Branching epithelial morphogenesis is closely linked to epithelial-to-mesenchymal transition (EMT), a process important in normal development and cancer progression. The miR-200 family regulates epithelial morphogenesis and EMT through a negative feedback loop with the ZEB1 and ZEB2 transcription factors. miR-200 inhibits expression of ZEB1/2 mRNA, which in turn can down-regulate the miR-200 family that further results in down-regulation of E-cadherin and induction of a mesenchymal phenotype. Recent studies show that the expression of miR-200 genes is high during late pregnancy and lactation, thereby indicating that these miRs are important for breast epithelial morphogenesis and differentiation. miR-200 genes have been studied intensively in relation to breast cancer progression and metastasis, where it has been shown that miR-200 members are down-regulated in basal-like breast cancer where the EMT phenotype is prominent. There is growing evidence that the miR-200 family is up-regulated in distal breast metastasis indicating that these miRs are important for colonization of metastatic breast cancer cells through induction of mesenchymal to epithelial transition. The dual role of miR-200 in primary and metastatic breast cancer is of interest for future therapeutic interventions, making it important to understand its role and interacting partners in more detail. Caenorhabditis elegans [Branching morphogenesis is a conservative process seen in several organs such as lung, prostate, kidney, salivary glands and breast. The unique role of branching morphogenesis is to increase surfaces of the epithelium/endothelium so it can deliver specific functions such as gas exchange and transport , urine formation/filtration (kidneys), and secretion from exocrine glands . Developmental events underlying branching morphogenesis in epithelial tissues are closely related to pathways important for cancer progression and metastasis such as epithelial-to-mesenchymal transition (EMT) and its reverse process mesenchymal-to-epithelial transition (MET) ,2,3,4. B elegans . To date elegans . miRs ar elegans . Pre-miR elegans . In the  elegans . The dou elegans . A 6–8 n elegans . miRs pl elegans . Accumul elegans ,19,20. F elegans .miRs are now increasingly recognized as master regulators of protein expression through their ability to bind and degrade mRNA of various critical regulatory and signaling proteins, such as transcription factors involved in gene regulation.CACUG  with ZEEpigenetic factors play an important role in regulating cell type specific gene expression, including miRs. Epigenetic mechanisms such as DNA methylation and histone modifications have been associated with regulation of the miR-200 family. A study comparing global miR expression and epigenetic states of human mammary fibroblasts and epithelial cells showed that fibroblasts repress the miR-200c/141 locus by both DNA methylation and dimethylation of histone 3 at lysine 9 (H3K9me2) and the miR-200ba/429 locus by promoter trimethylation of histone H3 at lysine 27 (H3K27me3). In contrast, the human mammary epithelial cells had their promoter regions occupied by trimethylation of histone H3 at lysine 4 (H3K4me3) and acetylation of histone H3 at multiple lysine residues (H3Ac), which are both marks of active transcription, while the fibroblasts were devoid of these marks .et al. showed in 2011 that miR-200 were silenced with promoter hypermethylation and repressive chromatin marks in muscle-invasive bladder tumors and undifferentiated bladder cell lines [Both miR-200 clusters are associated with local CpG enrichments. Wiklund ll lines . DNA metll lines . Promotell lines . In humall lines . In a rell lines . The miRIn summary, transcription factors together with epigenetic modulation demonstrate the complex network of factors controlling the miR-200 family. Furthermore, the interactions of miR-200 with their targets show how complex the reciprocal communication between miR-200 and their regulators and targets are.Branching morphogenesis is a highly conserved developmental process that occurs in many organs through similar signaling pathways and transcriptional regulation . EMT hasThe mammary gland is unique in that most of its development occurs postnatally, and the epithelial remodeling can be followed from the onset of puberty, during each menstruation cycle and in more dramatic ways through each period of pregnancy and lactation . Althouget al. have shown that all miR-200 members are down-regulated in human and mouse mammary epithelial stem/progenitor cells and up-regulated in differentiated epithelial cells [in vivo. Recently, Song et al. demonstrated that miR-22 regulates side branching of mammary duct in vivo in transgenic mice through silencing of TET (Ten eleven translocation) family of methylcytosine dioxygenases, resulting in demethylation of the miR-200 promoter. Interestingly, the cooperation between miR-22 and the miR-200 family resulted in EMT, an elevated pool of stem cells and later increased tumorigenesis [et al. demonstrated that seven temporally co-expressed miRNA clusters were implicated in regulating murine mammary gland development [et al. focused on miRs involved in mammary reproductive cycles, indicated that the miR-200 family is highly expressed during lactation [There are several studies that have focused directly or indirectly on the role of miRs-200 in mammary gland morphogenesis. Analyses of miR-200 expression in tissue from reduction mammoplasty show that the expression of miR-200 members are predominantly in the luminal epithelial compartment and to a much lesser extent in myoepithelial cells . Althougal cells . In thisigenesis . Howeverelopment . Global elopment . Interesactation . Additioactation . These set al. [The role of miR-200 family members in other branching organs has received little attention so far. Rebustini et al.  screenedet al. . In mouset al. .Although only few studies have shown any relevance for miR-200 family in branching morphogenesis in the mammary epithelium, they do suggest that miR-200 play a major regulatory role in maintaining epithelial integrity in the mammary gland. Most of these studies, however, have been conducted in the mouse mammary gland. There are substantial differences between mice and humans regarding mammary epithelial branching and the interaction between mammary epithelial and stromal cells. A better understanding of the role of these miRs within the human breast gland is needed and 3D culture models of human breast epithelial cells can substantially contribute to this field. We have generated a human breast epithelial cell line, D492, with stem cell properties. D492 generates luminal and myoepithelial cells and in 3D culture it is able to form elaborate bilayered branching structures with both myoepithelial and luminal cells reminiscent of the terminal duct alveolar unit in the human female breast gland ,58. WhenIn recent years, miRs have increasingly been linked to functions that are either tumor promoting and/or tumor suppressing, and altered expression of miRs has been noted at various stages of cancer progression in different tissues ,62,63,64in situ (DCIS) and almost absent in TN cancers [In breast cancer, previous cluster analysis of gene expression data has identified several different subtypes including luminal (A and B), ErbB2 over-expressing and basal-like breast cancer ,71. The  cancers ,76.et al. recently demonstrated that the miR-200 family was important in distal metastasis of breast cancer cells by facilitating epithelial colonization in lung through induction of mesenchymal to epithelial transition (MET) [et al. worked with four isogenic mouse breast cancer cell lines that differ in their ability to metastasize [et al. transduced 4TO7 with a vector stably expressing the miR-200c/141 cluster. The 4TO7 cells stably expressing miR-200c/141 formed metastases at the same rate as the 4T1 cells, showing that miR-200c/141 expression was sufficient to make these cells metastatic [Although the miR-200 family is increasingly recognized as a family of tumor suppressors there are indications that its expression can improve survival of metastatic potential in cancer . Korpal on (MET)   . In contastasize . The onltastatic .in vivo cycling strategy, Banyard et al. selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors [Using an e tumors . Repeatee tumors .Taken together, these data imply that complete loss of miR-200 family members, e.g., by genomic deletions as observed for classical tumor suppressor genes, may actually predict a favorable clinical outcome. The genomic location of the miR-200 family genes is on chromosome 1p36 (miR-200ba/429), a locus associated with a moderate frequency of genomic deletions, and chromosome 12p13 (miR-200c/141), associated with equally moderate frequency of DNA copy number gains and deletions in human breast cancer .et al. showed recently that the drug tetraindole SK228 induces miR-200c expression in breast cancer cells followed by repression of ZEB1 and ZEB2 and re-expression E-cadherin [in vivo studies indicate reduced breast cancer growth making this drug a potential agent for future cancer therapy. Direct administration of miR-200 family members for therapeutic purposes can at least be considered since miR-based drugs already have entered clinical trials [et al. showed that miR-200 family members directly target the pro-angiogenic cytokines IL-8 and CXCL1. A systemic miR-200 delivery resulted in marked reductions in metastasis had anti-angiogenic effect in several different cancer mouse models. Importantly, Peco et al. [In line with the above discussion, induction of miR-200 family expression as a strategy for therapy needs to be directed at early phases of the disease. Wang cadherin . The cell trials . Recentll trials . In thiso et al.  found tho et al. . In addio et al. . Recentlo et al. . AlternaThese data along with the above discussion suggest a dual role for miR-200 family members in breast cancer. In the initial phase of breast cancer, while the tumor is still local, the miR-200 members could be potential therapeutic agents by inhibiting or reversing the malignant progression. In contrast, if the tumor cells have already metastasized to distinct organs, the miR-200 members could possibly accelerate the tumor progression by helping cells to colonize in distant environments/organs. Further studies on the multiple functions of the miR-200 family are warranted before they can be claimed relevant as potential agents or targets of cancer therapy.miRs are major regulators of cell fate through their ability to inhibit gene expression. It is becoming clear that the miR-200 family is associated with epithelial tissue, and their role is to maintain the epithelial integrity in normal tissue through stimulation of cell-cell interactions, including induction of E-cadherin expression. In the breast gland, miR-200 family members are predominantly expressed in the luminal epithelial compartment. The fact that miR-200 expression goes down during EMT and up again during MET, make the miR-200 family an interesting candidate as a regulator of cancer progression and a possible therapeutic target. In terms of cancer progression, it is important to look at the expression in the temporal context, where expression in primary tumors may be beneficial in order to retain epithelial integrity, while expression in distal metastasis may facilitate colonization and tumor growth.Studying the functional role of miRs in normal tissue morphogenesis, including branching morphogenesis and epithelial integrity in distinct organs including the breast gland, have the opportunity to shed light on how miRs contribute to normal development and disease progression. Further studies of the miR-200 family in the epithelial tissues are warranted due to their potential to retain and restore the epithelial integrity. It will be interesting to see how miR-200 family members regulate epithelial stem and progenitor cells. Are epithelial stem cells lacking miR-200 expression? What happens when miR-200 family members are introduced into breast epithelial cells with stem cell properties? Does this drive cells into luminal epithelial differentiation and/or block cell proliferation?in situ hybridization. This is, however a difficult task in most cases because of the short sequence of each miR and the difficulties of getting the probes to bind in a specific manner. Compared to protein expression, which can be easily identified by specific antibodies, an ongoing challenge is to improve techniques to visualize expression patterns of distinct miRs in tissues and identify subcellular expression patterns. Recently Renwick et al. made important advances in this area in a study where they enhanced miRs detection in cancer samples by increasing linker lengths and signal amplification [Cellular localization of miRs can be visualized using fication .There is a gap in our current knowledge of the role of the miR-200 family in branching morphogenesis in the human breast, and there is a need for functional models to study its role in developmental processes in branching organs such as the breast. In that regard, organtypic 3D cell culture models could be of help. Even though the miR-200 family is recognized as a master regulator of epithelial integrity in cells, we also propose a need for expressional and functional analysis of different miR-200 members in distinct cell populations of the breast gland.et al. showed in 3D culture that blocking overexpressed β1-integrin in a HMT3522-T4 cancer cell line could revert the phenotype to more polarized normal-like acini [The ultimate goal of clinical cancer research is to reverse the malignant phenotype in tumors preventing the metastatic process. Although intrinsic factors such as the miR-200 family are powerful effectors of epithelial integrity, the extrinsic factors are also important inducers of epithelial fates. Extrinsic variables like growth factors or the extracellular matrix are strong initiators of signal transduction that in turn result in activation of intracellular factors such as miRs. It is of great importance to identify the extrinsic factors that influence the transcription regulation of different miRs. There are a number of agents that have been shown to revert the malignant phenotype towards a more benign state. Weaver ke acini . They lake acini .et al. [Truong et al.  found reet al. . Many ofIt is becoming clear that the existence of miRs has revolutionized our understanding of various processes in cell and molecular biology. By increasing our understanding of the functional role of each miR, we are now better able to put signaling pathways and transcription regulation into broader context. miRs are stepping in as master regulators of many developmental processes, and our understanding of the complexity of their function is also rapidly expanding. The miR-200 family is being recognized as a guardian of epithelial integrity in normal tissue including the breast. Its role in preventing EMT has been solidly evidenced and, in normal breast and primary cancer, miR-200 has been considered a tumor suppressor. The recent data demonstrating that miR-200 is up-regulated in breast cancer metastasis needs to be taken into contextual evaluation before therapy for knock in or out miR-200 members is initiated.


It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at  Copy-number variants (CNVs) are a major form of genetic variation in mammals –4 and a Using genome-wide single-nucleotide polymorphism (SNP) arrays –25, alleRecent advances in high-throughput sequencing (HTS) –33 are pWhen analyzing HTS data, it is critical to correct for various sources of experimental bias that distort the quantitative relationship between read-depth and true copy number, hindering the ability for accurate CNV detection ,53,54. Whttps://sourceforge.net/projects/asgenseng/.The aim of this study was to develop an integrated method, named AS-GENSENG, that can (1) detect CNVs by jointly exploiting patterns in total- and allele-specific read count, (2) estimate ASCN and 3) be applicable to both WGS and WES data. For bias correction, we inherited the one-step approach used by GENSENG  and leve be appliHTS captures multiple sources of information in one experiment. Inspired by the successful integration of probe intensity and SNP genotypes in array-based CNV calling, here we develop an analogous method for HTS-based CNVs detection. Figure Furthermore, ASReC is useful for detecting common CNVs (i.e. >5% frequency) from WES data because it accurately identifies the reference copy-number 2 group without prior assumption of CNV frequency. Figure A brief description is provided below and detailed information can be found in the Supplementary materials.We used WGS and WES data from HapMap individuals sequenced as a part of the 1000GP ,34. The oA) and oas)( = oA)( + oB). Note that oas). For WES data, we computed TReC and AsReC in each exon target. Although each region has only one TReC value, the ASReC in each region consists of two values: For sensitivity evaluation, we used previously published high-confidence CNVs in the same samples. For WGS samples, we used the high-confidence deletions (2200 for NA12891 and 2055 for NA12892) established by 1000GP ,34,55 s, we intea posteriori probability. In comparison with other segmentation methods such as circular binary segmentation (We developed an HMM classifying each genomic region (a window for WGS data or an exon target for WES data) to a copy-number state based on maximum entation , the useentation . A briefThe total number of hidden states is an input parameter and can be specified by users. For data sets used in this study, we set seven hidden states that, respectively, represent copy numbers of 0, 1, 2, 3, 4, 5 and 6 or more. In this work, the duplications with 6 or more copies were collapsed into one state because they were difficult to differentiate. To model ASCN, we defined several possible allelic configurations for each state  element is the probability of transition from state i to state j. We set the transition probability matrix according to our intuition that the copy number state is unlikely to change for nearby genomic regions but is likely to change for genomic regions that are far apart. Thus the self-transition probability  is much larger than the transition probability of transiting to other states. We assumed that most windows would have copy-number 2. Thus, the self-transition probability for state 2 would be higher than that of other states. In addition, the probability of transiting to state 2 would also be larger than the probability of transiting to other states. To handle the problem of varying distance between targets in WES data, we further modified each element in the transition matrix as suggested in Fromer et al.  element i, j) and , as d is the distance between two targets and D is the average distance between all targets.We model the state transitions using a first-order time-homogeneous Markov process (i.e. the state in one genomic region is affected only by the immediately previous region). Under this setting, the transition probability describes the probability of having a copy number change between two adjacent genomic regions. The transition probability is characterized as a square matrix, of which the dimension is the number of states and the , given the overall ASReC and underlying states.Emission probability specifies the likelihood of observing the TReC and ASReC inputs given the underling copy number and covariate information at the region. Given the underlying state, TReC and ASReC are independent and thus the likelihood can be factorized. The first factor is the probability of observing the TReC given the covariates and underling states, and the second factor is the probability of observing the ASReC from allele A . θ is a dispersion parameter. If there is no overdispersion, then θ = 0 and oA), which is an extension of a binomial distribution to allow for possible overdispersion . Specifimentclass2pt{minime is the emission probability of the ith genomic region given the underlying copy number iq = j (0 > j > 6); c is the proportion of random uniform component which is constant for all states; R is the maximum read count; 1/R is the uniform density; ith genomic region, respectively; and ix is the input covariates for the ith genomic region. For data sets used in this study, c was set at 0.01 and was determined empirically by initializing the model with varying values of c and identifying the maximizer of the data likelihood.Taken together, the emission probability becomes The quantitative relationship between underlying copy number and read-count data is additionally distorted by target- and sample-specific biases in exome capture, which requires data normalition prior to computing emission probability. Our normalization belongs to the reference-set category of methods but we developed a new procedure of using ASReC for accurate identification of the reference-set. This procedure consists of five steps: (i) for each sample and each target, compute the ratio between TReC of the given target and the sum of TReC of all targets given the sample. This results in a target-by-sample matrix of normalized TReC values; (ii) for each target but cross all samples, use R/MixTools to cluster samples based on the normalized TReC. (iii) Use ASReC to compute the probability of being copy-number 2 for each sample by dividing the BetaB likelihood of being copy-number 2 with the sum of Beta likelihoods of being each copy number . The BetaB likelihood is computed as before (see Emission probability for WGS data: likelihood of ASReC). (iv) For each cluster, compute the average probability of being copy-number 2 of all samples belonging to the cluster. Compare and choose the cluster with the largest probability of being copy-number 2 as the reference group. (v) Compute the median of the reference group as the best estimate of the expected TReC. At this end, we are ready to incorporate the expected TReC into the HMM framework as before (see Emission probability for WGS data: likelihood of TReC) and use of all available information in the data to infer the underlying copy number.HMM training provides the maximum-likelihood estimate of the HMM parameters. To improve computational efficiency, transition-probability parameters were specified using prior knowledge and user preference , and emiWe assign the most likely ASCN given the most likely copy number call. For example, if the most likely copy number for a variant is 3, AS-GENSENG chooses between ABB and AAB. It first selects windows with ASReC larger than a threshold (10 in this study) in the region and computes the average ASReC of selected windows. If no window were selected, we would not infer ASCN because the ASReC is not informative. Otherwise, we would compute the likelihood of AAB and ABB using BetaB distribution and choose the one with the largest likelihood as the inferred ASCN.A short description is provided below and detailed procedures can be found in the Supplementary materials.We used both simulation and empirical data to assess the performance of AS-GENSENG in comparison with state-of-the-art WGS methods including GENSENG , CNVnatoTwo sets of simulation were conducted. In the first simulation, we generated read-count data (i.e. TReC and ASReC) in a single sample by using the chromosome 1 WGS data from NA12891 as the template and implanting 200 CNVs by modifying read-counts within CNVs. Briefly, TReC was simulated using a negative binomial regression model taken into account the effects of GC content and mapability, and ASReC was simulated using a beta-binomial distribution. In the second simulation, we generated paired-end reads from a pair of CNV-containing hypothetical chromosomes, created by implanting 200 artificial CNVs into chromosome 1 of the human reference genome (hg19). Artificial CNVs were created by modifying the sequence within each variant according to its copy number. Based on the hypothetical chromosomes, we applied SAMTools's wgsim with default values to generate 100-bp paired-end reads. In total, 50 millions read pairs were generated and yielded ∼40× coverage. We simulated allele-specific reads using heterozygous SNPs from NA12891. In each simulated data set, the 200 implanted CNVs included 60% deletions with copy numbers 0–1 and 40% duplications with copy numbers 3–6, median size 3000 bp.To evaluate sensitivity and (FDR), we focused on autosomal CNVs and intersected the predicted CNVs of different methods with the known CNVs. The known CNVs  are either the simulated ground-truth CNVs for the simulated data or the 1000GP-released high-confidence deletions for the empirical data. Sensitivity was calculated as the proportion of known CNVs overlapped by predicted CNVs with the correct CNV type. Following 1000GP, we defined CNV type as deletions (integer copy-number 0 or 1) or duplications (integer copy number ≥3). Sensitivity for detecting duplications in the two HapMap individuals was not evaluated because of the lack of high-confidence duplications . The FDRCNV detection performance depends on sequencing coverage, especially for read-depth-based methods. In the comparative analyses conducted in this study, both simulated (40×) and the 1000GP data (>30×) had high coverage. Thus we carried out a computational experiment in order to identify the lower bound on sequencing coverage that AS-GENSENG can handle. In this experiment, we first used Picard's DownSampleSam.jar tool to down-sample the high coverage data to varying coverage of 30×, 20×, 10× and 5× and next applied AS-GENSENG to each resulting data set and evaluated the performance using the same metrics.A short description is provided below and detailed procedures can be found in the Supplementary materials.We used both simulation and empirical data to assess the performance of AS-GENSENG in comparison with state-of-the-art WES methods including Conifer , XHMM 4 and ExomWe used chr11 WES data of HapMap sample HG00264 as the template and simulated read-counts (i.e. TReC and ASReC) for 100 WES samples. This simulated data set contained 1000 deletions and 1000 duplications with allele frequencies of 1 and 5% and 200 deletions and 200 duplications with allele frequencies of 10 and 20%. See Supplementary Figure S18 for detailed description of the simulation pipeline.In addition to metrics used for WGS data, here we further applied the SuperArray Validation (SAV) ,34 to evet. al.  in order to identify Mendelian inconsistencies. DNA for each of these samples was acquired from the Coriell repository (see the Web Resources section) and used as input for the NanoString nCounter CNV assay, according to the manufacturer's instructions. In short, 600 ng of genomic DNA was fragmented to ∼500 bp by digestion with AluI and subject to a multiplex hybridization reaction involving all probes. We designed a custom NanoString probe set (using NanoString's nDesign Gateway software) targeting 11 deletions and 14 duplications predicted by AS-GENSENG, with each locus targeted by a single custom probe. The custom probes were 70–100 bp in length, each was placed in the middle of a targeted CNV and all satisfied the internal design parameters used by NanoString, such as good GC-content and not-overlapping segmental duplication or repetitive elements. The probe set also included eight negative control probes that target artificial sequences, and 10 normalization probes that target autosomal loci that are invariant in copy number. Data analysis was conducted as in  if the ASReC was >10. Supplementary Figures S1 and S2 show examples of simulations, including simulated copy number, TReC at each simulated genomic region, simulated ASCNs and covariates (GC content and mappability). For each simulation, we implanted 200 CNVs (122 deletions and 78 duplications on average); 27 were ASCNVs (12 deletions and 15 duplications) on average. We estimated sensitivity and FDR by intersecting the AS-GENSENG-predicted CNVs with the implanted CNVs . Results are detailed in Supplementary Table S6 and are summarized below.On average, AS-GENSENG predicted 180 ground-truth CNVs (90% sensitivity) from each simulated data set, including 113 deletions (92% sensitivity) and 68 duplications (86% sensitivity). Regarding ASCNVs, AS-GENSENG predicted 25 ground-truth ASCNVs (93% sensitivity), including 11 AS deletions (98% sensitivity) and 14 AS duplications (90% sensitivity). Therefore, we observe slightly higher sensitivity for detecting AS events and, furthermore, the FDR for ASCNVs is 0.3%, much lower than that for standard CNVs (14.5%). This lower FDR results from the fact that read-count signals alone are vague for differentiating between copy-number 2 and copy-numbers 3 or 1. Without ASReC, the algorithm could make false-positive calls. However, the difference in allele-specific proportion is much clearer between copy-number 2 and copy-numbers 3 or 1 . As a result, the FDR of ASCNVs is much lower.We next simulated sequencing reads affected by implanted CNVs. We simulated 200 CNVs (119 deletions and 81 duplications), 62 were ASCNVs (15 deletions and 47 duplications), and estimated sensitivity and FDR using the same criterion. We first compared AS-GENSENG's performance for standard CNVs versus ASCNVs and then compared AS-GENSENG to CNVnator and ERDS for the ability to detect standard CNVs. Results are detailed in Supplementary Tables S7 and S8 and summarized below. For standard CNVs, AS-GENSENG predicted 193 ground-truth CNVs resulting in 97% sensitivity (117 deletions with 98% sensitivity and 76 duplications with 94% sensitivity). For ASCNVs, AS-GENSENG predicted 61 ground-truth AS-CNVs resulting in 98% sensitivity (15 AS deletions with 100% sensitivity and 46 AS duplications with 98% sensitivity), slightly higher than that for standard CNVs. Further, AS-GENSENG's FDR for ASCNVs is 4%, lower than that for standard CNVs (7%). Compared to other methods on the ability to detect standard CNVs, AS-GENSENG had the highest sensitivity and lowest FDR .In summary, simulation results suggested that incorporating ASReC improves the sensitivity and specificity of CNV detection.To further evaluate the performance of our method for WGS data, we analyzed 1000GP ,34 data.First, we compared AS-GENSENG to GENSENG and CNVnator, both relying only on TReC for CNV detection. As shown in Table We then compared the specificity of various methods. Because the high-confidence CNV data set does not provide information on the true negatives for assessing specificity, we used the volume  of the predicted CNVs as a surrogate measurement of specificity. As shown in Table As shown in Table In summary, when applied to high-coverage WGS data, AS-GENSENG outperforms existing methods for detecting deletions that are >1 kb. It gives the best sensitivity  and among the best specificity . These results suggest that incorporating AS information improves the accuracy of CNV detection. Further, in regard to ASCNVs, AS-GENSENG is the only method that can predict ASCN call from WGS data in germline DNA samples. In this experiment, AS-GENSENG predicted 576 AS deletions and 205 AS duplications in NA12891, 664 AS deletions and 173 AS duplications in NA12892.As expected, we find that the higher the sequencing coverage, the better the performance for AS-GENSENG to detect CNVs. The lowest bound of sequencing coverage that AS-GENSENG still achieves a reasonable sensitivity is 10× (Supplementary Tables S9 and S10). At a low coverage of 5×, AS-GENSENG's sensitivity is remarkably reduced .In order to calibrate the performance of our method for WES data, we applied AS-GENSENG to simulated data sets and evaluated the sensitivity and FDR by comparing the predicted CNVs with the implanted ground-truth CNVs (Supplementary Table S11). In particular, we evaluated AS-GENSENG's ability to detect CNVs at varying allele frequencies. Following the criterion of rare CNVs (<5% in the population ), we simNext, using the same simulated WES data sets, we compared AS-GENSENG with three state-of-the-art methods, XHMM , Conifera priori.Presumably, at higher CNV frequencies, CNV signals may have stronger contributions to the very variance components that are excluded by the SVD method with an arbitrary threshold ; and TReTo further evaluate the performance of CNV detection in WES data, we applied AS-GENSENG, Conifer , XHMM 4 and ExomFirst, AS-GENSENG demonstrated the highest overall sensitivity for detecting high-confidence exonic deletions . Second, the sensitivity estimates of AS-GENSENG are consistent across CNV frequency categories, whereas the sensitivity estimates of the other three methods varied considerably between rare and common deletions. Third, for rare deletions, XHMM had the highest sensitivity; and for common deletions, AS-GENSENG had the highest sensitivity. Fourth, we note that the relatively low sensitivities of all methods observed in our evaluation are not surprising; similar results have been reported in recently published independent studies ,77. ThisP-value >0.5 based on the Wilcoxon Rank Sum test, which yielded an FDR of 19.2%. Similarly, the FDR was 12.8% in Conifer calls set , 44.4% in XHMM calls set  and 95.5% in ExomeDepth calls set . For duplications, there were 169 predicted duplication regions  in the AS-GENSENG calls set and 20 regions had P-value >0.5, which yielded a 23.7% FDR. Similarly, the FDR was 50.5% in Conifer calls set , 14.9% in XHMM calls set  and 85.8% in ExomeDepth calls set . With SAV, the FDR is not defined for CNVs in individual samples but rather to CNV regions in the collection of all samples, and therefore we did not evaluate FDR stratified by frequency as we did in the sensitivity comparison.To evaluate FDR, we followed the SAV approach developed by 1000GP ,34  to validate randomly selected deletions and duplications predicted from WES data. First, we compared AS-GENSENG calls in sample NA12272 with the relative copy number estimated by NanoString (following the method in ). It is et. al. . Among tet. al. . Among tet. al. data set  that exploits the rich information in both total (TReC) and allele-specific read-depth (ASReC) to detect CNVs and ASCNVs from both WGS and WES data. We use HMM to infer the underlying integer copy numbers and combine the joint analysis of TReC and ASReC with simultaneous bias correction in data likelihood. The WGS module of AS-GENSENG is applicable to a single genome, while the WES model is applicable to large-scale exome data. To our knowledge, AS-GENSENG is the first tool capable of detecting ASCNVs from HTS data in germline DNA samples.Analogous to the previous success with array-based CNV calling, we have demonstrated that joint analysis of TReC and ASReC not only allows the estimation of ASCN but also improves the estimation of total copy number . We show through numerous examples, using both WGS and WES data, that incorporating ASReC improves the performance of CNV detection. In addition, one novel component of AS-GENSENG is the use of beta-binomial distribution to incorporate allele-specific information. This approach, applied to model both deletions and duplications rather than only deletions, does not restrict the analysis to inbred genomes  and doesWe are aware of several limitations with AS-GENSENG and have recommend alternative strategies. First, we focused on the accurate detection of simple CNVs and computed TReC using reads with unambiguous mapping in the reference genome. This approach results in lower power to detect complex CNVs within repeated sequences. For detecting CNVs in repeat-rich region, we recommend the use of specialized methods that are capable of considering all mapping positions and handling the uncertainty of read mapping ,64,78–81We aimed to conduct a comprehensive evaluation by comparing the performance of AS-GENSENG to multiple state-of-the-art methods. In order to provide an unbiased evaluation, we applied each method using its recommended parameters and quality control filters. Through independent evaluations conducted by the 1000GP, Genome STRiP  was regaWe used multiple approaches  to evaluate FDR as it is more challenging to estimate without the knowledge of true false negative CNVs in the genome. For AS-GENSENG, although the absolute FDR observed in real data is higher than that observed in simulation, the relative FDR is still lower than other methods under comparison.In this study, all analyses were performed in a high-throughput cluster-computing environment where each computing node had a shared memory of 48 GB. Sequencing data were split into individual chromosomes and chromosome-wise data were then analyzed in parallel on multiple computing nodes. Thus, the running time of a method is determined by the most time-consuming chromosome. Given read-depth data from WGS, AS-GENSENG can call CNVs for a sample with ∼30× coverage within 2 h, while ERDS and CNVnator in <1 h. For normalized read-depth data from WES, all three competing methods  can call CNVs within 1 h for 300 samples and 200K exon targets.In sum, we have developed a novel method AS-GENSENG with the following distinguishing features: (i) joint analysis of both TReC and ASReC while accounting for various experimental biases in sequence data, (ii) ability to detect both CNVs and ASCNVs from both WGS data and WES data and (iii) ability to leverage ASReC and large-scale nature of WES projects for effective data normalization and accurate detection of common CNVs with various frequencies. Through rigorous assessment using simulation, empirical data and independent technology, we have demonstrated the superior performance of AS-GENSENG in numerous examples. We conclude that AS-GENSENG not only predicts accurate allele-specific CNV calls but also improves the accuracy of total copy number calls.https://sourceforge.net/projects/asgenseng.The AS-GENSENG software and source code are freely available at ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/data/.The 1000GP alignment files: http://hapmap.ncbi.nlm.nih.gov/downloads/phasing/2009--02_phaseIII/HapMap3_r2/CEU/TRIOS/.SNP genotypes from the HapMap project: http://www.bios.unc.edu/∼weisun/software/asSeq.htm.The R/asSeq package: ftp://ftp.broadinstitute.org/pub/svtoolkit/misc/1kg/NGPaper/.The 1000GP released deletion set: http://ccr.coriell.org.The Coriell repository: http://cran.r-project.org/web/packages/ExomeDepth/vignettes/ExomeDepth-vignette.pdf.The R/ExomeDepth package: http://gatkforums.broadinstitute.org/discussion/1490/frequently-asked-questions.The Genome STRiP FAQ: Supplementary Data are available at NAR Online.


CASR) gene on 3q13  in the African-American pediatric cohort, a locus previously reported in Caucasians. We also confirmed the association result in our pediatric European-American cohort (P = 1.38 × 10-4). We further replicated two other loci associated with serum calcium levels in the European-American cohort . In addition, we replicated a previously reported locus on 1q21, demonstrating association of serum magnesium levels with MUC1 . Moreover, in an extended gene-based association analysis we uncovered evidence for association of calcium levels with the previously reported gene locus DGKD in both European-American children and African-American children. Taken together, our results support a role for CASR and DGKD mediated calcium regulation in both African-American and European-American children, and corroborate the association of calcium levels with GCKR and GATA3, and the association of magnesium levels with MUC1 in the European-American children.Calcium, magnesium, potassium, sodium, chloride and phosphorus are the major dietary minerals involved in various biological functions and are commonly measured in the blood serum. Sufficient mineral intake is especially important for children due to their rapid growth. Currently, the genetic mechanisms influencing serum mineral levels are poorly understood, especially for children. We carried out a genome-wide association (GWA) study on 5,602 European-American children and 4,706 African-American children who had mineral measures available in their electronic medical records (EMR). While no locus met the criteria for genome-wide significant association, our results demonstrated a nominal association of total serum calcium levels with a missense variant in the calcium –sensing receptor ( Calcium, magnesium, potassium and sodium cations as well as chloride and phosphorus anions are major dietary minerals which cannot be manufactured by the human body. They play important roles in various chemical and biological functions and are essential to sustain life and maintain optimal health. As a result, these minerals are commonly measured in the blood serum to monitor a range of health conditions. Sufficient mineral intake is especially important for children and adolescents due to their rapid growth.CASR, CYP24A1, DGKD, GCKR, CARS, DGKH, GATA3, VKORC1L1), influencing serum calcium levels [MUC1, ATP2B1, DCDC5, TRPM6, SHROOM3, and MDS1) were also shown to be associated with serum magnesium levels [Consistent with the high heritability estimates of serum mineral concentrations  [m levels . Common m levels . HoweverIn this study, we performed a genome-wide association study to explore the contribution of potential common genetic variations to serum concentrations of the six major dietary minerals in children of both European Caucasian and African-American ancestry. A total of 10,308 children  were included in the analysis. All samples were genotyped on the Illumina HumanHap550 or Quad610 arrays, having ~530,000 SNPs in common. The average serum levels of the six minerals were tested for associations by linear regression as implemented in PLINK . The SNPThis study was approved by the Research Ethics Board at the Children's Hospital of Philadelphia (CHOP) and written informed consent was obtained from all research subjects and/or their parents by nursing and medical assistant staff under the direction of CHOP clinicians.). Details of the European-American and African-American cohorts used in this study are summarized in S1 Table. The serum levels of calcium and magnesium displayed normal distribution in both European-American and African-American cohorts . Subjects with missing data or measurement beyond 3SD of the mean were removed from the study. For the calcium levels, 5,261 European-American children and 3,817 African-American children were included after quality controlling the data. For the magnesium levels, 2,317 European-American children and 1,283 African-American children were included after quality control.10,308 subjects were recruited from The Children’s Hospital of Philadelphia (CHOP) Health Care Network. To identify genetically inferred European-American and African-American children, we first plotted the first two principal component vectors generated by EIGENSTRAT and then used k-means (k = 5) clustering to partition the subjects into subgroups  (S1 and All subjects were genotyped by Illumina 550K SNP array and Human610-Quad version 1 array following the Illumina standard protocols as previously described . Samples). We used age, gender and the first ten PCs calculated by EIGENSTRAT as covariates and the genomic inflation factors were 1 for all the analysis .The association analyses were carried out in PLINK using linear regression models. The additive model was used for the association test. The meta-analysis was performed using fixed effects models in PLINK.  calculated by VEGAS to the reference network. The subnetwork was constructed by the significant genes and the interactions among them. The subnetwork contains a few connected components and many singletons. We then extracted the largest connected component (LCC) of the subnetwork. The same list of genes will generate exactly the same LCC. Normally, the LCC is much bigger than other connected components. To investigate the biological significance of the genes in the LCC, we carried out a gene function enrichment analysis against the KEGG and BIOCARTA database using DAVID [We next mapped the nominally significant genes (ng DAVID .S2 Table).We carried out a GWA study on our pediatric cohort including 5,602 children of European-American ancestry and 4,706 children of African-American ancestry, where we investigated the associations between genotyping markers and six dietary mineral traits, including calcium, magnesium, potassium and sodium cations, and chloride and phosphorus anions. Although no genome-wide-significant signal was detected due to the lack of power, we confirmed a few previously identified loci (CASR) gene on 3q13 . The SNP was previously reported to associate with serum calcium levels in both European Caucasian and Indian-Asian populations, but no previous studies have been conducted in African-American population or pediatric populations [P = 1.38 × 10–4, P–adj = 5.52 × 10–4, Beta = 0.05, minor allele T, Table 1). Thus, our results extend for the first time the previous observations of CASR mediated calcium regulation in adults of European ancestry to children of both African-American and European-American origin. We further replicated two other loci associated with serum calcium levels in the European-American cohort  located in MUC1 on 1q21. In contrast, the rs4072037 variant was not associated with the magnesium level trait in the African-American children (Table 1). All the directions of the associations confirmed in our study were consistent with those reported in previous studies. For the other dietary minerals , no significant locus was found to be associated in our dataset.In the African-American children, we report the association of total serum calcium levels with a missense variant in the calcium-sensing receptor (S3 Table). For the gene-based test, we found that the previously identified genome-wide significant locus on 2q37.1 (DGKD) was significant in both the European-American and African-American children (S3 Table).We next sought to infer the gene-level significance from our SNP associations and utilize the prior knowledge from protein-protein interaction networks , 16. To S8 Fig) in the subnetwork were enriched in ‘TGF-beta signaling pathway’ . For the magnesium trait, we identified a subnetwork containing 782 nodes and 270 edges, and we found that the most enriched pathway of the largest connected component  was ‘Cell cycle’ . However, none of the p values of the enriched pathways was less than 0.05 after multiple testing (S4 Table).The significant genes from both European-American and African-American cohorts were further mapped to the reference PPI network. For the calcium trait, we identified a subnetwork containing 815 nodes and 290 edges, and we found that genes of the largest connected component , while gain-of-function mutations can give rise to autosomal-dominant hypocalcemia or type 5 of Bartter syndrome [CASR are associated with serum calcium levels in Caucasians and Indian Asians [CASR and serum calcium level not only in European-American children but also in African-American children, further demonstrating that the CASR gene plays an important role in calcium regulation across the age span and major ethnicities.The product of the he blood . Previousyndrome . Two pren Asians . Here, wCASR with serum calcium levels in the CoLaus European cohort  and the Indian Asian cohort . In comparison, the associations identified in our cohorts are less robust . Since the sample sizes are similar, the variability in developing children of different ages could result in a lack of power to identify genome-wide significance. For example, the age covariance is very significant and much more significant than gender and the PCs indicating a strong association between age and serum mineral levels in our study. It is well known that the serum mineral levels are correlated with the age of children due to their rapid growth, and become stabilized in adult. In addition, environment factors such as diet and physical activity could have a greater influence on the mineral levels in children than adults.In a previous study , the autDGKD, GCKR and GATA3 were identified in a recent meta-analysis of GWAS in European ancestry individuals [DGKD, GCKR and GATA3 influence the serum calcium level is still unclear [GCKR and GATA3 gene loci in European-American children, but not in African-American children, suggesting these loci may be specific to European ancestries. Although not replicated at the SNP level, DGKD was significant in both the European-American and African-American children in the gene-level tests, indicating DGKD may also influence the serum level of calcium in the African-American children.ividuals . However unclear . Here, wMUC1 is a member of the mucin family, which encodes a membrane bound, glycosylated phosphoprotein named mucin 1. A previous GWA study has reported common variants at this genomic region that are associated with serum magnesium levels in individuals of European descent [MUC1 locus may be specific to European ancestries. descent . We confSince relatively few mineral-related loci have been discovered by the classical GWA approach, we employed a network-based pathway analysis method to determine if functionally related genes may collectively contribute to certain serum mineral traits , 12. We CASR mediated calcium regulation in both African-American and European-American children, and corroborate association of calcium levels with GCKR and GATA3 and association of magnesium levels with MUC1 in European-American children. Moreover, in an extended gene-based analysis, we uncovered evidence for association of calcium levels with DGKD in both European-American and African-American children. We anticipate that future pediatric studies with greater sample sizes will be able to uncover additional associations that will further delineate the genetic underpinnings of these traits.In conclusion, our results support a role for S1 FigThe first two principal component vectors generated by EIGENSTRAT were plotted. European-American cohort were colored in blue. African-American cohort were colored in red. In general, black, brown and green dots denote Asian, Hispanic and Multiracial Americans.(PDF)Click here for additional data file.S2 FigThe first two principal component vectors generated by EIGENSTRAT were plotted. European-American cohort were colored in blue. African-American cohort were colored in red. In general, black, brown and green dots denote Asian, Hispanic and Multiracial Americans.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


The hippocampus has been reported to be structurally and functionally altered as a sequel of very preterm birth (<33 weeks gestation), possibly due its vulnerability to hypoxic–ischemic damage in the neonatal period. We examined hippocampal volumes and subregional morphology in very preterm born individuals in mid- and late adolescence and their association with psychiatric outcome.baseline and follow-up) from 65 ex-preterm adolescents (mean age = 15.5 and 19.6 years) and 36 term-born controls (mean age=15.0 and 19.0 years). Hippocampal volumes and subregional morphometric differences were measured from manual tracings and with three-dimensional shape analysis. Psychiatric outcome was assessed with the Rutter Parents’ Scale at baseline, the General Health Questionnaire at follow-up and the Peters Delusional Inventory at both time points.Structural brain magnetic resonance images were acquired at two time points  larger right hippocampal volume and bilateral anterior surface deformations at This study suggests a dynamic association between cross-sectional hippocampal volumes, longitudinal changes and surface deformations and psychosis proneness. Very preterm birth  is associated with an increased risk of brain damage and consequent neurological disorders, neuropsychological, and behavioural impairments in childhood and later in life –5.Long-lasting and widespread structural brain alterations have been described in VPT samples , 6, 7, sHippocampal volume decrements in VPT individuals compared to controls have been described in the first two decades of life, from infancy  to adoleAt a cognitive level, the hippocampus has been associated with processes involved in general intelligence , learninSmaller hippocampal volumes in VPT born samples have been associated with low intelligence quotient  and withth week of gestation [In terms of development, the cytoarchitecture of the hippocampus is formed by the 34estation . Howeverestation , 40. Sucestation . Structuestation –44. Longestation , 46.Trajectories of development have described differential time-dependent courses for hippocampal subregions , which sbaseline (age 15 years), VPT individuals would have smaller hippocampal volumes than controls, based on our previous findings [follow-up (age 19 years) there would be diminished between-group differences in hippocampal volume and shape, on the basis of previous findings in other regions of interest  [The primary aim of this study was to examine hippocampal volume and shape, which reflects subregional atrophy , in VPT line age  years, Vfindings . A variefindings , and thefindings . We furtallosum) . In termallosum) .baseline assessment).VPT participants were recruited from a cohort of individuals born before 33 weeks’ gestation between 1982 and 1984 who were admitted to the neonatal unit of University College London Hospital within 5 days of birth. From this population, 302 survived and were recruited as part of a long-term follow-up study –56. At abaseline assessment. Inclusion criteria were full-term birth (38–42 weeks) and birth weight >2500 grams. These same individuals were invited back for follow-up assessment. Successful MRI scanning was carried out in 36 (51%) individuals at both time-points. Exclusions criteria for both VPT individuals and controls were a history of neurological conditions including meningitis, head injury and cerebral infections, or any contra-indications to MRI scanning.A term-born comparison group of 71 individuals was recruited by advertisements in the press for the follow-up could not be analysed due to signal artefacts. Thus, 61 preterm adolescents and 35 controls, none of which had received a clinical psychiatric diagnosis, were included in the data analysis. Data for an additional control participant was excluded from shape analysis due to processing error.Participants were excluded from subsequent data analyses for the following reasons: one control participant had been diagnosed with depressive disorder, one VPT participant had cerebral palsy and two had severe hearing or visual impairment. The structural MRI scan for one VPT participant at follow-up and from an accompanying parent at baseline assessment.The Institute of Psychiatry, King’s College London Ethical Committee (Research) approved the study and the consent procedure used. Written informed consent was obtained from all participants at The Rutter Parents‘ Scale was used to assess emotional, attentional and conduct problems . This scThe Peters Delusion Inventory (PDI) , a self-We used a 12-item version of the General Health Questionnaire (GHQ-12)  to assesfollow-up assessment.The PDI was administered again at Magnetic resonance imaging was performed using a 1.5 Tesla system . Three-dimensional T1-weighted spoiled gradient-echo (SPGR) sequences with 124 1.5 mm slices, in-plane resolution 1.5mm x 1.5mm, TR = 35 ms, effective TE = 5 ms, flip angle = 3 were acquired axially. The same image acquisition parameters were used on the same MRI scanner for both baseline and follow-up assessments.baseline and follow-up assessments were used as covariates in subsequent analyses. In brief, SPGR images were masked to exclude non-brain tissues, and each voxel subsequently classified as grey matter or white matter, or other classes of tissue , by an automated segmentation algorithm. Total brain grey and white matter volumes were derived from the images in native space. Whole brain volume was calculated as the sum of total grey and white matter.Whole brain volume (WBV) at both time-points was calculated using Statistical Parametric Mapping software . Whole brain volumes at Pre-processing was conducted in accordance with the protocol established by Narr and colleagues . For allWe used a Direct Hippocampal Mapping procedure , impleme2 tests , t-tests  and repeated measures ANOVAs . Correlation analyses between the two time points were performed for cognitive and behavioural variables. Volumetric analysis of hippocampal volume and WBV was carried out using a multivariate analysis of covariance for cross-sectional analysis at baseline and follow-up, with left and right hippocampal volume as the dependent variables, group as the experimental factor and sex as a covariate. As hippocampal volume and WBV are correlated [10 transformed, which reduced both skewness and kurtosis of the data (Shapiro-Wilk test p > 0.05).Statistical analysis of demographic and volumetric data was conducted using SPSS . Cross-sectional and longitudinal analysis of demographic, cognitive and behavioural data between groups utilised χrrelated , both rabaseline (T1) and follow-up (T2) was quantified by using longitudinal change in WBV as an error term (WBVerror = WBVT1/WBVT2) [Change between 1/WBVT2) . This erbaseline and follow-up  being the within subject factor and group being the between-subject factor. Sex was the only covariate included in each ANCOVA despite the groups not being well-matched for age at scan nor IQ, as this would have been statistically inappropriate [Paired-samples t-tests were used to test for within-group longitudinal changes in raw hippocampal volume and WBV. A repeated-measures ANCOVA was used to assess group effects on longitudinal change over time, with volume at ropriate  as both ropriate  correctibaseline  and at follow-up (GHQ-12 and PDI scores) were explored using a multiple linear regression approach. Again FDR correction with q = 0.05 was carried out, this time correcting for the five clinical or behavioural outcome variables tested.Associations between cross-sectional and longitudinal normalised hippocampal measures and clinical measures , behavioural/psychiatric outcomes at baseline and follow-up assessments, as well as longitudinally within each group. To correct for the multiple comparisons across the 2000 atlas vertices, 50,000 permutations were run by randomising group membership and comparing significance maps of real and randomised groups. This process generated a corrected p-map that represents statistical differences in hippocampal shape between groups. Three-dimensional hippocampal shape was further investigated in relation to group and PDI ‘caseness’, as our previous work demonstrated selective associations between behavioural outcome and regional brain morphology (e.g. hyperactivity scores and caudate volume) in VPT-born individuals but not in controls [The feature selected for three-dimensional hippocampal shape analysis was the tail-to-head trend of the hippocampus, which is represented by a graph comprised of a node at the centroid of each contour, thus giving a measure of the distance between the medial core and each surface vertices of the structure. This measure is analogous to that used in our previous work , but is controls .Mean gestational age for the VPT group was 28.8 weeks , for controls it was 40.0 weeks ; mean birth weight for the VPT group was 1248.3 grams , for controls it was 3250.3 grams .baseline and follow-up are shown in baseline and follow-up assessments (p = 0.40). At both baseline and follow-up VPT adolescents were significantly older and had lower IQ score than controls. IQ scores at baseline and follow-up were significantly correlated .Demographic, cognitive and behavioural data for VPT adolescents and controls at baseline, behavioural/psychiatric scores did not differ significantly between the two groups . At follow-up assessment, results were similar and no statistically significant between-group differences were detected . Correlations between Rutter Total scores and PDI scores at baseline and between GHQ-12 scores and PDI scores at follow-up were non-significant. A significant correlation was observed between PDI total scores at baseline and follow-up assessments .At p = 0.55) between VPT individuals and controls, after adjusting for sex. The preterm group had significantly smaller WBV compared to controls .Baseline assessment: There was no significant difference in left (p = 0.62) or right ‘normalised’ hippocampal volume (p = 0.26) or right hippocampal volume (p = 0.29) between VPT individuals and controls. Again, the VPT group had reduced WBV compared to controls . Descriptive statistics for bilateral hippocampal volumes in VPT adolescents and controls are given in Follow-up assessment: There was no significant difference in ‘normalised’ left , equating to approximately 28% of the surface of left and right hippocampi see . There wFollow-up assessment: There were still substantial numbers of significant surface contractions in the preterm group. However, there was a decrease in the number of surface points differing between groups and the deformations were now almost entirely concentrated in the tail of the left and right hippocampi (equating to the subiculum and CA1 subfield), while hippocampal head and body were no longer significantly different see .baseline and follow-up and that right hippocampal volume increased 14.7%. Both increases were statistically significant after FDR correction . In controls left hippocampal volume increased significantly by a magnitude of 12.5% , but the 8.2% increase in right hippocampal volume was not significant (p = 0.19). Mean longitudinal hippocampal increases were calculated using WBVerror to correct for measurement error over time [p>0.05). Furthermore, the effect of time on normalised bilateral hippocampal volumes was not significant across groups . There were no longitudinal changes in WBV or in intracranial volume for either group.Within-group analysis in ex-preterm adolescents indicated that left hippocampal volume increased 13.9% between ver time . RepeateWhen comparing longitudinal changes in hippocampal shape within each group, there were minor expansions in both left and right hippocampus in the VPT group, predominantly in the tail of the structure and accounting for approximately 10% of the total surface see . In contBaseline assessment: Linear regression analyses were performed to assess the association between bilateral hippocampal volumes  and outcome measures in the whole sample (VPT participants and controls), as well as within group. Bilateral normalised hippocampal volumes were not significantly associated with PDI and Rutter scores.Gestational age and birth weight were not significantly associated with cross-sectional bilateral hippocampal volumes in the VPT group .follow-up in the whole sample . Furthermore, those individuals who scored as a PDI ‘case’ at follow-up had larger normalised right hippocampal volume at baseline than those scoring as ‘non-case’ . Left hippocampal volume at baseline was not significantly associated with PDI scores at follow-up. Bilateral normalised hippocampal volumes were not significantly associated with continuous or dichotomous GHQ scores .Follow-up assessment: Right and left normalised hippocampal volumes were not significantly associated with PDI scores. However, baseline right hippocampal volume was significantly positively associated with PDI total score at mple see  as well Longitudinal hippocampal volume change: Volumetric bilateral hippocampal changes between the two time-points were not significantly associated with PDI scores, Rutter scores, GHQ scores, or birth weight and gestational length . see .p>0.05).There were no significant associations between continuous or dichotomous GHQ scores and cross-sectional and longitudinal bilateral hippocampal volumes and in the whole sample and within group  in the whole sample , whereby PDI ‘cases’ showed surface deformations compared to PDI ‘non-cases’, especially in the subiculum and CA1 subfield . No statistically significant association between hippocampal shape at follow-up and PDI scores were found. Distinct areas of the prefrontal cortex receive projections from neurons in the head of the hippocampus, therefore it could be speculated that these results support the idea that psychiatric disorders characterized by delusional beliefs such as schizophrenia involve alterations in fronto-temporal circuitry [follow-up and behavioral scores collected at the same time-point may be explained by the fact that shape differences were then mostly observed in the posterior hippocampal segment.Right hippocampal shape deformations at ircuitry . Variatiircuitry , 93 and ircuitry . The lacin vivo, but they do not achieve the precision obtained by the study of neuropathological data, which allows assessment at a microscopic level and can differentiate between the outer  and inner (e.g. dentate gyrus) subfields. Furthermore, only one rater conducted the hippocampal segmentation. Ideally multiple raters would be available to increase the robustness of the segmentations and to allow testing of how different raters may influence results. Nevertheless the rater in the current study achieved high reliability with a historical dataset on which the protocol was defined as well as excellent intra-rater reliability, meaning that additional raters are unlikely to have greatly influenced the current findings. Another limitation of the study may be the representativeness of our control sample, a large proportion of whom scored as a PDI ‘case’ at baseline (23%) and follow-up assessments (43%). Psychotic experiences and beliefs have been reported in the general population with prevalence up to 40% [Limitations of this study include the limited sample size, and the fact that the neurodevelopmental profile of VPT individuals who were born in the 1980s may differ from that of younger cohorts who may have received more advanced forms of neonatal care. Regarding the study methodology, a potential problem to the interpretation of morphological analysis is its limited anatomical accuracy. To date, surface shape-based such as the method we used, provides the most valid way of investigating changes p to 40% . Howeverp to 40% . The ex-p to 40% . Finallyp to 40% , somethiTo conclude, although this study did not reveal significant differences in hippocampal volume between VPT individuals and controls in mid- and late adolescence, a substantial percentage of the total hippocampal surface showed subregional deformations in the VPT group, suggestive of atrophy, predominantly in the posterior and anterior regions. Cross-sectional and longitudinal hippocampal volumes were dynamically associated with delusional ideation scores in young adulthood. These results further our understanding of the structural correlates underlying long-term neurodevelopmental consequences of very preterm birth. They also support the idea of a dynamic association between brain structure and function throughout neurodevelopment.S1 FileThis contains all the raw data used in the analysis. This includes demographic, group status, brain volume, hippocampal volume and behavioural measures. Also included are a number of derived metrics included in the study, such as normalised volumes, longitudinal measure error and change in volume or behavioural measure over time. Not included are the values comprising the 3D hippocampal shape maps as these high dimensional data require specific software for visualisation and analysis. The authors will make these available on request, alongside the details of the necessary additional software packages.(XLSX)Click here for additional data file.


Cross-sectional studies detected associations between physical fitness, living area, and sports participation in children. Yet, their scientific value is limited because the identification of cause-and-effect relationships is not possible. In a longitudinal approach, we examined the effects of living area and sports club participation on physical fitness development in primary school children from classes 3 to 6.One-hundred and seventy-two children  were tested for their physical fitness . Living area  and sports club participation were assessed using parent questionnaire.p = 0.009, ES = 0.16) and lower-extremity strength . Further, significantly better performance development were found for endurance  and lower-extremity strength  for children continuously participating in sports clubs compared to their non-participating peers.Over the 4 year study period, urban compared to rural children showed significantly better performance development for upper- (Our findings suggest that sport club programs with appealing arrangements appear to represent a good means to promote physical fitness in children living in rural areas. Physical fitness is an important health determinant that is related to several physiological functions. Recent studies regarding the health burden of chronic diseases revealed significant inverse associations between physical fitness and various cardiovascular risk factors  in children and adolescents -3. FurthSeveral non-modifiable   and modiBesides the subcategory living area, sports participation represents another factor that is related to physical fitness ,15,16. FTherefore, this study used a longitudinal approach and we aimed at investigating differences in physical fitness levels over time  between children living in urban as compared to rural areas and between sports club participating children and their non-participating peers. Based on the literature -13, it iN = 24) that were randomly selected from urban  and rural  areas of the federal state Brandenburg (Germany). This classification was made according to the federal statistics report  = 4.2, p = 0.009, ES = 0.16) and for the triple hop test  . Additionally, the main effect of living area was significant for the 50-m sprint test . In sixth graders, post-hoc analyses indicated significantly better performances in 4 out of 6 physical fitness tests  in favor of children living in urban compared to rural areas  and for the 9-min run test   but 2 physical fitness test . Additionally, the main effect of sports club participation turned out to be significant  in all but 1 physical fitness tests . Post-hoc analyses indicated significantly better performances in all physical fitness tests for children participating in sport clubs compared to those who did not. This is particularly prevalent for fifth- and sixth-graders  over the 4-year study period, children living in urban as compared to rural areas showed a significantly better performance development for upper-  and lower-extremity muscle strength (triple hop test) and (2) children participating in sport clubs showed a significantly better performance development over the 4-year study period for measures of endurance (9-min run) and lower-extremity muscle strength (triple hop test) as compared to their non-participating peers.In addition to the already existing cross-sectional studies, the present investigation provided data originating from a longitudinal study approach over a 4-year experimental period. As a result, it was found that living area has a positive effect on the development of physical fitness in children in terms of significantly better performance increments for children living in urban compared to rural areas. This was detected in significant findings for upper-  and lower-extremity muscle strength (triple hop test).It is of interest to note that our results regarding better physical fitness in urban as compared to rural children are in contrast to most of the studies reported in the literature -14,23,24The results of this study illustrated that, irrespective of living area, children participating in sports clubs showed better physical fitness development than their non-participating peers. The findings of our longitudinal approach are in accordance with that of cross-sectional studies concerning a positive association of sports club participation and physical fitness ,15,16. FOur finding regarding better physical fitness development in sports club participating children may be attributed to the formal and structured organizational frame in which physical activity takes place. In fact, Silva et al.  reportedIn summary, due to the link between physical fitness and several health-related outcomes in youth ,36 as weFindings from the present study indicate that the development of physical fitness is positively affected by living area and sports club participation. More specifically, children living in urban areas and children participating in sports clubs were fitter and fitness progressed faster than in their counterparts in terms of endurance (9-min run), upper-  and lower-extremity muscle strength (triple hop test). From a health perspective, these are important findings because deficits in muscle strength represent intrinsic injury and fall-related factors in youth ,39. AddiThe authors declare that they have no competing interests.TM, KG, and UG analyzed the data and wrote the manuscript. DW, KG, MH, and UG contributed to the study design, data collection and critical review of draft manuscripts. TM, UG, MH, and DW assisted with the statistical analysis and interpretation of data. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/499/prepub


Low-income women of reproductive age are at increased risk for obesity and resulting increases in the risk of maternal/fetal complications and mortality and morbidity. Very few weight-loss interventions, however, have been targeted to this high-risk group. Based on the high prevalence of social media use among young and low-income individuals and previous successes using group formats for weight-loss interventions, the use of social media as a platform for weight-loss intervention delivery may benefit low-income women of reproductive age.Examine the feasibility of delivering group-based weight-loss interventions to low-income women of reproductive age using face-to-face meetings and Web-based modalities including social media.Participants attended a family planning clinic in eastern North Carolina and received a 5-month, group- and Web-based, face-to-face weight-loss intervention. Measures were assessed at baseline and 20 weeks.2. Among the 12 women who completed follow-up, mean weight change was -1.3 kg. Participation in the intervention was modest and retention at 5 months was 30%. Returnees suggested sending reminders to improve participation and adding activities to increase familiarity among participants.Forty participants enrolled, including 29 (73%) African American women. The mean body mass index of enrollees was 39 kg/mEngagement with the intervention was limited and attrition was high. Additional formative work on the barriers and facilitators to participation may improve the intervention’s feasibility with low-income women of reproductive age. Low-income women of reproductive age (WRA) are at increased risk for obesity , which iWeb-based delivery of intervention components may reduce barriers to intervention participation experienced by low-income WRA  . For exaAlthough there is a growing body of research examining Web-based weight-loss interventions,  few haveThis feasibility study employed a single group pre-post design. We piloted a weight-loss intervention among female participants enrolled from a county health department family planning clinic and assessed outcome measures at baseline and 5 months.2 were considered eligible for the weight-loss study. Although the United States Preventive Services Task Force recommends that patients with a BMI greater than 30 take part in weight-loss interventions [The Integrated Screening and Health Assessment, Prevention, and Evaluation (InShape) Study  screenedventions , those iParticipants who expressed interest in the weight-loss study were contacted by phone and invited to an enrollment visit. To have sufficient numbers for the group format component of the intervention, the intervention did not begin until all participants were enrolled. The Institutional Review Board of the University of North Carolina at Chapel Hill approved and monitored this study.The InShape weight-loss intervention content was based on the Weight-Wise program, a group-based, 16-session, behavioral weight-loss intervention targeting low-income, midlife (40-64 years old) women . The WeiAt the enrollment visit, conducted at the study research office, the interventionist first obtained informed consent and completed baseline measures . Then, dOnce a sufficient number of participants completed the enrollment and counseling visit, the remaining intervention activities were initiated. Five face-to-face group sessions (70-120 minutes) were held over a 20-week period at the health department on days and times that participants indicated were convenient (each session was offered 2 times during the week). The mean interval between the enrollment visit and the first group session was 85 (6-148) days. During each session, participants reflected on previous session content; received new intervention content, including hands-on activities such as PA or food preparation demonstrations; and completed an action plan for the coming week’s goals.At the onset of intervention activities, participants were invited to “like” a Facebook fan page created for the study, where participants could model healthy behaviors and support others in their dietary and PA behavior changes . Study sUsing Google docs, a Web-based document sharing program, we provided participants with hyperlinks (via email) to 8 Web-based nutrition and PA educational lessons every 2 weeks for 16 weeks, beginning 1 month after intervention activities were initiated . Each leThe interventionist sent participants weekly emails containing a link to a brief, 6-question online self-monitoring questionnaire where participants were asked to self-report weight, frequency of keeping a food record, fruit and vegetable intake, amount of moderate physical activity, and number of steps. Tailored tips for improving or maintaining healthy behaviors were delivered to participants based on their questionnaire responses.After completion of the intervention, we conducted 15-minute in-person and telephone qualitative interviews to assess intervention acceptability and barriers to participation. Incentives ($20 gift cards) were given at measurement visits (baseline and 5 months) and upon completion of qualitative interviews, and also were available based on “InShape points,” which were earned by participating in intervention activities. We conducted 5 monthly drawings for points-based incentives. The number of entries/participant equaled the number of their InShape accumulated points.Trained personnel collected data at baseline and 5 months. Weight and height (baseline visit only) were measured twice without shoes using an electronic scale and portable stadiometer and then averaged. Blood pressure was measured 3 times  with an Omron HEM-907XL automated blood pressure monitor and averaged.We assessed multiple other outcomes through self-report at baseline and 5 months. Total walking time was documented with the RESIDE PA survey  and dailUsing the Qualtrics survey program  we recorded the frequency of participants’ use of the online self-monitoring questionnaires and completion of the Web-based lesson questionnaires. The interventionist manually recorded Facebook fan page contributions and logged participant attendance at group sessions. At the end of the intervention period, participants also completed an acceptability questionnaire and a qualitative interview to solicit their thoughts about and experiences with the intervention and suggestions for improving it.Descriptive statistics were used to describe baseline characteristics and the frequency of participant involvement in intervention activities. Due to the limited sample size at 5 months, we did not use statistical tests to examine differences in measures at baseline and 5 months. Statistical analysis was performed with SAS software . Analysis of qualitative interviews included identifying key themes and quantifying instances of specific responses.2, 51 (20%) expressed an interest in the weight-loss study and 119 (47%) in either the lifestyle or the weight-loss study. Of these 170 eligible and initially interested participants, we successfully contacted 108 (64%). Of those contacted, 64 (59%) expressed continued interest in the weight-loss study with 40 (63%) completing the baseline visit to comprise the study sample.Of the 251 (54%) participants in the InShape Screening Study who were eligible for the weight-loss intervention based on a BMI greater than 27.5 kg/m2. Over half were categorized as having class II or III (extreme) obesity. Most participants reported using the Internet (95%) and Facebook (83%).The majority of participants (N=40) were under 30 years of age, African American, low-income, and uninsured . One-quaTwelve participants (30%) completed 5-month measures (study returnees). At the end of the intervention period (5 months), the difference in mean weight among the 12 study returnees was -1.3 kg (SD=4.4 kg) with a range in weight change of -9 to 5 kg. Of note, 7 of 12 participants lost weight. Mean change in walking time was 116.3 minutes/week (SD=191.6 minutes/week) with a range of 0 to 675 minutes/week. Mean change in servings/day of fruit and vegetables was 0.5 servings/day (SD=1.5 servings/day) with a range of -2 to 3 servings/day.Intervention activity participation was modest at the beginning and declined over time . A totalQualitative analysis (n=11) suggested that participants had hectic lives and work schedules that interfered with intervention participation and behavior change. Some returnees reported lack of access to the Internet, lack of transportation, and a desire for additional tools like digital calorie counting applications and food scales. Returnees wanted more frequent group sessions and more meeting times and suggested more text and email reminders. Suggestions for improving the Facebook component of the program included having team building exercises, enlisting friends, and providing a list of participants to each person to facilitate more face-to-face interaction with other participants. Participants also suggested more moderator posts, reminders, and greater frequency of providing incentives.Despite the need for weight loss among our sample of overweight and obese participants, only a small percentage were interested in joining the study and 30% of participants completed the intervention. On average, minimal changes in weight were observed among returnees.Use of Web-based components was low and eroded over time. Despite the high levels of self-reported Internet and social media use, it is possible that participants’ Internet access was episodic rather than continuous, which may have hindered participation in Web-based components. Lack of familiarity with other participants prior to using the Facebook fan page may have limited engagement in the social media intervention component. Attrition in the current study was greater than previous Web-based weight-loss intervention studies that mostly included older populations, including some that used Facebook for intervention delivery ,18. PartFuture social media interventions should plan for this contingency by having rapid recruitment procedures in place. It may also be necessary to provide some intervention components or other content to participants in this lag period to maintain interest. In addition, having multiple intervention components on different platforms  may have produced too much burden for participants. Retention could possibly be improved by delivering all intervention components through 1 platform. Other factors endemic to our study population may have also played a role in limited engagement and high attrition. For instance, young adults cite different weight loss motivations  compared to older adults  . Our empSeveral suggestions for improving levels of engagement in future studies targeting low-income WRA can be drawn from our results. Based on participant feedback about the need for reminders and the high penetration of cellular phones in low-income populations,  incorporThis study has several limitations. There was a significant lag between many participants expressing interest and beginning the intervention. Given the small number of returnees, we did not undertake statistical testing. Misclassification is possible since we measured PA by self-report, which is subject to overreporting,  and our Although use of the Web and social media in particular is prevalent among young, lower income women, participation in the Web-based components of our intervention was limited. Additional formative research with larger samples of low-income WRA should be conducted in order to develop novel intervention strategies for weight loss among low-income women of reproductive age who are at high risk for both chronic disease and poor pregnancy outcomes.


Smoke-free legislation is associated with improved early-life outcomes; however its impact on perinatal survival is unclear. We linked individual-level data with death certificates for all registered singletons births in England (1995–2011). We used interrupted time series logistic regression analysis to study changes in key adverse perinatal events following the July 2007 national, comprehensive smoke-free legislation. We studied 52,163 stillbirths and 10,238,950 live-births. Smoke-free legislation was associated with an immediate 7.8%  reduction in stillbirth, a 3.9%  reduction in low birth weight, and a 7.6%  reduction in neonatal mortality. No significant impact on SIDS was observed. Using a counterfactual scenario, we estimated that in the first four years following smoke-free legislation, 991 stillbirths, 5,470 cases of low birth weight, and 430 neonatal deaths were prevented. In conclusion, smoke-free legislation in England was associated with clinically important reductions in severe adverse perinatal outcomes. Fifty years after the official recognition of its adverse health effects, tobacco smoking remains the primary cause of preventable mortality worldwide1141Population-level reductions in SHS exposure and active smoking can effectively be achieved by implementing legislation to prohibit smoking in enclosed public places and the workplace7910111213We sought to address this by investigating the association between the 2007 national introduction of smoke-free legislation in England and various indicators of early-life mortality – namely stillbirth, neonatal mortality, and SIDS – in a national birth cohort of over 10 million singleton births. Given the conflicting evidence from previous studiesDuring the study period, 10,291,113 singleton births were registered in England, including 52,163 stillbirths 0.5%; . Among tStillbirth and early-life mortality were highly associated with low birth weight categories and low socioeconomic status, and less strongly with other demographic characteristics . ImportaIntroduction of smoke-free legislation was associated with an immediate 7.8%  reduction in odds of being stillborn, a 3.9%  reduction in odds of having low birth weight, and a 7.6%  decrease in odds of neonatal mortality over-and-above the underlying temporal trend . No signThe reduction in neonatal mortality was primarily attributable to an impact on late neonatal deaths: −13.7%  may have resulted in unexplained variation43Whereas we identified an overall association between smoke-free legislation and reduced infant mortality, we were surprised by the finding that SIDS was not affected1The impact of smoke-free legislation on perinatal and early-life health is likely to have been mediated via several routes. A number of previous studies have demonstrated important drops in maternal smoking during pregnancy following implementation of smoke-free legislation110111213474849Our findings add an important new dimension to the emerging evidence on the benefits of smoke-free legislation to child health971821Since the majority of the burden of early-life morbidity and mortality occurs in low- and middle-income countries18In conclusion, we present evidence that implementation of smoke-free legislation in England was associated with substantial perinatal and early-life benefits, with over 5,000 cases of low birth weight and almost 1,500 deaths averted within four years.This study was performed according to a pre-specified and registered research protocol . Using a national dataset of all singleton births registered in England between 1995 and 2011, we analysed the association between the July 2007 implementation of smoke-free legislation and the odds of stillbirth, low birth weight, and early-life mortality.This study was reviewed by the National Health Services (NHS) South East Scotland Research Ethics Service and The University of Edinburgh’s Centre for Population Health Sciences Ethics Review Group. Both committees provided an exemption from formal ethical assessment based on the use of anonymised, unidentifiable data.The intervention under study was the implementation of smoke-free legislation in England on 1 July 2007Four primary outcomes were evaluated: low birth weight ; stillbirth (intrauterine death from 24 weeks of gestation); neonatal death (death in the first 28 days of life); SIDS  10-U R95, or R99 with no other specification).In addition, we considered the following secondary outcomes: very low birth weight ; early neonatal death (death in the first week of life); late neonatal death (death between 7 and 28 days of life); post-neonatal death (death between 28 days of life and the first birthday); and infant death (death within the first year of life).a priori not to involve preterm birth as an outcome or intermediate variable in our analyses.Data on gestational age were not available through the Office for National Statistics before 2006, restricting the pre-legislation period to only 1.5 years. Given the recognised seasonal variation in gestational age we considered this period insufficient to estimate underlying trends pre-legislation, and therefore decided Individual-level data on all registered singleton births in England between 1 January 1995 and 31 December 2011 were obtained via the government’s Office for National Statistics. Data were linked by the Office for National Statistics to death certificates for stillbirths and for all infants dying before their first birthday. To minimise the risks of individuals being identified, individual-level continuous variables were categorized as follows: month and year of birth; month and year of death; timing of death ; sex; birth weight ; maternal marital status (married /not married); maternal parity; and maternal age (<20 years/20–24 years/25–29 years/30–34 years/35–39 years/≥40 years). The individual-level data also included Government Office Region, urbanisation level , and socioeconomic status measured using the Index of Multiple Deprivation (in quintiles), these being derived from maternal post code of residence. The Index of Multiple Deprivation is a relative measure of deprivation at the small-area levelThe patterning of demographic data was initially tabulated by timing of death and birth weight categories. Separate logistic regression models using individual-level data were then developed to investigate the association between the introduction of smoke-free legislation and the odds of developing each outcome5151The following categorical covariates were included in each model to account for potential individual-level confounding: sex; maternal age; maternal marital status; Index of Multiple Deprivation quintile; region; and urbanisation level. All mortality-related outcomes were also adjusted for birth weight.Among the model covariates, data on parity were missing for a substantial portion of the study population (41.3%), as this variable had been recorded for married women only. All other covariates had 0–3% missing data. Since logistic regression analysis needs to be undertaken on cases with complete data for all covariates, parity was excluded from the primary models to maximise power. We performed two sets of pre-specified sensitivity analyses to investigate potential residual confounding resulting from exclusion of parity as a covariate. In the first set, models were re-run with parity included, analysing individuals with complete data only. In the second set, multiple imputations were performed using chained equations to impute missing data for parity, creating five unique datasetsA change in odds of developing low birth weight following the introduction of smoke-free legislation is potentially on the causal pathway between smoke-free legislation and any observed impact on early-life mortality . We therIn order to estimate the absolute impact of smoke-free legislation for outcomes that were significantly affected, we developed counterfactual scenarios51All analyses were undertaken using Stata SE version 12.0 .As we used the maximum time span and population available, sample size calculations were redundant. Given the national nature of this evaluation, we estimated that we would have adequate power to detect clinically relevant temporal changes in the outcomes of interest11121323242527How to cite this article: Been, J. V. et al. Impact of smoke-free legislation on perinatal and infant mortality: a national quasi-experimental study. Sci. Rep.5, 13020; doi: 10.1038/srep13020 (2015).