Headache Characteristics of Pediatric Sport-Related Concussion.

BACKGROUND: Headache is among the most common symptoms following concussion, yet headache after concussion (HAC) remains poorly characterized. This study describes headache characteristics over the first four weeks following pediatric sport-related concussion. METHODS: This is a retrospective case series of 87 athletes (mean: 14.9 years; range: 8.4-18.8 years; 38% female) treated in a specialty sports concussion clinic within 28 days of injury. Primary outcomes of headache consistency, frequency, duration, and associated migrainous symptoms were assessed at immediate (0 to 48 h) and weekly time points over the first 28 days post-injury. Generalized mixed linear models compared headache characteristics across time points. Secondary analyses compared each outcome by as-needed analgesic use. RESULTS: During the immediate post-injury period, headache was more often constant (p = 0.002) and associated with migrainous symptoms (p < 0.001). By the third week post-injury, episodic headache was more prevalent (p < 0.001). Most patients (54%) transitioned from constant, migrainous headache to episodic, non-migrainous headache. This finding was uninfluenced by as-needed analgesic medication use. CONCLUSIONS: These findings document the trajectory of HAC. Future studies should assess relationships between initial headache characteristics and recovery.

Conservation and divergence of canonical and non-canonical imprinting in murids.

BACKGROUND: Genomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behavior. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we use low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extraembryonic tissues at allele-specific resolution. RESULTS: We identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse reveal that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extraembryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extraembryonic imprinting. Meta-analysis of novel imprinted genes reveals multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the appearance of ZFP57 binding motifs, and the insertion of endogenous retroviral promoters. CONCLUSIONS: In summary, we provide an expanded list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

Profiling Histone Methylation in Low Numbers of Cells.

Chromatin immunoprecipitation (ChIP) enables the study of DNA-protein interactions. When coupled with high-throughput sequencing (ChIP-seq), this method allows the generation of genome-wide profiles of the distribution of specific proteins in a given cellular context. Typical ChIP-seq experiments require millions of cells as input material and thus are not ideal to study many in vivo cell populations. Here, we describe an ultra-low-input native ChIP-seq method, ULI-NChIP-seq, to profile histone modification patterns in as low as 150 cells.

Epigenetic Mechanisms Governing Female and Male Germline Development in Mammals.

BACKGROUND: DNA methylation (DNAme) and histone posttranslational modifications (PTMs) play an integral role in the transcriptional regulation of specific sets of genes and retrotransposons. In turn, these chromatin marks are essential for cellular reprogramming, including during germline development. While DNAme is stably propagated in most somatic tissues, this epigenetic mark undergoes cycles of widespread erasure and re-establishment in the early embryo as well as in the germline. SUMMARY: De novo DNAme occurs at distinct developmental stages in male and female germ cells; before birth in prospermatogonia (PSG) and after birth in growing oocytes. Furthermore, while only ∼40% of the mouse genome is methylated in mature oocytes, ∼80% of the genome is methylated in mature sperm. Here, we review recent epigenome studies which reveal a complex interplay between histone PTMs and de novo DNAme in shaping the sexually dimorphic profiles of DNAme observed in mature gametes in the mouse, including in intergenic regions as well as at imprinted gametic differentially methylated regions (gDMRs). We discuss the dynamics and distribution of key histone PTMs in male and female germ cells, including H3K36me2/me3, H3K4me3, and H3K27me3, and the implications of positive and negative crosstalk between these PTMs and the DNAme machinery. Finally, we reflect on how the sex-specific epigenetic landscapes observed in the mouse germline impact transcriptional regulation in both the gametes and the early embryo. KEY MESSAGES: Investigation of the roles of chromatin modifying enzymes and the interplay between the chromatin marks that they deposit in germ cells has been facilitated by analyses of conventional or germline-specific knockout mice, combined with low-input genome-wide profiling methods that have been developed in recent years. While clearly informative, these findings generally reflect "snapshots" of chromatin states derived from analyses of cells analyzed in bulk at a specific period in development. Technological advances and novel experimental models will be required to further refine our understanding of the underlying mechanism and order of establishment of chromatin marks and the impact of sexually dimorphic epigenetic patterning on transcription and other nuclear processes in germ cells, the early embryo and beyond.

Interplay between chromatin marks in development and disease.

DNA methylation (DNAme) and histone post-translational modifications (PTMs) have important roles in transcriptional regulation. Although many reports have characterized the functions of such chromatin marks in isolation, recent genome-wide studies reveal surprisingly complex interactions between them. Here, we focus on the interplay between DNAme and methylation of specific lysine residues on the histone H3 tail. We describe the impact of genetic perturbation of the relevant methyltransferases in the mouse on the landscape of chromatin marks as well as the transcriptome. In addition, we discuss the specific neurodevelopmental growth syndromes and cancers resulting from pathogenic mutations in the human orthologues of these genes. Integrating these observations underscores the fundamental importance of crosstalk between DNA and histone H3 methylation in development and disease.

Factor Structure for the Sport Concussion Assessment Tool Symptom Scale in Adolescents After Concussion.

OBJECTIVE: To examine the factor structure of the Sport Concussion Assessment Tool-5 (SCAT5) symptom scale in adolescents on their initial presentation to a concussion clinic within the typical recovery period after concussion (ie, <30 days). We hypothesize that the SCAT5 symptoms represent various clinically meaningful groups. A secondary purpose was to examine the effects of sex on the factor structure of the SCAT5 symptom scale. STUDY DESIGN: Retrospective cross-sectional analysis. SETTING: Tertiary, institutional. PATIENTS: Nine hundred eighty-one adolescents (45% women) aged between 13 and 18 years. INDEPENDENT VARIABLES: Adolescents completed the SCAT5 symptom scale. MAIN OUTCOME MEASURES: The factor structure of SCAT5 examined using a principal axis factor analysis. RESULTS: A 5-factor structure model explained 61% of the variance in symptoms. These 5 factors are identified as Energy (17%), Mental Health (13%), Migrainous (13%), Cognitive (9%), and Vestibulo-Ocular (9%). A similar 5-factor model emerged for each sex, and the proportion of variance in symptoms explained by the 5-factor model was comparable between the sexes. CONCLUSIONS: The findings of this report indicate that the SCAT5 symptoms aggregated into 5 delineated factors, and these factors were largely consistent across the sexes. The delineation of symptoms into 5 factors provides preliminary validation for the presence of different concussion phenotypes. Confirmatory factor analysis is warranted to examine the applicability and clinical utility of the use of the 5-factor structure in a clinical setting.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Does Exercise Increase Vestibular and Ocular Motor Symptom Detection After Sport-Related Concussion?

BACKGROUND AND PURPOSE: Postconcussive vestibular and ocular motor symptoms are common and contribute to longer recovery. The Vestibular/Ocular Motor Screening (VOMS) is used to detect such symptoms, but a VOMS performed at rest may miss symptoms that are only provoked by exertion. Supervised exercise challenges (SECs) have been shown to detect concussion-related symptoms provoked by physical exertion. The purpose of this study was to determine whether athletes undergoing an SEC will exhibit greater symptom provocation with the VOMS compared to a VOMS performed at rest prior to an SEC. METHODS: Thirty-six athletes (58.3% male) between ages 10 and 18 years and within 30 days of concussion were included. All participants completed VOMS assessments at rest and immediately after an SEC. VOMS total symptom score increases were calculated for both pre- and post-SEC assessments compared using Wilcoxon ranked sum tests. The frequencies of positive assessments for each VOMS item were compared using McNemar's test. RESULTS: There were significant increases in post-SEC symptom provocation scores compared with pre-SEC scores for all VOMS items. The post-SEC VOMS identified 29 participants (80.6%) as positive in at least 1 VOMS item compared with 21 participants (58.3%) identified as positive pre-SEC (P = 0.008). For all VOMS items, the post-SEC VOMS identified participants who were previously negative on a pre-SEC VOMS but became positive after the SEC. DISCUSSION AND CONCLUSIONS: An SEC performed prior to a VOMS assessment may increase the detection of vestibular and ocular motor symptoms that may be missed if the VOMS was performed only at rest.Video Abstract available for more insight from the authors (see the Video Supplemental Digital Content 1, available at: http://links.lww.com/JNPT/A342).

Do male and female adolescents report symptoms differently after concussion?

We investigated sex-based differences in the presence and severity of aggregated symptom indicators as well as individual concussion symptoms.Materials and Methods: A cross-sectional examination of sex differences in symptoms reported by adolescents upon initial concussion evaluation at a concussion clinic. Nine hundred and eighty-six adolescents completed the Sport Concussion Assessment Tool (SCAT5) symptom checklist. Chi-square and Mann-Whitney U tests were used for differences in the presence and severity of symptoms, respectively. Sex differences in global indices of symptom distress were compared.Results: Females endorsed more symptoms (Female: Median (M)=15, Interquartile range (IQR):9-18 vs. Male: M=11, IQR: 6-15, p<0.001) and a greater total symptom score (Female: M=37, IQR:16-45 vs. Male: M=20, IQR:8-39). After False Discovery Rate (FDR) adjustment, females endorsed the presence of 21 of 22 individual symptoms more frequently than males (p≤ 0.039), with greater symptom severity for 20 of 22 individual symptoms (p≤0.036). Moderate ESs were observed for sex-based differences in the total symptom score and the global severity index. Small ESs was observed for differences in most individual symptoms.Conclusions: The greater frequency and severity of concussion symptoms reported by female adolescents highlights the importance of considering sex as a modifier for the management of concussion.

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) diet alters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.

Transcription shapes genome-wide histone acetylation patterns.

Histone acetylation is a ubiquitous hallmark of transcription, but whether the link between histone acetylation and transcription is causal or consequential has not been addressed. Using immunoblot and chromatin immunoprecipitation-sequencing in S. cerevisiae, here we show that the majority of histone acetylation is dependent on transcription. This dependency is partially explained by the requirement of RNA polymerase II (RNAPII) for the interaction of H4 histone acetyltransferases (HATs) with gene bodies. Our data also confirms the targeting of HATs by transcription activators, but interestingly, promoter-bound HATs are unable to acetylate histones in the absence of transcription. Indeed, HAT occupancy alone poorly predicts histone acetylation genome-wide, suggesting that HAT activity is regulated post-recruitment. Consistent with this, we show that histone acetylation increases at nucleosomes predicted to stall RNAPII, supporting the hypothesis that this modification is dependent on nucleosome disruption during transcription. Collectively, these data show that histone acetylation is a consequence of RNAPII promoting both the recruitment and activity of histone acetyltransferases.

Symptom Provocation During Aerobic and Dynamic Supervised Exercise Challenges in Adolescents With Sport-Related Concussion.

CONTEXT: Supervised exercise challenges (SECs) have been shown to be safe and beneficial in the early symptomatic period after concussion. Thus far, most in-clinic SECs studied have included a form of basic aerobic exercise only. An SEC that also includes dynamic forms of exercise mimics all steps of a standard return-to-play progression and may enhance the detection of concussion symptoms to guide in-clinic management decisions. OBJECTIVE: To determine whether an SEC that includes a dynamic SEC (DSEC) uncovered symptoms that would not have been identified by an SEC involving an aerobic SEC (ASEC) alone in adolescent patients with sport-related concussion. DESIGN: Retrospective case series. SETTING: Multidisciplinary sport concussion clinic at a tertiary care center. PATIENTS OR OTHER PARTICIPANTS: A total of 65 adolescent athletes (mean age = 14.9 ± 2.0 years, 72.3% males) who underwent an in-clinic SEC within 30 days of concussion. MAIN OUTCOME MEASURE(S): Presence of pre-exercise symptoms and symptom provocation during the SEC were recorded, with exercise-provoked symptoms categorized as occurring during ASEC or DSEC. RESULTS: Of the total patient sample, 69.2% (n = 45/65) experienced symptom provocation at some point during the SEC. Symptoms were provoked in 20 patients during the ASEC, whereas 25 completed the ASEC without symptom provocation before becoming symptomatic during the subsequent DSEC and 20 completed the SEC without any symptom provocation. Of the 65 patients in the total sample, 46 were asymptomatic immediately before the SEC. Of these previously asymptomatic patients, 23.9% (n = 11/46) experienced symptom provocation during the ASEC, and an additional 37.0% (n = 17/46) remained asymptomatic during the ASEC but then developed symptoms during the DSEC. CONCLUSIONS: The ASEC alone may not detect symptom provocation in a significant proportion of concussion patients who otherwise would develop symptoms during a DSEC.

Use of Supervised Exercise During Recovery Following Sports-Related Concussion.

OBJECTIVE: To assess the safety of supervised exercise (SE) in acute sport-related concussion (SRC) and its influence on recovery. DESIGN: Retrospective cohort study. SETTING: University SRC clinic at a tertiary care center. PATIENTS: One hundred ninety-four consecutive new patient charts were reviewed. Patients were included if they were seen within 30 days of sustaining a SRC, and their medical records included all required data elements. One hundred twenty-six patients were included in the analysis. INTERVENTIONS: Symptomatic patients who initiated SE within 16 days of SRC (n = 24) were compared with those who did not undergo SE or initiated SE after postinjury day 16 (n = 84). Age, sex, history of previous concussions, injury severity, relevant comorbidities, and other treatments received were included in the analysis. MAIN OUTCOME MEASURES: The association between early SE and clearance for return to sport was determined using a hazard ratio (HR). The number of days from SRC until clearance for return to sport and the number of days symptomatic from concussion were also compared between early SE and nonearly SE cohorts. RESULTS: No serious adverse events occurred in the early SE group. Early SE was associated with earlier return to sport (HR = 2.35, P = 0.030). The early SE group had fewer days from SRC until clearance for return to sport (mean 26.5 ± 11.2 days vs 35.1 ± 26.5 days, P = 0.020). There was a trend toward fewer symptomatic days in the early SE group (P = 0.054). CONCLUSION: Early SE performed in the symptomatic stage of SRC was safe and associated with earlier return to sport.

Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo.

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

NSD1-deposited H3K36me2 directs de novo methylation in the mouse male germline and counteracts Polycomb-associated silencing.

De novo DNA methylation (DNAme) in mammalian germ cells is dependent on DNMT3A and DNMT3L. However, oocytes and spermatozoa show distinct patterns of DNAme. In mouse oocytes, de novo DNAme requires the lysine methyltransferase (KMTase) SETD2, which deposits H3K36me3. We show here that SETD2 is dispensable for de novo DNAme in the male germline. Instead, the lysine methyltransferase NSD1, which broadly deposits H3K36me2 in euchromatic regions, plays a critical role in de novo DNAme in prospermatogonia, including at imprinted genes. However, males deficient in germline NSD1 show a more severe defect in spermatogenesis than Dnmt3l-/- males. Notably, unlike DNMT3L, NSD1 safeguards a subset of genes against H3K27me3-associated transcriptional silencing. In contrast, H3K36me2 in oocytes is predominantly dependent on SETD2 and coincides with H3K36me3. Furthermore, females with NSD1-deficient oocytes are fertile. Thus, the sexually dimorphic pattern of DNAme in mature mouse gametes is orchestrated by distinct profiles of H3K36 methylation.

Changes in Vestibular/Ocular-Motor Screen Scores in Adolescents Treated With Vestibular Therapy After Concussion.

PURPOSE: To examine interrelationships among Vestibular/Ocular-Motor Screen (VOMS) items and to characterize the recovery of VOMS performance in a sample of adolescents treated with vestibular physical therapy (VPT) after concussion. METHODS: Seventy-seven patients with concussion and 77 participants without concussion completed the study. Adolescents with concussion received an individualized VPT intervention consisting of targeted exercises for gaze stability, postural stability, ocular-motor control, habituation, and aerobic activities. The exercises were performed during a weekly clinic visit and via a home exercise program. RESULTS: Except for near-point convergence distance, all VOMS items were significantly interrelated. Over the course of VPT, significant improvements in VOMS performance were observed, and discharge scores were similar to scores observed in adolescents without concussion. CONCLUSIONS: The VOMS measured moderately related functions and captured changes over the course of VPT. Clinicians should consider the contextual risk of "false positive" in their interpretation of VOMS.

Inter-Strain Epigenomic Profiling Reveals a Candidate IAP Master Copy in C3H Mice.

Insertions of endogenous retroviruses cause a significant fraction of mutations in inbred mice but not all strains are equally susceptible. Notably, most new Intracisternal A particle (IAP) ERV mutagenic insertions have occurred in C3H mice. We show here that strain-specific insertional polymorphic IAPs accumulate faster in C3H/HeJ mice, relative to other sequenced strains, and that IAP transcript levels are higher in C3H/HeJ embryonic stem (ES) cells compared to other ES cells. To investigate the mechanism for high IAP activity in C3H mice, we identified 61 IAP copies in C3H/HeJ ES cells enriched with H3K4me3 (a mark of active promoters) and, among those tested, all are unmethylated in C3H/HeJ ES cells. Notably, 13 of the 61 are specific to C3H/HeJ and are members of the non-autonomous 1Δ1 IAP subfamily that is responsible for nearly all new insertions in C3H. One copy is full length with intact open reading frames and hence potentially capable of providing proteins in trans to other 1Δ1 elements. This potential "master copy" is present in other strains, including 129, but its 5' long terminal repeat (LTR) is methylated in 129 ES cells. Thus, the unusual IAP activity in C3H may be due to reduced epigenetic repression coupled with the presence of a master copy.