricted Monarch sperm proteins using BLAST searches across 12 insect species. Based on the analysis above, our a priori expectation was that a substantial number of proteins with identifiable homology amongst Lepidoptera would be absent from more divergent insect species. This analysis identified a total of 45 proteins unique to Monarch, 140 proteins (23.9% of the sperm proteome) with no detectable homology to proteins in non-Lepidopteran insect taxa and 173 proteins conserved across all species surveyed Fig. . Proteinome Fig. . The aveDichotomous spermatogenesis in Lepidoptera, and in particular the production of sperm which do not fertilize oocytes, has intrigued biologists for over a century. Despite widespread interest, little is known about the functional roles fulfilled by apyrene sperm or why they have been retained in a nearly ubiquitous fashion during the evolution of Lepidoptera. Our comparative proteomic analysis of heteromorphic sperm, a first of its kind, provides important perspective and insights regarding the functional and evolutionary significance of this enigmatic reproductive phenotype. First, our analyses indicate that a substantial number of novel sperm genes are shared amongst Lepidoptera, thus distinguishing them from other insect species without dichotomous spermatogenesis, and suggest they are associated with heteromorphic spermatogenesis and the diversification of apyrene and eupyrene sperm. This observation can be attributed, at least in part, to the rapid evolution of Lepidoptera specific sperm genes. It is also possible that de novo gene gain may contribute to this observed genetic novelty, although it is not possible to assess this directly with the genomic and transcriptomic resources currently available in Lepidoptera. Our comparative and quantitative analyses, based on protein abundance measurements in both species, further suggests that some of these proteins contribute to apyrene sperm function and evolution. Given that apyrene sperm constitute the vast majority of cells in our co-mixed samples, it is reasonable to speculate that higher abundance proteins are either present in both sperm morphs or specific to apyrene cells. Confirmation of this will require targeted proteomic analysis of purified apyrene and eupyrene cell populations and will result in a refined set of candidates for further study in relation to apyrene sperm functionality. Ultimately, the comparative analysis of morph-specific sperm proteomes is critical to understanding the functional diversification of the fertilization incompetent apyrene sperm morph and the evolutionary maintenance of dichotomous spermatogenesis.Our results indicate that the origin of heteromorphic spermatogenesis early in Lepidoptera evolution and/or the subsequent evolution of this system is associated with a burst of genetic novelty that is distinct from patterns of diversification across the remainder of the genome. The evolution of dichotomous spermatogenesis has therefore had a marked impact on Lepidoptera molecular evolution and suggests that focused studies of other reproductive transitions may inform our broader understanding of the evolution of reproductive genetic systems and their contribution to genomic novelty.Additional file 1:- Predicted functions of Monarch proteins curated using Uniprot and Blast2GO. (XLSX 29 kb)Functional InformationAdditional file 2:- Proteomic data including full Monarch sperm proteome, MS/MS results by replicate, PTM information, orthology relationships and rates of molecular evolution. (XLSX 100 kb)Mass Spectrometry DataAdditional file 3:- APEX protein abundance estimates and differential abundance analyses. (XLSX 54 kb)Protein Abundance and Quantitative AnalysesAdditional file 4:- Phylogeny exhibiting the evolutionary relationship of the thirteen insect species utilized in this study. (PDF 129 kb)Phylogenetic Results


Accurately measuring antibody repertoire sequence composition in a small amount of blood is challenging yet important for understanding repertoire responses to infection and vaccination. We develop molecular identifier clustering-based immune repertoire sequencing (MIDCIRS) and use it to study age-related antibody repertoire development and diversification before and during acute malaria in infants (< 12 months old) and toddlers (12–47 months old) with 4−8 ml of blood. Here, we show this accurate and high-coverage repertoire-sequencing method can use as few as 1000 naive B cells. Unexpectedly, we discover high levels of somatic hypermutation in infants as young as 3 months old. Antibody clonal lineage analysis reveals that somatic hypermutation levels are increased in both infants and toddlers upon infection, and memory B cells isolated from individuals who previously experienced malaria continue to induce somatic hypermutations upon malaria rechallenge. These results highlight the potential of antibody repertoire diversification in infants and toddlers. Somatic hypermutation of antibodies can occur in infants but are difficult to track. Here the authors present a new method called MIDCIRS for deep quantitative repertoire sequencing with few cells, and show infants as young as 3 months can expand antibody lineage complexity in response to malaria infection. Although we and others have developed methods to control for artifacts from high amplification bias and sequencing error rates through data analysis9, obtaining accurate sequencing information has now been made possible by the use of molecular identifiers (MID)13. MIDs serve as barcodes to track genes of interest through amplification and sequencing. They are short stretches of nucleotide sequence tags composed of randomized nucleotides that are usually tagged to cDNA during reverse transcription to identify sequencing reads that originated from the same mRNA transcript. Despite these advancements, the large amount of input RNA required and low diversity coverage make it challenging to analyze small numbers of cells, such as memory B cells from dissected tissues or blood draws from young children, using IR-seq because these samples require many PCR cycles to generate enough material to make sequencing libraries, thus exacerbating PCR bias and errors.V(D)J recombination creates hundreds of billions of antibodies and T cell receptors that collectively serve as the immune repertoire to protect the host from pathogens. Somatic hypermutation (SHM) further diversifies the antibody repertoire, which makes it impossible to quantify this diversity with nucleotide resolution until the development of high-throughput sequencing-based immune repertoire sequencing (IR-seq)11. Given the wide use of IR-seq in basic research as well as clinical settings, we believe the method outlined here will serve as an important guideline for future IR-seq experimental designs.Here we report the development of MID clustering-based IR-seq (MIDCIRS) that further separates different RNA molecules tagged with the same MID. Using naive B cells, we demonstrate that MIDCIRS has a high coverage of the diversity estimate, or different types of antibody sequences, that is consistent with the input cell number and a large dynamic range of three orders of magnitude compared to other MID-based immune repertoire-sequencing methodsPlasmodium falciparum infection. Although the antibody repertoire in fetuses14, cord blood15, young adults6, and the elderly16 has been studied, infants and toddlers are among the most vulnerable age groups to many pathogenic challenges, yet their immune repertoires are not well understood. Infants are widely thought to have weaker responses than toddlers to vaccines because of their developing immune systems17. Thus, understanding how the antibody repertoire develops and diversifies during a natural infection, such as malaria, not only provides valuable insight into B cell ontology in humans, but also provides critical information for vaccine development for these two vulnerable age groups. Using peripheral blood mononuclear cells (PBMC) from 13 children aged 3–47 months old before and during acute malaria, with two of the children followed for a second year and nine additional pre-malaria individuals we show that infants and toddlers use the same V, D, and J combination frequencies and have similar complementarity determining region 3 (CDR3) length distributions. Although infants have a lower level of average SHM than toddlers, the number of SHMs in reads that mutated in infants is unexpectedly high. Infants have a similar, if not higher, degree of antigen selection strength, assessed by the likelihood of amino acid-changing SHMs, compared with toddlers. Remarkably, during acute malaria, antibody lineages expand in both infants and toddlers, and this expansion is coupled with extensive diversification to the same degree as in young adults in response to acute malaria19. Furthermore, informatically reconstructing antibody clonal lineages using sequences from both pre-malaria and acute malaria samples from the same individuals shows that infants are capable of introducing SHMs upon a natural infection. This two time point-shared lineage analysis reveals that memory B cells isolated from pre-malaria samples in malaria-experienced individuals continue to induce SHMs upon acute malaria rechallenge and most IgM memory B cells maintain IgM, whereas a small fraction switch isotypes. In summary, using an accurate and high-coverage IR-Seq method, we discover features of the antibody repertoire that were previously unknown in infants and toddlers, shedding light on the development of the immune system and its interactions with pathogens.As a proof of principle, we use MIDCIRS to examine the antibody repertoire diversification in infants (<12 months old) and toddlers (12–47 months old) from a malaria endemic region in Mali before and during acute 13. They can be designed with sufficient length, and thus diversity, to tag each individual molecule uniquely. However, this requires knowledge of the total number of RNA molecules in the sample, which requires a significant amount of time and effort to measure for each sample before library preparation. Longer MIDs are likely to decrease the reverse transcription efficiency21. Therefore, we fixed the MID length at 12 random nucleotides and developed a generalized approach to identify each individual transcript using a sequence similarity-based clustering method to separate a group of sequencing reads with the same MID into sub-groups. Consensus sequences are then built by taking the average nucleotide at each position within a sub-group, weighted by the quality score. Each consensus sequence represents an RNA molecule, and identical consensus sequences are merged into unique consensus sequences, or unique RNA molecules  to test the dynamic range of MIDCIRS. The resulting diversity estimates, or different types of antibody sequences, display a strong correlation with cell numbers at 83% coverage . Previous studies have shown that about 80% of naive B cells express distinct heavy chain genes23, thus our method achieves a comprehensive diversity coverage that is much higher than other MID-based antibody repertoire-sequencing techniques13.We used sorted naive B cells with varying numbers  from PCR and sequencing errors (1 in 200 nucleotides24)  are able to generate a significant number of mutations in response to infection, which would demonstrate their capacity to diversify the antibody repertoire33.SHM is an important characteristic of antibody repertoire secondary diversification due to antigen stimulation34  and around 20 in toddler IgG and IgA sequences . To minimize any possible inflation of SHMs, we excluded all sequences that were mapped to novel alleles, which were identified by both TIgGER35 and inspecting IgM sequences (Methods). These putative novel alleles account for 8% of all unique sequences on average , including infants. These results demonstrate high levels of SHM that exceed what have been documented previously34.Here, we show that infants (< 12 months old) and toddlers (12–47 months old) reach an unexpectedly high level of SHMs in all three major isotypes, particularly IgG and IgA34 Fig. . AlthougP. falciparum during the annual malaria season26, we hypothesized that the SHM load would increase with age. However, we found that the SHM load rapidly increases with age in infancy and then appears to plateau around 12 months of age in an initial smaller set of children with paired pre-malaria and acute malaria PBMC samples , the parts of the antibody responsible for proper folding. The unexpectedly high level of SHMs observed in infants prompted us to ask whether those SHMs have characteristics of antigen selection, as seen in older children and adults. As previous studies have shown that infants have limited CD4 T cell responses and neonatal mice exhibit poor germinal center formation17, we hypothesized that infant antibody sequences would display weaker signs of antigen selection. Here, we use a recently published tool, BASELINe37, to compare the selection strength. BASELINe quantifies the likelihood that the observed frequency of replacement mutations differs from the expected frequency under no selection; a higher frequency implies positive selection and a lower frequency implies negative selection, and the degree of divergence from no selection relates to the selection strength. Surprisingly, despite infants harboring fewer overall mutations, these mutations are positively selected in the CDRs and negatively selected in the FWRs in both IgG and IgA ), suggesting that the significant increase in SHM is antigen-driven and selected upon. To compare with a large amount of historical adult data, we calculated replacement to silent mutation ratios (R/S ratios), which are about 2-3:1 in FWRs and 5:1 in CDRs for both infants and toddlers , we previously demonstrated that B cell clonal lineages could be informatically defined and contain pathogen-specific antibody sequences6. In addition, the clonal lineage analysis also highlighted the lack of antibody diversification in the elderly after influenza vaccination6. Using the same approach and a similar threshold44, we aimed to answer whether infants and toddlers are able to diversify antibody clonal lineages in response to infection and, if so, whether they have a similar ability to do so, which was previously impossible to answer due to technical limitations. To do this, we first visualized structures of informatically defined clonal lineages for the entire antibody repertoire . This analysis effectively captures five parameters that quantify lineage complexity in a sample: number of total clonal lineages (number of circles), diversity of each lineage , size of each lineage , SHM load of each lineage .The densely packed lineages could result from large lineage sizes (one unique RNA molecule with many copies), large lineage diversities (many unique RNA molecules), or a combination of the two. To closely examine the possible differences in the degree of this intra-clonal lineage expansion and diversification between infants and toddlers, especially upon acute febrile malaria, we projected the global lineage structure  and numbers of unique RNA molecules (x axis). Lineage “c” , indicating extensive clonal expansion of particular sequences in addition to diversification. Changing lineage forming threshold from 90 to 95% does not change the overall structure of the lineages . We hypothesized that the lower absolute SHM load of infants would imply a defect in the ability to diversify clonal lineages in response to infection, leading the slope change from pre-malaria to acute malaria to be low  or even negative (pink dashed line is closer to y axis than blue dashed line). Surprisingly, our analysis shows that infants diversify their clonal lineages in a similar manner as toddlers in response to acute malaria  and toddlers  . To our surprise, infants were able to induce more SHMs compared to toddlers . COLT52 analysis on these pre-malaria memory B cell-containing lineages shows that 53% contain traceable progeny sequences from the acute malaria PBMCs . Overall, there is a significant increase of SHM in these acute malaria progenies compared with their ancestor pre-malaria memory B cells  supplemented with 1% β-mercaptoethanol (Sigma). The following antibody clones were obtained from Biolegend and used as 1:25 dilution: Alexa Fluor 488 OKT3 (CD3), Alexa Fluor 700 RPA-T4 (CD4), Alexa Fluor 488 HCD14 (CD14), APC-Cy7 2H7 (CD20), PE-Cy7 O323 (CD27), APC HIT2 (CD38), Alexa Fluor 488 MEM-188 (CD56), and Brilliant Violet 605 IA6-2 (IgD). The following antibody clones were obtained from BD Biosciences: PE-CF594 RPA-T8 (CD8), Brilliant Ultraviolet 395 SJ25C1 (CD19), and Brilliant Ultraviolet 737 B-ly4 (CD21). Flow cytometry and cell sorting were performed on BD FACS Aria II.Frozen PBMCs were thawed, washed once in RPMI, and then processed for cell staining and sorting following standard FACS staining protocol6 to partial Illumina P7 adaptor. Full Illumina adaptors were added during the second PCR step along with library indexes. Total RNA was purified using All Prep DNA/RNA kit (Qiagen) following the manufacturer’s protocol. cDNA synthesis was done using Superscript III (Life Technologies) with 30% of the RNA as input. After free primer removal by Exonuclease I digestion (New England Biolabs) according to the manufacturer’s protocol, Takara Ex Taq HS polymerase (clone Tech) was used for both PCR reactions. The first PCR was performed with the following program: initial denature at 95°C for 3 min, followed by 20 cycles of 95°C for 30 s, 57°C for 30 s, and finally 72°C for 2 min with a 4°C hold. All cDNA was used for first PCR and about 1% of 1st PCR product was used for second PCR. The second PCR was performed with the following program: initial denature at 95°C for 3 min, followed by 10 cycles of 95°C for 30 s, 57°C for 30 s, and finally 72°C for 2 min with a 4°C hold. The second PCR products were gel purified and quantified by qPCR Library Quantification Kit . 0.009 pmol of the QCed second PCR product were sequenced on Illumina Miseq with paired-end 250 bp read mode  MS-102-2003). The list of primers for RT and PCR can be found in Supplementary Table MIDs were added during the reverse transcription step through the use of fusion primers, which contain the partial Illumina P5 sequencing adaptor followed by twelve random nucleotides and primers to the constant region of five antibody isotypes. We fused eleven forward primers that were previously designedhttps://github.comjstjohn/SeqPrep). The merged reads were filtered with specific V-gene and constant region primers to determine immunoglobulin (Ig)-sequencing reads. The primers were then truncated from the reads. The retained reads were further truncated to 320 bp for the naive B cells in method verification experiments and 270 bp for samples from malaria cohort. Read numbers after each filter are listed in Supplementary Tables Raw reads from Illumina MiSeq PE250 were first cleaned up following steps outlines in Fig. https://github.com/utjianglab/MIDCIRS.Raw reads were split into MID groups according to their 12 nucleotide barcodes. For each MID group, quality threshold clustering was used to cluster similar reads. This process groups reads derived from a common template RNA molecule together, whereas separating reads derived from distinct RNA molecules. A Levenshtein distance of 15% of the read length was used as the threshold. This was calibrated using RNA controls with known sequences  is the Levenshtein distance between read i and the consensus sequence in MID sub-group I, NI is the number of reads in MID sub-group I, and L is the read length.The difference between the consensus sequence for an RNA molecule and the raw reads associated with it represent the errors generated in either PCR or sequencing. The error rate can be calculated using the following formula :1\documeNI is the number of reads in MID sub-group I, and L is the read length.To estimate the improved error rate using MIDCIRS, we equally divided the raw reads from one library into two data sets. The same MID sub-group generating process was performed on both data sets. By comparing the differences between the consensus sequences with identical MID between these two data sets, we can calculate the improved error rate for using MID sub-groups as:6. From the International ImMunoGeneTics information system database 62, human heavy chain variable gene segment sequences  were downloaded. Each unique sequence was first aligned to all 249V gene alleles. The specific V-allele with a maximum Smith–Waterman score was then assigned. In some cases, newly identified germline alleles, defined either by TIgGER35 or our method (below), were added to the template sequences. J-segments and D-segments were then similarly assigned. The number of mutations from germline sequence was counted as the number of substitutions from the best aligned V and J templates as previously described6. The CDR3 was omitted due to the difficulty in determining the germline sequence. The germline sequences of V, D, and J gene segments were grouped by combining similar alleles into families using IMGT designation in VDJ correlation plots. In total, 58V, 27D, and 6J families were used.As described in previous work, similar methods were used to define the V, D, and J gene segments for all sequencesTo address the possibility of novel germline alleles inflating the observed number of mutations, new germline alleles were assembled. In short, IgM sequences for each individual were aligned and assigned to the traditional V-gene alleles in the IMGT database. If novel alleles exist in individuals, parts of unique RNA sequences will be assigned as mutations when they are actually derived from differences between novel and traditional alleles. The ratios of unmutated unique RNA molecules to those with one, two, three, and four mutations compared to the IMGT germline were determined, and if any were found to be less than 2 to 1, the alleles were flagged for further inspection. Unique RNA molecules were used to minimize the contributions of clonal expansion, and IgM sequences were used to minimize the contributions of SHM. Sequences within flagged alleles were then aligned to the closest IMGT germline to determine if the mutations were truly polymorphisms. When identical mutation patterns were observed in a minimum of 80% of all sequences in a flagged allele family, it was deemed a novel germline allele. For individuals with sorted naive B cells, novel alleles were generated from the naive B cell BCR sequences to complement those found in the bulk IgM sequences.35. TIgGER compares the mutation rate at a specific position to the overall number of mutations for sequences within the same assigned V-gene allele. Outliers within the low mutation region suggests the existence of a novel allele, and the shape of the curve can effectively distinguish between individuals homozygous and heterozygous for the novel allele.TIgGER was used as previously reported as another method to discover novel allelesOur new method and TIgGER have a 90% percent overlap in newly identified alleles. Discrepancies between the two methods were treated with a conservative estimation on the number of SHM, meaning we liberally included novel alleles as part of the germline gene segments. Non-overlapping novel alleles were manually inspected, and the union of novel alleles detected by TIgGER and our method was included as part of the germline gene segments. Sequences mapped to these novel alleles were excluded from our analysis, which accounts for an average 8% of all sequences (Supplementary Table 6. The unique RNA sequences were inputted to IMGT High V quest to translate into amino acid sequences. The boundary of the CDR3 is defined by IMGT numbering for Ig and two conserved sequence markers of “Tyr-(Tyr/Phe)-Cys” to “Trp-Gly”. CDR3 length was determined according to these anchor residues.Nucleotide sequences were translated into amino acid sequences based on codon translation as previously described37. The unique RNA molecules of PBMC, populations were inputted to BASELINe and compared with the closest IMGT germline alleles. The observed number of replacement and silent mutations were compared with the expected number of mutations for the assigned germline sequence. A selection strength as measured by the probability density function (PDF) was generated using BASELINe to indicate the direction and degree for CDR (CDR1 and 2) and FWR  regions for each unique RNA molecule first, then combined for each individual, and then further combined for each of the two groups, infants and toddlers. The PDFs were plotted and compared between infants and toddlers  and the ratio for silent vs. replacement mutation was calculated. The CDR3 and FWR4 were omitted due to the difficulty in determining the germline sequence. FWR1 for all sequences was also omitted because it was not covered entirely by some of the primers. Samples with 5,000,000 PBMCs were subsampled to 120,000 RNA molecules. Samples with fewer PBMCs were subsampled to proportionally fewer RNA molecules according to the PBMC number.According to the amino acid sequence translation results and V/D/J gene templates alignment results, we counted the number of nucleotide mutations resulting in amino acid substitutions  or no amino acid substitutions  in FWR region (FWR2 and 3) and CDR region (CDR1 and 2) using the IMGT/HighV-QUEST6:vdj refers to the combination of one v allele family from 58V-gene allele families ({V}), one d allele family from 27 D gene allele families ({D}), and one j allele family from 6J gene allele families ({j}). For the reads weighted correlation, Xvdj and Yvdj refer to the fraction of reads assigned to the respective vdj combination for individuals X and Y, respectively. vdj combinations. For the lineage weighted correlation, these parameters refer to the fraction of lineages for each vdj allele family combination. Samples with 5,000,000 PBMCs were subsampled to 120,000 RNA molecules. Samples with fewer PBMCs were subsampled to proportionally fewer RNA molecules according to the PBMC number. vdj combinations that were not detected in either sample being compared were omitted from the correlation calculation.The correlation of VDJ usage between infants and toddlers were calculated with Pearson correlation coefficient as the following formula  as in pr44, accounting for the larger size of the CDR3 and junction in humans as compared to zebrafish. RNA sequences with the same V and J allele assignments, the same junction length, and whose junction regions differed by no more than 10% on the nucleotide level were grouped together into a lineage. This is equivalent to a biological clone that underwent clonal expansion. To test the robustness of this threshold, we also tried the threshold of 95% similarity for CDR3 region44, and it did not change the overall position of each lineage in the diversity-size plot, nor did it change the linear fits of lineage distribution generated from these two threshold  and validated manually. We implemented a lineage visualization tool, COLT-Viz . In short, COLT considers constraints  along with mutational patterns to build lineage trees. The height of each node is proportional to the number of RNA molecules associated with the unique sequence (size), the color of each node relates to the number of SHMs, and the distance between nodes is proportional to the Levenshtein distance between the node sequences.Representative lineages were selected to visualize the lineage structures and the evolution of antibody sequences. Lineage structures were generated using COLTTo determine the fate of the pre-malaria memory B cells upon acute malaria infection, two time point-shared lineages were formed as described above, and lineages containing sequences from both FACS-sorted pre-malaria memory B cells and acute malaria PBMCs were isolated for further analysis. COLT was used to generate lineage tree structures. Pre-malaria memory B cells that served as parent nodes to acute malaria sequences, as exemplified (Supplementary Fig. References or links for all software tools used are listed in the relevant “Methods” sections. All other relevant data are available from the authors.Sequencing data that support the findings of this study have been deposited in dbGaP with the accession code phs001209.v1.p1.Supplementary InformationPeer Review File


The ADH@TiO2 NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD) coenzyme between NADH and NAD+ was realized by enzymatic regeneration of NADH from NAD+ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD+ under visible light. This bioinspired ADH@TiO2 NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD+/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH) was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO In recent years, the design and construction of enzyme@capsule microsystems inspired from the fundamental functions of subcellular organelles has attracted significant attention ,2,3. The3O4 .,19.2 NPsembranes e. The AD µL·mg−1 .2 is a well-known n-type semiconductor that has long been used for photocatalysis . Th. Th2 NPs+ and NADH catalyzed by ADH@TiO2 NP microreactors is shown in +-NADH regeneration, NAD+ was catalytically reduced to NADH by the spatially confined ADH. In the second step of cycling, NADH oxidation in reaction system was initialized upon exposure to visible light irradiation (+ to NADH without visible light illumination (+/NADH coenzyme was realized in the synthetic functional microreactors under visible light irradiation. The catalytic stability of the ADH@TiO2 NP microreactors was also investigated (2 NP microreactors were sufficiently stable even after 13 consecutive cycles.The cycling performance of NADadiation b. To commination c. Accordmination a–c, suststigated d. The re2 NP microreactors were developed to facilitate the mimicking of sustainable nicotinamide coenzymes of NAD+/NADH cycling in a synthetic functional microcompartment under visible light irradiation. The designed microreactors represent a model of a simple nonbiological microsystem that is related to enzymatic catalysis and inorganic photochemical reactions. Thus, our research provides new opportunities in visible light energy capture and transduction. Furthermore, it is possible to synthesize this simple microsystem easily and on a large scale. Consequently, the proposed membrane-bounded microcompartments with core-shell hierarchical structure have great potential in photoactive microreactor design and drug delivery.In summary, novel bioinspired photoresponsive ADH@TiO


We investigated the plasma levels of tumor-specific cell-free DNA (cfDNA) in 17 stage I–II (early) and IV (advanced) non-small cell lung cancer (NSCLC) patients who underwent radiotherapy. Digital polymerase chain reaction (PCR) and targeted sequencing showed that total and tumor-specific cfDNA levels increased in response to radiotherapy in both early- and advanced-stage NSCLC patients. We detected high copy numbers of epidermal growth factor receptor mutations (L858R and T790M) in the cfDNA samples from stage IV NSCLC patients who underwent stereotactic body radiation therapy to treat brain metastasis related to tyrosine kinase inhibitor (TKI) treatment failure. In conclusion, our study demonstrates that radiotherapy increases tumoral cfDNA levels in the plasma and shows potential to serve as an indicator for diagnosing drug-resistant tumor-related gene mutations in early-stage NSCLC patients or those undergoing molecular targeted therapy. Diehlrs long) , 8, 12, rs long) , 13. Thers long) –11, thouCfDNA analysis has been used to assess drug resistance in non-small cell lung cancer (NSCLC) . A phaseNext-generation sequencing of cfDNA samples from untreated stage IV NSCLC patients has shown promise as a diagnostic tool , 17. HowRadiotherapy is widely used for cancer treatment and palliative care because targeted irradiation increases tumor cell apoptosis. NSCLC patients undergo stereotactic body radiotherapy (SBRT), chemo radiotherapy, or palliative radiotherapy at different stages of the disease. Since higher levels of cell-free DNA and CTC have been observed in the plasma of patients during chemo radiotherapy , 20, we Radiation doses vary widely among cell lines, animal models, and clinical settings. In a laboratory setting, cancer cell lines are irradiated with 2–20 Gy/fraction (fr) and undergo apoptosis within 24–48 h , 22. In EGFR mutations and showed new brain metastasis, suggesting failure of tyrosine kinase inhibitor (TKI) treatment. The 11 stage I primary NSCLC patients were treated with curative SBRT or 3D-CRT, whereas, the six stage IV NSCLC patients received palliative SBRT against brain metastasis. A high-radiation dose was delivered specifically to the cancer tissue as routine therapy for all patients as shown in We enrolled seventeen patients with suspected NSCLC that underwent curative or palliative radiotherapy between July 2013 and July 2015. Eleven stage I NSCLC patients rejected surgery and chose radiotherapy, whereas, the remaining 6 stage IV NSCLC patients were positive for P < 0.05; Figure We detected cfDNA in all patients, and the total circulating cfDNA increased in response to radiotherapy in 12 out of 17 patients Table . The medEGFR, a dominant driver in lung adenocarcinomas. We performed digital PCR analysis of cfDNA from four patients diagnosed with adenocarcinoma bearing the EGFR L858R mutation (n = 2) or EGFR exon 19 deletion (n = 2). The copy numbers for these EGFR mutations increased from 0, 20866, 44194, and 64282 copies/mL before radiotherapy to 10562, 139579, 363269, and 2122718 copies/mL, respectively after radiotherapy  showed these mutations in their pre-radiotherapy samples. Furthermore, we detected EGFR G719C mutation in the post-radiotherapy sample from patient (case 1), whose biopsy was not performed and the EGFR mutation status was unknown previously. In case 16, we detected the EGFR L747S mutation, which is known to confer resistance to TKIs. We also observed an increase in allele frequency and tumoral DNA content in 5 out of 6 cases .The allele frequencies for the EGFR gene mutations in the cfDNA of stage IV NSCLC patients that experience distal metastasis recurrence as a result of drug failure.Tumoral cfDNA analysis is useful for making cancer diagnoses and for assessing cancer-related drug resistance. However, this strategy is not viable in early stage cancer patients and those undergoing cancer treatments because the concentration of cfDNA is limited. In this study, therefore, we demonstrated that radiotherapy increases tumoral cfDNA levels in the plasma of stage I and IV NSCLC patients. We also demonstrated drug resistance-related We subjected patients to radiotherapy  and analyzed total cfDNA levels in pre-RT, RT and post-RT plasma samples daily. We observed higher total and tumoral cfDNA levels at 24 h after irradiation than in pre-RT samples in stage I–II NSCLC patients. The total and tumoral cfDNA levels peaked at 7 days after irradiation. This suggested that cancer tissue apoptosis occurs within 24 h after radiotherapy and is similar to the kinetics of apoptosis in the cancer cell lines. Clinically, tumor reduction is observed at 4–8 weeks after radiotherapy as defined response evaluation criteria in solid tumours:Revised RECIST guideline (version 1.1). This large time lag between effects at a cellular level and at an anatomical level is probably due to radiation-induced inflammation and edema, which delays visual detection  of tumor reduction. We also observed elevated cfDNA levels after irradiation with a total dose of 30 Gy or less in 12 cases. Low irradiation doses of 8 Gy/1 fr to 30 Gy/10 fr are used for palliative treatment –30.Radiation-induced apoptosis generates 50–300 kb fragments of DNA in cell lines . HoweverEGFR mutations than patients with wild-type EGFR [Radiotherapy is as effective as surgery for patients with stage I–II NSCLC . Howeverype EGFR , 35. ThiEGFR mutations. However, cancer cells with EGFR T790M and ALK L1196M mutations acquire drug resistance. The second-generation ALK repressor (Alectinib) for the ALK L1196M mutation and the third-generation drug for the EGFR T790M mutation are approved for some instances of resistance after TKI treatment [Molecular targeted therapy is preferred for stage IV patients with anaplastic lymphoma kinase (ALK) fusion or reatment , 37. ScrEGFR mutations and underwent TKI treatment. These patients received SRS with CyberKnife to treat brain metastasis as a result of treatment failure or emergence of drug resistance. In previous studies, cfDNA analysis showed increased drug-resistant mutations in the plasma of patients with a failed TKI treatment [EGFR T790M. Our study detected EGFR T790M mutation in two out of the four patients. Most patients with NSCLC are associated with brain metastasis and undergo radiotherapy with or without TKI treatment [In this study, we performed digital PCR and sequence analysis in four patients  that were diagnosed with NSCLC bearing reatment , 39. Thereatment . In patiWhen local failure occurs during systemic chemotherapy, radiotherapy is preferred for palliative care. Our study suggests that cfDNA detection combined with radiotherapy could replace rebiopsy to determine the tumor genotype.et al. and Cheg et al. evaluated total cfDNA levels using traditional irradiation methodology and suggested that irradiation induces cancer cell apoptosis, thereby increasing tumoral DNA in the plasma :646–50.) [) the cancer tissue using stereotactic radiosurgery (SRS) and stereotactic radiotherapy (SRT) and confirmed tumoral cfDNA by digital PCR and sequence analysis. While our study speculates that the tumoral cfDNA was derived from apoptotic tumor cells, we confirm that irradiation increases tumoral cfDNA substantially. Importantly, we also clarified the time course of cfDNA elevation with clinical dose irradiation. The time course of apoptosis with irradiation is investigated previously in cell line and animal experiment, however the dose and fraction are clearly different as is described in introduction.Leon 646–50.) , 42. In 646–50.) . This inCfDNA analysis was approved by Food and Drug Administration (FDA) in 2017 and has been used clinically for diagnostic and prognostic applications. Our study investigated the influence of radiation on cfDNA quantity and composition.Our study has several limitations. First, we performed digital PCR and targeted sequencing in a single experiment because of low recovery. Second, the sample size in our study was small. For that reason, we cannot completely exclude the possibility of technical error. However, to overcome clinical and biological bias, we selected only cases that were treated using pinpoint irradiation methods to avoid normal tissue contamination and chemotherapy effects. We also performed daily blood tests during radiotherapy and analyzed the digital PCR and targeted sequencing. We conclude that our results reflect tumor cfDNA kinetics during and after irradiation in the 17 NSCLC patients.Our study demonstrates that radiotherapy increases total and tumoral cfDNA levels in early-stage and advanced stage NSCLC patients. Moreover, we demonstrate the utility of tumoral cfDNA analysis after irradiation for assessing drug-resistant mutations, which can help when preparing strategies for subsequent molecular targeted therapy.The study protocols were carried out as approved by the Ethics committee of the Tokyo Metropolitan Cancer and Infectious Diseases Centre at Komagome Hospital (#2013-1233). We obtained written informed consent from all the study subjects for the use of blood and resected tumor tissue for research purposes. All samples and medical data used in this study were anonymous to protect patient information.We enrolled 17 patients diagnosed with NSCLC between July 2013 and July 2015 at the Komagome hospital. The patients underwent curative or palliative radiotherapy. The main endpoint of this study was to evaluate cfDNA levels after radiotherapy. Tumors were diagnosed as NSCLC based on the histopathology of biopsy specimens and the patients were graded according to the classification of the Union for International Cancer Control 7th edition. Tumor genotyping was performed by PCR. The clinicopathological features of the patients are summarized in Table EGFR mutation positive and presented with brain metastasis after TKI treatment failed.As shown in Figure We used the computed tomography (CT) datasets for these patients, including fully delineated targets and organs-at-risk (OAR) to strategize subsequent radiotherapy. The treatment plans included a prescribed dose of 50 Gy/4 fr or 75 Gy/25 fr for primary stage I–II NSCLC patients and 23 Gy/1 fr or 35 Gy/3 fr to treat NSCLC patients with brain metastasis. We reduced the radiation dose to 45 Gy/4 fr in case 11 to avoid complications because of OAR. For stage IV disease, we performed only stereotactic radiosurgery (SRS) to the brain and did not use radiation therapy at other sites.We prescribed 50 Gy/4 fr SBRT for NSCLC patients with negative lymph nodes of 3 cm or less and three-dimensional (3D) conformal radiotherapy (3D-CRT) at 75 Gy/25 fr for patients with 3–5 cm tumors in the primary site with adjacent risk organs. The doses were determined according to standard rules as is described in the National Comprehensive Cancer Network (NCCN) and Japan guideline. Stereotactic body irradiation therapy (SBRT) to the brain was performed using Cyber Knife , and radiation treatment of the lungs was performed using Vero4DRT  .g. Cleared plasma was stored in cryostat tubes at –80° C until use. Plasma isolation was performed within 30 min of blood collection to prevent DNA contamination from blood cells.Whole blood was collected in 7-mL vacutainer tubes containing EDTA and centrifuged for 5 min at 800 × We concentrated 1 mL patient plasma using Maxwell rapid sample concentrator  and extracted nucleic acids using the Maxwell blood purification kit  according to the manufacturer’s instructions. DNA was purified using the QIAamp Circulating Nucleic Acid Kit  according to the manufacturer’s instructions. We eluted the DNA in 50 μL double distilled water and stored at –20° C. The DNA concentration was quantified with a Quantus fluorometer  following the manufacturer’s instructions.EGFR, EGFR exon 19 deletion as well as EGFR L858R and T790M mutations using a TaqMan SNP Genotyping kit  according to the manufacturer’s instructions (10 ng of cfDNA per reaction). Patient and standard samples were processed in duplicates, and the mean cycle threshold (Ct) value of duplicates was determined. The percentage of mutant EGFR alleles was calculated as the ratio of the copy number of mutant EGFR relative to the total copy number of EGFR (wild-type and mutant).Allele-specific qRT-PCR was performed in a LightCycler 480 Real-Time PCR System . We determined the copy number of mutant and wild-type alleles of For the sequencing assays, we used two commercial and validated panels: the Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion AmpliSeq Colon and Lung Cancer Panel . The IonWe aligned FASTQs to the human genome (hg19), and identified point mutations using Torrent Suite Software v3.0 and the Ion Torrent Variant Caller v4.0 Plug-in using somatic high stringency parameters and the targeted and hotspot pipelines. We set a 5% allele frequency (AF) threshold and a 500× minimum coverage to identify de novo mutations; we also selected 0.1% AF to identify previously characterized mutations during treatment . All idet-test to compare cfDNA amounts at pretreatment versus the highest point under radiotherapy with EZR [We used Student’s with EZR and the f


Despite evidence that genetic factors contribute to gestational length and pretermbirth, robust associations with genetic variants have not been identified. Wehypothesized that analyzing larger data sets with gestational length information bygenomewide association would reveal trait-influencing variants.P < 5×10-8) or suggestiveassociation (P < 1×10-6).We performed a genomewide association study in a discovery data set of 43,568 women ofEuropean ancestry from 23andMe, Inc., for gestational length as a continuous trait andfor term or preterm (<37 weeks) birth as a dichotomous outcome. We used three Nordicdata sets  for replication of 14 genomic loci achieving either genomewide achieved genomewidesignificance, all of which were replicated in the Nordic data sets. Functional analysisof the WNT4 locus indicated the likely causative variantalters the binding of ESR1. ADCY5 and RAP2C, which had suggestive significance in the discovery stage, weresignificantly replicated and achieved genomewide significance in joint analysis. Commonvariants in EBF1, EEFSEC andAGTR2 were also associated with preterm birth withgenomewide significance. Analysis of mother-infant dyads indicated that these findingslikely resulted from maternal genome actions.In the discovery stage, for gestational length, four loci (Our study is the first to identify maternal genetic variants robustly associated withgestational length and preterm birth. Roles of these loci in uterine development,maternal nutrition, and vascular control support their mechanistic involvement andcreate opportunities to investigate new risk factors for prevention of pretermbirth. Preterm birth (defined as birth before 37 completed weeks of gestation) affects 9.6% ofpregnancies in the United StatesPreterm birth, and gestational length in general, is a complicated phenotype withcontributions from two genomes – maternal and fetal – that may have separateor interacting contributions. Furthermore, different genotypes may predispose to pretermbirth at different gestational ages. Finally, defining preterm birth as a dichotomous traitbased upon a somewhat arbitrary cutoff of 37 weeks, rather than time of birth for aspecified level of fetal maturity or as a continuous trait, limits data interpretation.Therefore, defining the genetic variants associated with gestational length (a quantitativetrait) as well as preterm birth (a dichotomous trait), will both yield important newinsights. Further, analyzing gestational length as a continuous trait increases the power todetect associations that is limited when traits are dichotomized.P <1×10-6) and performed replication analyses for gestational length andpreterm birth in three data sets of Nordic women . Further, we provide evidenceindicating the observed effect was due to an action in the maternal genome and providefunctional data implicating the causative SNP underlying the WNT4 locus.To overcome previous sample size limitations, we leverage data on gestational length andpreterm birth in a large sample of women of European ancestry collected as part of genotyping and phenotyping efforts by 23andMe, Inc., a geneticscompany. We then selected the top loci .http://www.eandireview.com).Unrelated women of European ancestry who self-reported gestational length of their firstlive singleton birth were included in the analysis. Categories of preterm birth addressedon the survey were 1) spontaneous preterm labor, 2) planned or required delivery formedical reasons, 3) cervical problems, 4) other, or 5) none of the above. Women with amedical indication for their preterm delivery were excluded from the study; those that didnot specify a medical indication on the survey were retained to optimize sample size.Preterm birth status was determined based on dichotomization of self-reported gestationallength . For those in the preterm group,96.8% of women responded to the question regarding mode of delivery. For the term birthgroup, we ascertained information on aggregate outcomes of all pregnancies, and could notunambiguously determine spontaneous or medically indicated birth at more than 37weeks.Women in the discovery data set were participants in 23andMe’s research program.All women provided informed consent and answered surveys online following a human subjectsprotocol, reviewed and approved by Ethical & Independent Review Services, a privateinstitutional review board . The SNP with smallest P value withineach region was chosen as the index SNP. Regions that achieved suggestive significance(P < 1×10-6) were tested in thereplication stage.We clustered SNPs into association regions (or loci). Specifically, we definedassociation regions by first identifying SNPs with We used the data of 8,643 mothers from three independent Nordic birth studies  of singlGenotyping of the Nordic studies was conducted using various SNP arrays as previouslydescribed.P values (inflation adjusted) combining resultsfrom the three Nordic data sets were calculated using the fixed-effects inverse-variancemethod. Significant replication P values and the samedirection of effect at the index or other significant SNPs  in the region or theirclose proxies (r2 > 0.8) were regarded as statistical evidence of replication of a putativelocus. The significance level of each region was corrected by the effective number ofindependent SNPs tested in the region and the total number of regions that underwentreplication attempts  were also performed to examine possible dominance effect. Thereplication attempts . A regioWe also performed association tests in 4,090 infant samples and joint maternal/fetalgenetic association analysis in 3,184  mother/infantpairs from the Nordic data sets to evaluate whether the observed significant associationswere likely to be of maternal or fetal origin.We checked whether the SNPs associated with gestational length or preterm overlap withpreviously reported genomewide association SNPs in the GWAS catalogWNT4locus, one of the most significant loci with plausible functional relevance in pregnancy.First we examined the expression level of WNT4 in humanendometrial stromal cells, before and after decidualization using mRNA-seq technology. Wepredicted specific transcription factors binding using a Bioinformatic approach andstudied the presence of H3K4me3 marks and open chromatin domains overlapping thehypothetical causal SNP by ChIP-seq and ATAC-seq, respectively. We performedelectrophoretic mobility shift assays (EMSA) to determine whether the variantdifferentially affected specific transcription factor binding. Detailed description ofthese analyses can be found in the WNT4 locus).We performed experimental functional follow-up of the P =2.3×10-41) with older mothers having shorter gestational length. Of these, four had an association P< 5×10-8  shared associationloci for preterm birth risk. Altogether, 14 independent loci were taken forward forreplication. To confirm the robustness of the association signals, we conducted similarassociation tests in a subset of discovery subjects who explicitly checked“spontaneous delivery” in the questionnaire  and the results weresimilar to those obtained from the full discovery data sets  at the index SNPand other SNPs with P < 1×10-6 in thediscovery stage, and their close proxies (r2 > 0.8). Six loci . In addition, associations ofthe BOLA3 locus with gestational length, and the TEKT3 and the TGFB1 loci withpreterm birth showed marginal significance (P < 0.05). Atthe EBF1, EEFSEC, AGTR2, WNT4 and RAP2C loci, the most significant SNPs in the replication stage were eithersame as or in substantial LD (r2 > 0.6) with the most significant SNPs in discovery stage. However, at theADCY5 locus, the LD between the most significant SNPs inreplication stage and the discovery stage is less substantial (r2 < 0.4). SNPs at the EEFSEC locus showednominally significant dominant effects (P.dom < 0.05). r2 >0.8) with SNPs significantly associated with gestational length orpreterm birth in the discovery stage. Within these loci, there are SNPs reportedlyassociated with complex traits  have also been associated with increased risk of prostatecancer (rs10934853-A)WNT4 locus werepreviously associated with endometriosis,cis-expressionQTLs) based on GTEx dataA number of SNPs with potentially functional impact  are encompassed by the loci we identified as potentially important . HoweverADCY5 locus have been reported to be associatedwith birth weightADCY5, WNT4 and EBF1 loci that are associated with birth weight.ADCY5 and WNT4 loci appear to influence birth weight through the fetal genome and noneof them were in close LD with the SNPs showing significant association with gestationallength; while the SNP (rs7729301) at the EBF1 locus seems toinfluence birth weight through the maternal effect, and the allele (G) associated withreduced birth weight was also associated with shorter gestational length  prior todecidualization in vitro to 29.5 TPM after decidualization in samples run in duplicatefrom two different endometrial stromal cell lines. The genetic loci we identified fall in noncoding regions of the genome, suggesting thatthey will affect gene regulation rather than protein function. To dissect the consequencesof these variants, knowledge of cell-type context in which they are active is essential.The lization. UsingRWNT4 that might be altered by variants associated withgestational length. To this end, we used the CisBP web serverWNT4 locus. These analyses indicate that rs3820282 (r2=0.94 with the index SNP rs56318008), which is located in the first intron ofWNT4 is capable of altering the binding of the estrogenreceptor (ESR1). Specifically, the underlying quantitative data from protein bindingmicroarray (PBM) assaysWNT4 locus is modulation (viars3820282) of the binding of ESR1. rs3820282 is also strongly associated with epithelialovarian cancerWe next sought to identify particular regulatory mechanisms controlling the expression ofOur genomewide association study is the first to identify human genetic polymorphisms thatare significantly and reproducibly associated with gestational length and preterm birth, thesingle greatest contributor to mortality in children younger than five years and a commonsource of morbidity throughout life for those who survive. Our approach demonstrates theutility of using data collected as part of direct-to-consumer genotyping and phenotyping torapidly assemble large data sets capable of revealing contributing loci in particularlycomplex phenotypes such as preterm birth where both maternal and fetal genomes, and manygenes, are likely to contribute to the outcome. By combining the power of a large43,568-person discovery data set and stringent replication by the well phenotyped Nordicdata sets, we identified and replicated six maternal genomic loci robustly associated withgestational length and three of them also associated with preterm birth with genomewidesignificance (P<5E-8) in the joint analysis.EBF1 (early B-cell factor 1),also achieving genomewide significance for preterm birth, has been demonstrated to beessential for normal B cell development,EBF1 confers its effect on birth timingthrough pregnancy-specific mechanisms, or by contributing to more general cardiovascular ormetabolic traits that influence gestation remains to be determined. In addition, theassociation between this locus and gestational length may explain the effect of this locuson birth weight reported by Horikoshi et al.The top four replicating genomewide significant SNPs for gestational length are inbiologically plausible genes. EEFSEC , also genomewide significant for both gestational length and preterm birthrisk, participates in the incorporation of selenocysteine into selenoproteins.Selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, servecritical cellular homeostatic functions in maintaining redox status and antioxidantdefenses, as well as modulating inflammatory responses.EEFSEC are in highLD with SNPs that have previously been associated with age of onset of menarche, expressionquantitative trait loci (eQTLs) for EEFSEC abundance, risk ofprostate cancerAGTR2 , the coding genenearest to a group of X chromosome SNPs achieving genomewide significance in the gestationallength analysis, had suggestive association with preterm birth discovery stage, andgenomewide significance for preterm birth in the joint analysis with robust association inthe Nordic replication. AGTR2 has been suggested to play arole in modulating uteroplacental circulation, and harbors variants that may contribute tothe risk of preeclampsia.EEFSEC, is a potential shared mechanism for thesegenes in spontaneous preterm birth.WNT4 (wingless-type MMTV integrationsite family member 4), with strong replication in the Nordic populations. WNT4 mutations have been found in women with Mullerian ductabnormalities, primary amenorrhea, and hyperandrogenism,WNT4, in high LD with our indexSNPs, are associated with risk for endometriosisWNT4, and the T allele generates a strong ESR1binding site, and as such likely alters estrogen-based regulation of WNT4 and/or adjacent genes. The role of estrogen signaling as the functionalconsequence of the polymorphism is further supported by the association of the same regionwith endometriosis and ovarian cancer, both hormone-responsive disorders. Further, theparallel of the spectrum of disorders associated with the WNT locus mirrors that of ARID1A,also critical for endometrial function early in pregnancy, with loss of function variantscausing atypical endometriosis and ovarian cancer, and enhanced estrogen activity. WNT4 did not achieve genomewide significance or suggestiveassociation in the preterm birth risk dichotomous trait analysis, suggesting its role may belargely exerted near term gestation. The final gene locus achieving genomewide significance in the discovery stage forgestational length was ADCY5 (adenylyl cyclase type 5) and RAP2C (member of the RAS oncogene family) achieved near genomewide significancein the discovery stage and were successfully replicated (ADCY5 locus have been reportedto be associated with birth weightRAP2C region (in geneFRMD7) was previously reported to be associated withspontaneous preterm delivery in Danish/Norwegian studies (the samples used in this previousstudy overlap with our replication samples).BOLA3, TEKT3 and TGFB1), while showing marginal evidenceof replication, remain suggestive and await the addition of further studies for analysis. plicated . SNPs atThe primary limitation of our study centers on the characteristics of our study data sets.The gestational length information of the 23andMe samples was self-reported, and 3.2% ofwomen in the preterm group did not respond as to whether the labor and delivery wasspontaneous or medically indicated. In the term group, we were not able to unambiguouslydetermine spontaneous from medically-indicated births. Despite these limitations, weincluded these samples in order to dramatically increase the sample size of the discoverystage, recognizing that our replication data sets would be more precisely phenotyped forspontaneous preterm birth. A previous study suggested that approximately 90% ofmother-reported gestational lengths agreed with their associated medical records.Our study demonstrates the utility of combining large samples with self-reportedphenotyping with more modestly sized but precisely phenotyped replication studies to revealmaternal loci associated with gestational length and preterm birth. With this foundation,future expansion of maternal and fetal genotyped samples associated with gestational lengthinformation is anticipated to further refine our understanding of human pregnancy, risk foradverse pregnancy outcomes, and targeting of new preventive strategies for preterm birth. Asthe National Institutes of Health expand “Precision Medicine” initiatives inthe years ahead, we would argue that the optimal time to advance human health is before andduring pregnancy. Our work suggests that integration of genomic information on women, andlikely their offspring, with birth timing, may allow development of new options forpreventative and therapeutic measures.Supplementary Appendix


The measurement of virus-specific neutralising antibodies represents the “gold-standard” for diagnostic serology. For animal morbilliviruses, such as peste des petits ruminants (PPRV) or rinderpest virus (RPV), live virus-based neutralisation tests require high-level biocontainment to prevent the accidental escape of the infectious agents. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of neutralising antibodies against animal morbilliviruses. By expressing the haemagglutinin (H) and fusion (F) proteins of PPRV on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Serological responses against the four distinct lineages of PPRV could be measured simultaneously and cross-neutralising responses against other morbilliviruses compared. Using this approach, we observed that titres of neutralising antibodies induced by vaccination with live attenuated PPRV were lower than those induced by wild type virus infection and the level of cross-lineage neutralisation varied between vaccinates. By comparing neutralising responses from animals infected with either PPRV or RPV, we found that responses were highest against the homologous virus, indicating that retrospective analyses of serum samples could be used to confirm the nature of the original pathogen to which an animal had been exposed. Accordingly, when screening sera from domestic livestock and wild ruminants in Tanzania, we detected evidence of cross-species infection with PPRV, canine distemper virus (CDV) and a RPV-related bovine morbillivirus, suggesting that exposure to animal morbilliviruses may be more widespread than indicated previously using existing diagnostic techniques. RPV is a morbillivirus, a close relative of measles virus (MeV) and peste des petits ruminants (PPRV). Like rinderpest, PPR is now being considered for global eradication by vaccination Goats vaccinated with RPV-derived vaccines resist PPRV infection ex vivo environment and the availability of potential viral receptors in vitro may alter the conformation of the viral haemagglutinin, modulating sensitivity to neutralising antibodies. If biologically-relevant neutralising antibody responses are to be quantified accurately, assays that utilize primary field strains of virus and their cognate receptors are required. By generating viral “pseudotypes” bearing both the haemagglutinin (H) and fusion (F) proteins of the morbillivirus The “gold standard” for measuring morbillivirus-specific antibodies is the virus neutralisation test In this study we examined the neutralisation of ruminant morbilliviruses by sera from animals exposed to either PPRV or RPV. We demonstrate that while cross-neutralisation is evident between the two morbilliviruses, the strength and breadth of the response against the two viruses differs markedly. Further, we identify an RPV-specific neutralising activity, indicative of the circulation of an RPV-related bovine morbillivirus.22.1HEK293 2.2luc) has been described SalI and NotI and cloned into the vector VR1012 . The nucleic acid sequences of all amplified cDNAs were determined externally . All oligonucleotide primers were obtained from Integrated DNA Technologies, Leuven, Belgium.The recombinant vesicular stomatitis virus (VSV) in which the glycoprotein (G) gene has been deleted (VSVΔG) and replaced with firefly luciferase (2.3luc (VSVG) as described 50)] was calculated using the Spearman-Kärber formula 293T cells were transfected with the H and F expression vectors from the respective morbillivirus, followed by super-infection with VSVΔG2.44 293-dogSLAM cells were plated into each well of a 96-well plate . Fourfold serum dilutions were prepared in triplicate in complete medium ranging from 1:8 to 1:32768. The diluted serum samples were then added to the 293-dogSLAM cells followed by 2.5 × 103 TCID50 of each VSVΔGluc pseudotype. Luciferase activity was measured at 48–72 h post-plating and antibody titres calculated by interpolating the point at which there was a 90% reduction in luciferase activity  2 × 102.5Virus neutralising activity in sera from cattle vaccinated with live attenuated RPV and PPRV vaccines was assessed in a 96-well plate based micro-neutralisation assay as per the standard OIE specified procedure 2.6Antibodies against PPRV H in African sera were detected by ELISA using a competitive ELISA  as per the manufacturers’ instructions. Briefly, inactivated PPRV is coated onto an ELISA plate prior to the addition of test and control sera in the presence of a monoclonal antibody against PPRV H. Antibodies within the test sample compete for binding with the anti-H monoclonal antibody, binding of which is then quantified using an anti-mouse IgG horseradish peroxidase (HRP) conjugated antibody. The percent inhibition (PI) of monoclonal antibody binding for each test sample is calculated in comparison with positive and negative control sera. The diagnostic threshold for the assay is set at 50% PI of the monoclonal antibody control. A PI value of <25% is considered negative.33.1Neutralising antibody titres were measured in sera from cattle infected previously per se. Titres induced by N75 infection were highest against the lineage 2 N75 (homologous strain) and Benin 2010 strains. Neutralisation of lineage 3 Kenya 2011 by sera from the N75 vaccinates , cattle (B. taurus), buffalo (S. caffer) and gazelle (G. Thomsonii and G. Grant)) from areas reported previously to harbour PPRV. The buffalo and gazelle sera had been screened previously by cELISA and classed as “negative” (<50% inhibition on cELISA), the sera which tested positive by ELISA having been sent to The Pirbright Institute for further study. Buffalo sera were collected across the Serengeti National Park (SNP) from 2005 to 2012. The goat sera, collected during 2012, came from the NCA, a region that had undergone a PPRV vaccination campaign in 2011–2012.The accurate identification of PPRV-exposed animals in the field is critical to eradication campaigns and serological tests can facilitate the identification of animal reservoirs of infection in cases where viral genomic material is undetectable in blood or body fluids. We screened a panel of Tanzanian ruminant sera  and two vaccinated with live attenuated PPRV vaccines (N75 and S96). Animals infected with IC89 developed significantly higher antibody titres, antibodies that cross-neutralized PPRV strains from each of the four lineages. These data suggest that an animal exposed to a virus such as IC89, and which had recovered and generated a strong humoral response, would be cross-protected from infection with viruses from all four lineages. In contrast, the neutralising responses of the sera from animals vaccinated with N75 or S96 generated significantly lower titres of neutralising antibodies in comparison with the IC89-infected animals. Lineage 2 N75 and lineage 4 S96-containing vaccines are licenced in several PPRV-endemic countries. Neutralising antibody titres induced by the two vaccines were highest against the vaccine strain N75, and lowest against lineage 3 Kenya 2011. Such observations may assist the design of future eradication strategies and retrospective analyses of sera from vaccine trials might provide an insight into whether the level of neutralising antibody induced by vaccination correlates with immunity to infection with viruses from distinct lineages. In this study, we were restricted to the examination of sera from cattle infected experimentally with PPRV. Future studies should examine sequential sera from goats and sheep, the primary targets for PPRV vaccination campaigns, as it is likely that the replication of live attenuated viruses will vary between target species and hence the strength and breadth of immunity induced will vary accordingly.The high sensitivity of the pseudotype-based assay facilitated the detection of a higher proportion of sero-positives than a widely used ELISA. While it should be noted that the ELISA was optimised for specificity, allowing PPRV exposure in a herd to be confirmed, it was evident that the assay sensitivity was low, underestimating the number of positive animals significantly. Indeed, three animals with high titre neutralising antibodies tested negative by ELISA. Presumably the humoral response in those animals targeted epitopes on PPRV H distinct to that recognised by the monoclonal antibody within the kit or targeted the PPRV F protein.We detected PPRV neutralising responses in sera from both buffalo and goats. The presence of neutralising antibodies in 38% of buffalo sera that had tested negative previously by ELISA suggests that PPRV exposure was under-reported in previous studies By comparing batches of experimental sera from animals exposed to either RPV or PPRV, we demonstrated that the neutralising responses were highest against the homologous virus, consistent with the original serology-based evidence for the distinction of RPV and PPRV If vaccination to eradicate PPR is to succeed, future studies should address which species act as reservoirs of infection and whether they may shed infectious virus. If species such as cattle shed infectious PPRV, this must be taken into consideration in a post-PPRV eradication environment. Continued immunosurveillance will ascertain whether PPRV is spreading more widely; broadening its host species in a post-RPV world. Finally, we should consider whether RPV eradication has created a vacated niche


Obesity is a heterogeneous disease with many different subtypes. Epigenetics could contribute to these differences. The aim of this study was to investigate genome-wide DNA methylation searching for methylation marks associated with obesity in children and adolescents. We studied DNA methylation profiles in whole blood cells from 40 obese children and controls using Illumina Infinium HumanMethylation450 BeadChips. After correction for cell heterogeneity and multiple tests, we found that compared to lean controls, 31 CpGs are differentially methylated in obese patients. A greatest proportion of these CpGs is hypermethylated in obesity and located in CpG shores regions. We next focused on severely obese children and identified 151 differentially methylated CpGs among which 10 with a difference in methylation greater than 10%. The top pathways enriched among the identified CpGs included the “IRS1 target genes” and several pathways in cancer diseases. This study represents the first effort to search for differences in methylation in obesity and severe obesity, which may help understanding these different forms of obesity and their complications. Data from U.S. population surveys demonstrate a significant increase in obesity prevalence among children age 2–19 years old, from 5.5% in 1976–19801insulin-like growth factor 2 (IGF2) promoter was higher in overweight infants than in lean infantsperoxisomal proliferator activated receptor-γ-co-activator-1α promoter in children blood predicts adiposity at adolescence independently of sex, age, pubertal timing and activityThere is growing evidence that DNA methylation might contribute to obesity. Indeed, candidate gene methylation studies in animal models and humans have demonstrated methylation changes in promoters of various genes that are implicated in obesity, appetite control and/or metabolism, insulin signaling, immunity, growth and circadian clock regulation678et al. discovered two obesity-associated inflammatory genes Ubiquitin Associated And SH3 Domain Containing A and Tripartite Motif Containing 3 (UBASH3A and TRIM3), and Almen et al. identified 20 CpGs differentially methylated between groups. A larger study looking at the impact of BMI on DNA methylation in different tissues using the HumanMethylation 450 BeadChip by Dick et al. found five probes correlated with BMI, three of which were in the intron of HIF3A 13et al. found 575 demethylated CGIs and 277 hypermethylated CGIs in obese children. Finally, Huang et al. identified 129 differentially methylated CpGs associated with 80 unique genes. None of the obesity-associated CpGs was common to all studies, which may be due to differences between the study populations, diverse genetic backgrounds, or heterogeneous metabolic phenotypes between different BMI categories.To identify novel genes and pathways related to obesity and obesity-induced complications, epigenome-wide association studies (EWAS) are needed. Two previous studies using the HumanMethylation27 BeadChip with 27,000 CpGs, primarily targeting gene promoters and CpG islands (CGIs), examined blood leukocytes of obese and lean adolescents10Given the paucity of research on the different BMI categories, the purpose of this study were twofold: (i) investigate DNA methylation marks in all obese children compare to lean controls; (ii) identify the differentially methylated CpGs associated with severe obesity in childhood. We hypothesized that patients with severe obesity could help at identifying epigenetic changes, as extreme phenotypes improve genetic association studies.The genome-wide methylation analysis was conducted in 20 obese children  and 17 controls . Three cBecause DNA methylation varies by cell type and could bias EWAS results conducted in blood samples, we estimated the cell type compositions in each sample using minfi, and found that cellular composition was not similar between cases and control subjects . To corrCHORDC1 gene (+13% in control subjects compare to obese children). This gene also showed two other probes differentially methylated in obese compare to lean children. Because of the relatively small numbers in the study and small differences in methylation levels for most of the significant CpG sites, we analysed the confounding variables age and sex. We found that the methylation of 5/31 and 1/31 of the identified CpG sites was correlated with respectively age or sex  between obese children and control subjects, 10 were hypermethylated, and 21 were hypomethylated in obese compared to lean children . The lare or sex  and 2.−4, Pearson’s Chi-squared test) ; only 16% of our obesity–associated CpGs were located in CGI, compared to 38% in shores (22% in S-Shore and 16% in N-shore)  . The gened test) .Since obesity is an extremely heterogeneous disease, we then chose to focus only on the extremely obese children (BMI z-score ≥ 3.5).LMX1A ACSF3 (Acyl-CoA Synthetase Family Member 3) that showed a decreased by 17% of methylation level in control subjects compared to obese children. Thirteen of the 151 CpGs were common to our first analysis comparing obese children all together, 18 seems specific to moderately obese children and 138 to severely obese children , 69 were hypermethylated, and 82 were hypomethylated in severely obese patients compared to lean controls . Of theschildren .Next, we performed a gene set enrichment analysis (GSEA) to explore the potential of shared biologically relevant pathways among the obesity-associated methylation events. Seven pathways showed a significant enrichment including “IRS1 Target Genes” and different cancer traits .The genomic distribution of these 151 differentially methylated CpGs in relation to CpG density  was not clearly different from the whole array CpG distribution and there was no significant enrichment within specific gene regions (data not shown).In this study we aimed to identify obesity related methylation marks in peripheral blood leukocytes using a genome wide approach in youth obese children. The primary finding of this study was that most of the epigenetic marks are different in moderate and severe obesity. We identified respectively 18 and 138 differentially methylated CpGs between moderate or severe obese children and lean controls. As observed for genetic association studies, sampling individuals with extreme phenotypes can enrich the presence of epigenetic variations and can therefore lead to an increase in detection of these differences. Moreover, most of the differentially methylated CpGs was found within open seas or intergenic regions, consistently with previous findings showing that DNA methylation may be more dynamic outside CGIs.ANKRD11: ankyrin repeat domain 11, AVPI1: arginine vasopressin-induced 1, CDK19: cyclin-dependent kinase 19, FOXK2: forkhead box K2, HDAC4: histone deacetylase 4, IFT140: intraflagellar transport 140, KIAA0753, LTBP1: latent transforming growth factor beta binding protein 1, MYOM2: myomesin 2, TBC1D8: TBC1 domain family, member 8). Among these genes, HDAC4 is an interesting candidate since recently, Abu-Farha et al. showed a reduced expression of HDAC mRNA and protein in human obese subjects both in blood cells and adipose tissueHDAC methylation level higher in obese children than in controls.Compare to previous studies1011121317via obesity-associated hormones, growth factor signalling, inflammation, vascular integrity processes, microenvironmental perturbations, and other mediators, which could enhance the cancer risk and/or progressionNumerous genes found in our severe obese analysis were also associated with cancer. Many epidemiological and clinical studies have demonstrated that early obesity is an established risk factor for many cancers in later lifeet al. have shown for example that methylation across the H19 DMRs  was similar across several tissues from divers embryonic originset al. also found that DNA methylation levels did not differ in blood and buccal cells, from mesodermal and ectodermal embryonic tissues, respectivelyet al.Most of the identified genes are not expressed or do not have a relevant function in blood cells; whether these epigenetic marks in blood may reflect or correlate with methylation in more relevant tissues is not known. However, several studies showed that DNA methylation measured in whole blood is a marker for less accessible tissues that are directly involved in disease. For instance, Murphy There were several limitations to this study. The first limitation was that we used DNA from whole blood. To correct our methylation data for this weakness, we used a Houseman correction algorithmThe second limitation is that we cannot conclude about the existence of these epigenetic marks before the establishment of the obesity. While this lack of interpretability is inherent to the design of the cross-sectional case control study, the finding of methylation marks associated with the early stages of severe obesity in young patients may be of pathogenic relevance to certain features or complications of this disease. Indeed, the marks that have been found here could be used in a longitudinal study of the young patients in order to gain both biomarker and mechanistic insights. The longitudinal sampling of cells from adolescence to adulthood should further allow which of these epigenetic changes follow the development of the long term overt phenotype of severe obesity and its complications.Our major strength was that all studied participants were children, aged from 3 to 13 years old, less subject to cofounding factors like medication or comorbidities, very common in adult obese patients.In conclusion, the identification of methylation changes in specific genes will provide important targets for further study into the underlying mechanisms and the therapeutic potential for childhood obesity.2 and 16.7 ± 2.2 kg/m2, respectively (Twenty obese children (5 to 13 years old) and equal numbers of control children (3 to 13 years old) were included in the study. The BMI for obese children  and lean children  was 26.0 ± 4.8 kg/mectively . Age andDNA was extracted from whole blood cells of 20 case and 20 control subjects using Gentra DNA extraction kit (Qiagen). Genomic DNA (1 ug) from each of the 40 subjects was bisulphite-converted using Zymo EZ DNA Methylation-Gold kit (ZymoResearch) and the DNA was analysed using the Infinium HumanMethylation450 platform  by The Genotyping National Center .DNA methylation status of case and control subjects was established using Illumina Infinium HumanMethylation450 BeadChips that cover 485,764 cytosine positions of the human genome. Preprocessing and normalization involved steps of probe filtering, color bias correction, background subtraction and subset quantile normalization as previously describedThe R package minfiTo correct our methylation data analysis for cell heterogeneity between samples, we used R package RefFreeEwasHow to cite this article: Fradin, D. et al. Genome-Wide Methylation Analysis Identifies Specific Epigenetic Marks In Severely Obese Children. Sci. Rep.7, 46311; doi: 10.1038/srep46311 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


AGTR1) may play an important role in the development of high-altitude pulmonary edema. We envisaged a role for AGTR1 gene variants in the pathogenesis of HAPE and investigated their potential associations with HAPE in a Han Chinese population. We genotyped seven AGTR1 polymorphisms in 267 patients with diagnosed HAPE and 304 controls and evaluated their association with risk of HAPE. Statistically significant associations were found for the single nucleotide polymorphisms (SNPs) rs275651  and rs275652 . Another SNP rs10941679 showed a marginally significant association after adjusting for age and sex in the additive genetic model . Haplotype analysis confirmed that the haplotype “AG” was associated with a 35% reduction in the risk of developing HAPE, while the haplotype “AA” increased the risk of developing HAPE by 44%. These results provide the first evidence linking genetic variations in AGTR1 with HAPE risk in Han Chinese individuals.Previous studies demonstrated that Angiotensin II Receptor 1 ( High-altitude pulmonary edema (HAPE), a lethal, non-cardiogenic form of pulmonary edema, is a severe form of acute mountain sickness occurring in susceptible individuals at altitudes >2,500 m above sea level. It is an acute idiopathic disease that presents within 2 to 4 days after arrival at high altitude , 2 and cACE), angiotensin (AGT), renin (REN), aldosterone synthase (CYP11B2), and angiotensin II receptor type 1 (AGTR1), have been reported to be involved in the physiological response to hypoxic conditions. These genes have been associated with high risk of susceptibility to HAPE in some populations AGTR1 genresponse . We specresponse . AGTR1 gobserved , and a nentified . Howeverentified , 16. In Several potential limitations should be taken into consideration. First, participants in this study were limited to the Han Chinese population; whether the results are also applicable to other ethnic groups is currently unknown. Second, the limited sample size of our study did not allow subgroup analysis. Third, when multiple comparisons were carried out, we did not apply Bonferroni correction to control potential type I errors because this correction method might have increased the chance of a type II error. Finally, additional studies including fine mapping and functional validation are needed to clarify these findings and the mechanisms by which this polymorphism increases HAPE risk.To sum up, we identified the first significant associations between SNPs rs275651, rs275652 and rs4524238 and HAPE susceptibility, which may provide important biological markers for early diagnosis and prognosis for HAPE patients.From March 2013 to October 2015, we consecutively recruited 267 HAPE patients (HAPE-susceptible cohorts) and 304 ethnically matched control subjects (HAPE-resistant cohorts) from the General Hospital of Tibet Military Region, Lhasa, China. All of the subjects were of genetically unrelated Chinese Han ancestry and informed consent was obtained from each individual enrolled. The patient was immediately admitted to the hospital if anoxic symptoms arose at high altitude. All HAPE patients were diagnosed and confirmed to suffer from HAPE following previously described criteria . A standAGTR1 gene with minor allele frequency (MAF) > 0.05 in the HapMap Asian population were obtained from PubMed databases and single-nucleotide polymorphism (SNP) databases [the dbSNP (NCBI) and the Japanese SNP database (JSNP)]. DNA was extracted from peripheral whole blood cells by the GoldMag-Mini Whole Blood Genomic DNA Purification Kit  following the manufacturer's protocols, and DNA concentration of each sample was determined by spectrometry . The PCR and extension primers were designed using the MassARRAY Assay Design 3.0 software . SNPs were genotyped with the Sequenom MassARRAY RS1000 according to the instructions of the manufacturer [SNPs within the facturer . We perfAGTR1 polymorphisms in controls were tested for deviation from Hardy–Weinberg equilibrium (HWE) using a goodness-of-fit χ2 test. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated by unconditional logistic regression after adjustment for age and sex. The genotypic associations of every polymorphism were compared under the dominant, recessive, and additive genetic models as suggested by previous studies using the same method [http://www.broadinstitute.org/haploview) and the SHEsis software platform  was used to analyze the linkage disequilibrium (LD) structure, calculate D′ to define haplotype blocks and estimate haplotype frequencies [Statistical analysis was performed using Microsoft Excel and SPSS 16.0. All hypothesis testing was two-sided with a p value of 0.05 deemed as significant. Observed genotype frequencies for e method , 21. Finquencies , 23.