Descriptive registry study on outcome and complications of external ventricular drainage treatment of intraventricular haemorrhage in a Danish cohort: a study protocol.

INTRODUCTION: Intraventricular haemorrhage (IVH) is associated with high morbidity and mortality. External ventricular drainage (EVD) has been shown to decrease mortality. Although EVD is widely used, outcome and complication rates in EVD-treated patients with IVH are not fully elucidated. This study aims to describe EVD complication rates and outcomes in patients with primary and secondary IVH at two university hospitals in Denmark. The study will provide a historical reference of relevant endpoints for use in future clinical trials involving patients with IVH. METHODS AND ANALYSIS: This descriptive, multicentre registry study included adult patients (age 18+) with primary or secondary IVH and treated with at least one EVD between 2017 and 2021 at Aarhus University Hospital or Odense University Hospital. Patients are identified using the Danish National Patient Register. Data are collected and recorded from patient medical records. Relevant descriptive statistics and correlation analyses will be applied. ETHICS AND DISSEMINATION: Ethical approval and authorisation to access, store and analyse data have been obtained (Central Denmark Region Committee on Health Research Ethics). The research lead will present the results of the study. Data will be reported according to the Strengthening the Reporting of Observational Studies in Epidemiology and results submitted for publication in peer-reviewed journals.

Effect of nationwide school policy on device-measured physical activity in Danish children and adolescents: a natural experiment.

Background: A new Danish school policy with a requirement for 45 min physical activity daily during school hours was introduced in 2014. The objective of this natural experiment was to evaluate the effect of this nationwide school policy on physical activity in Danish children and adolescents. Methods: Four historical studies completed between 2009 and 2012 comprised the pre-policy study population. Post-policy data were collected in 2017/18. All post-policy schools were represented in the four pre-policy studies. Age-groups and seasons were matched. In total, 4816 children and adolescents aged 6-17 were included in the analyses (2346 pre-policy and 2470 post-policy). Children and adolescents were eligible if they had accelerometer measurements and did not have any physical disabilities preventing activity. Physical activity was measured by accelerometry. Main outcome was any bodily movement. Secondary outcomes were moderate to vigorous physical activity and overall movement volume (mean counts per minute). Findings: The school policy interrupted a linear decreasing pre-policy trend in physical activity during school hours. All activity outcomes increased post-policy during a standardized school day (8:10 am-1 pm). Increases were more pronounced in the youngest children. Specifically, we observed a daily increase during a standardized school day in 2017/2018 of 14.2 min of movement (95% CI: 11.4-17.0, p < 0.001), 6.5 min of moderate to vigorous physical activity (95% CI: 4.7-8.3, P < 0.001), and 141.8 counts per minute (95% CI: 108.5-175.2, P < 0.001). Interpretation: A national school policy may be an important strategy to increase physical activity during school hours among children and adolescents. Funding: The Danish Foundation TrygFonden has funded the PHASAR project (ID 115606).

Treatment of Intraventricular Hemorrhage with External Ventricular Drainage and Fibrinolysis: A Comprehensive Systematic Review and Meta-Analysis of Complications and Outcome.

BACKGROUND: External ventricular drainage (EVD) is a key factor in the treatment of intraventricular hemorrhage (IVH) but associated with risks and complications. Intraventricular fibrinolysis (IVF) has been proposed to improve clinical outcome and reduce complications of EVD treatment. The following review and metaanalysis provides a comprehensive evaluation of IVH treatment with external ventricular drainage (EVD) and intraventricular fibrinolysis (IVF) with regards to complications and clinical outcomes. METHODS: The PRISMA guidelines were followed preparing this review. Studies included in the meta-analysis were compared using forest plots and the related odds ratios. RESULTS: After a literature search, 980 articles were identified and 65 and underwent full-text review. Forty-two articles were included in the review and meta-analysis. We found that bolted and antibiotic-coated catheters were superior to tunnelled/uncoated catheters (P < 0.001) and antibiotic- vs. silver-impregnated catheters (P < 0.001]) in preventing infection. Shunt dependency was related to the volume of blood in the ventricles but unaffected by IVF (P = 0.98). IVF promoted hematoma clearance, decreased mortality (22.4% vs. 40.9% with IVF vs. no IVF, respectively, P < 0.00001), improved good functional outcomes (47.2% [IVF] vs. 38.3% [no IVF], P = 0.03), and reduced the rate of catheter occlusion from 37.3% without IVF to 10.6% with IVF (P = 0.0003). CONCLUSIONS: We present evidence and best practice recommendations for the treatment of IVH with EVD and intraventricular fibrinolysis. Our analysis further provides a comprehensive quantitative reference of the most relevant clinical endpoints for future studies on novel IVH technologies and treatments.

Integrative analysis of clinical and epigenetic biomarkers of mortality.

DNA methylation (DNAm) has been reported to be associated with many diseases and with mortality. We hypothesized that the integration of DNAm with clinical risk factors would improve mortality prediction. We performed an epigenome-wide association study of whole blood DNAm in relation to mortality in 15 cohorts (n = 15,013). During a mean follow-up of 10 years, there were 4314 deaths from all causes including 1235 cardiovascular disease (CVD) deaths and 868 cancer deaths. Ancestry-stratified meta-analysis of all-cause mortality identified 163 CpGs in European ancestry (EA) and 17 in African ancestry (AA) participants at p < 1 × 10-7 , of which 41 (EA) and 16 (AA) were also associated with CVD death, and 15 (EA) and 9 (AA) with cancer death. We built DNAm-based prediction models for all-cause mortality that predicted mortality risk after adjusting for clinical risk factors. The mortality prediction model trained by integrating DNAm with clinical risk factors showed an improvement in prediction of cancer death with 5% increase in the C-index in a replication cohort, compared with the model including clinical risk factors alone. Mendelian randomization identified 15 putatively causal CpGs in relation to longevity, CVD, or cancer risk. For example, cg06885782 (in KCNQ4) was positively associated with risk for prostate cancer (Beta = 1.2, PMR  = 4.1 × 10-4 ) and negatively associated with longevity (Beta = -1.9, PMR  = 0.02). Pathway analysis revealed that genes associated with mortality-related CpGs are enriched for immune- and cancer-related pathways. We identified replicable DNAm signatures of mortality and demonstrated the potential utility of CpGs as informative biomarkers for prediction of mortality risk.

Sample size determination in method comparison and observer variability studies.

The comparison of two quantitative measuring devices is often performed with the Limits of Agreement proposed by Bland and Altman in their seminal Lancet paper back in 1986. Sample size considerations were rare for such agreement analyses in the past, but recently several proposals have been made depending on how agreement is to be assessed and the number of replicates to be used. We have summarized recent developments and recommendations in various situations including a distinction between method comparison and observer variability studies. These include current state-of-the-art analysis of and reporting guidelines for agreement studies. General recommendations close the paper.

Postoperative drainage for 6, 12, or 24 h after burr-hole evacuation of chronic subdural hematoma in symptomatic patients (DRAIN-TIME 2): study protocol for a nationwide randomized controlled trial.

BACKGROUND: Chronic subdural hematoma (CSDH) is a common acute or subacute neurosurgical condition, typically treated by burr-hole evacuation and drainage. Recurrent CSDH occurs in 5-20% of cases and requires reoperation in symptomatic patients, sometimes repeatedly. Postoperative subdural drainage of maximal 48 h is effective in reducing recurrent hematomas. However, the shortest possible drainage time without increasing the recurrence rate is unknown. METHODS: DRAIN-TIME 2 is a Danish multi-center, randomized controlled trial of postoperative drainage time including all four neurosurgical departments in Denmark. Both incapacitated and mentally competent patients are enrolled. Patients older than 18 years, free of other intracranial pathologies or history of previous brain surgery, are recruited at the time of admission or no later than 6 h after surgery. Each patient is randomized to either 6, 12, or 24 h of passive subdural drainage following single burr-hole evacuation of a CSDH. Mentally competent patients are asked to complete the SF-36 questionnaire. The primary endpoint is CSDH recurrence rate at 90 days. Secondary outcome measures include SF-36 at 90 days, length of hospital stay, drain-related complications, and complications related to immobilization and mortality. DISCUSSION: This multi-center trial will provide evidence regarding the shortest possible drainage time without increasing the recurrence rate. The potential impact of this study is significant as we believe that a shorter drainage period may be associated with fewer drain-related complications, fewer complications related to immobilization, and shorter hospital stays-thus reducing the overall health service burden from this condition. The expected benefits for patients' lives and health costs will increase as the CSDH patient population grows. TRIAL REGISTRATION: ISRCTN Registry ISRCTN15186366 . Registered in December 2020 and updated in October 2021. This protocol was developed in accordance with the SPIRIT Checklist and by use of the structured study protocol template provided by BMC Trials.

Remote ischemic postconditioning has a detrimental effect and remote ischemic preconditioning seems to have no effect on small intestinal anastomotic strength.

BACKGROUND: The effect of remote pre- and postconditioning on anastomotic healing has been sparsely studied. The aim of our study was to investigate whether remote ischemic conditioning (RIC) applied before and after the creation of a small bowel anastomosis had an effect on anastomotic healing on postoperative day five evaluated by a tensile strength test and histological analysis. MATERIALS AND METHODS: Twenty-two female piglets were randomized into two groups. The intervention group (n = 12) received RIC on the forelimbs consisting of 15 min of ischemia followed by 30 min of reperfusion before the first end-to-end ileal anastomosis was created. The RIC procedure was repeated and the second and more distal anastomosis was performed. The control group (n = 10) had two similar anastomoses with similar time intervals but without RIC. On postoperative day five, the anastomoses were subjected to macroscopic evaluation, tensile strength test and histological examination. RESULTS: Mean tensile strength when the first transmural rupture appeared (MATS-2) was significantly lower in the first anastomosis in the intervention group compared to the control group (11.4 N vs 14.7 N, p < .05). Similar result was found by the maximal strength (MATS-3) as defined by a drop in the load curve (12.3 N vs 15.9 N, p < .05). Histologically, a significantly higher necrosis score was found in the anastomosis in the intervention group (1.4 vs 0.8, p < .05). No other significant differences were found. CONCLUSIONS: In conclusion, post-anastomotic remote ischemic conditioning had a detrimental effect and pre-anastomotic conditioning seems to have no effect on small intestinal anastomotic strength.

Hepatitis C time trends in reported cases and estimates of the hidden population born before 1965, Denmark and Sweden, 1990 to 2020.

BackgroundAccording to the World Health Organization, hepatitis C virus (HCV) infection should be under control by 2030.AimOur aim was to describe the size and temporal changes in reported cases of chronic HCV infection in Denmark and Sweden and to estimate the size of the hidden (undiagnosed) population born before 1965.MethodsWe extracted all HCV infections reported to national surveillance systems in Denmark and Sweden from 1990 to 2020. Prediction of the size of the hidden HCV-infected population was restricted to the cohort born before 1965 and cases reported up to 2017. We applied a model based on removal sampling from binomial distributions, estimated the yearly probability of diagnosis, and deducted the original HCV-infected population size.ResultsDenmark (clinician-based) reported 10 times fewer hepatitis C cases annually than Sweden (laboratory and clinician-based), peaking in 2007 (n = 425) and 1992 (n = 4,537), respectively. In Denmark, the birth year distribution was monophasic with little change over time. In recent years, Sweden has had a bimodal birth year distribution, suggesting ongoing infection in the young population. In 2017, the total HCV-infected population born before 1965 was estimated at 10,737 living persons (95% confidence interval (CI): 9,744-11,806), including 5,054 undiagnosed, in Denmark and 16,124 (95% CI: 13,639-18,978), including 10,580 undiagnosed, in Sweden.ConclusionsThe reporting of HCV cases in Denmark and Sweden was different. For Denmark, the estimated hidden population was larger than the current national estimate, whereas in Sweden the estimate was in line with the latest published numbers.

National randomized clinical trial on subdural drainage time after chronic subdural hematoma evacuation.

OBJECTIVE: Placement of a subdural drain reduces recurrence and death after evacuation of chronic subdural hematoma (CSDH), but little is known about optimal drainage duration. In the present national trial, the authors investigated the effect of drainage duration on recurrence and death. METHODS: In a randomized controlled trial involving all neurosurgical departments in Denmark, patients treated with single burr hole evacuation of CSDH were randomly assigned to 24 hours or 48 hours of postoperative passive subdural drainage. Follow-up duration was 90 days, and the primary study outcome was recurrent hematoma requiring reoperation. Secondary outcome was death. In addition, complications and length of hospital stay were recorded and analyzed. RESULTS: Of the 420 included patients, 212 were assigned 24-hour drainage and 208 were assigned 48-hour drainage. The recurrence rate was 14% in the 24-hour group and 13% in the 48-hour group. Four patients died in the 24-hour group, and 8 patients died in the 48-hour group; this difference was not statistically significant. The ORs (95% CIs) for recurrence and mortality (48 hours vs 24 hours) were 0.94 (0.53-1.66) and 2.07 (0.64-7.85), respectively, in the intention-to-treat analysis. The ORs (95% CIs) for recurrence and mortality per 1-hour increase in drainage time were 1.0005 (0.9770-1.0244) and 1.0046 (0.9564-1.0554), respectively, in the as-treated sensitivity analysis that used the observed drainage times instead of the preassigned treatment groups. The rates of surgical and drain-related complications, postoperative infections, and thromboembolic events were not different between groups. The mean ± SD postoperative length of hospital stay was 7.4 ± 4.3 days for patients who received 24-hour drainage versus 8.4 ± 4.9 days for those who received 48-hour drainage (p = 0.14). The mean ± SD postoperative length of stay in the neurosurgical department was significantly shorter for the 24-hour group (2 ± 0.9 days vs 2.8 ± 1.6 days, p < 0.001). CONCLUSIONS: No significant differences in the rates of recurrent hematoma or death during 90-day follow-up were identified between the two groups that randomly received either 24- or 48-hour passive subdural drainage after burr hole evacuation of CSDH.

Genetic meta-analysis of twin birth weight shows high genetic correlation with singleton birth weight.

Birth weight (BW) is an important predictor of newborn survival and health and has associations with many adult health outcomes, including cardiometabolic disorders, autoimmune diseases and mental health. On average, twins have a lower BW than singletons as a result of a different pattern of fetal growth and shorter gestational duration. Therefore, investigations into the genetics of BW often exclude data from twins, leading to a reduction in sample size and remaining ambiguities concerning the genetic contribution to BW in twins. In this study, we carried out a genome-wide association meta-analysis of BW in 42 212 twin individuals and found a positive correlation of beta values (Pearson's r = 0.66, 95% confidence interval [CI]: 0.47-0.77) with 150 previously reported genome-wide significant variants for singleton BW. We identified strong positive genetic correlations between BW in twins and numerous anthropometric traits, most notably with BW in singletons (genetic correlation [rg] = 0.92, 95% CI: 0.66-1.18). Genetic correlations of BW in twins with a series of health-related traits closely resembled those previously observed for BW in singletons. Polygenic scores constructed from a genome-wide association study on BW in the UK Biobank demonstrated strong predictive power in a target sample of Dutch twins and singletons. Together, our results indicate that a similar genetic architecture underlies BW in twins and singletons and that future genome-wide studies might benefit from including data from large twin registers.

Does diabetes modify the effect of heparin on plasma proteins? - A proteomic search for plasma protein biomarkers for diabetes-related endothelial dysfunction.

AIM: Heparin administration affects the concentrations of many plasma proteins through their displacement from the endothelial glycocalyx. A differentiated protein response in diabetes will therefore, at least partly, reflect glycocalyx changes. This study aims at identifying biomarkers of endothelial dysfunction in diabetes by statistical exploration of plasma proteome data for interactions between diabetes status and heparin treatment. METHODS: Diabetes-by-heparin interactions in relation to protein levels were inspected by regression modelling in plasma proteome data from 497 patients admitted for acute angiography. Analyses were conducted separately for all 273 proteins and as set-based analyses of 44 heparin-relevant proteins identified by gene ontology analysis and 42 heparin-influenced proteins previously reported. RESULTS: Seventy-five patients had diabetes and 361 received heparin before hospitalization. The proteome-wide analysis displayed no proteins with diabetes-heparin interaction to pass correction for multiple testing. The overall set-based analyses revealed significant association for both protein sets (p-values<2*10-4), while constraining on opposite directions of effect in diabetics and none-diabetics was insignificant (p-values = 0.11 and 0.17). CONCLUSIONS: Our plasma proteome-wide interaction approach supports that diabetes influences heparin effects on protein levels, however the direction of effects and individual proteins could not be definitively pinpointed, likely reflecting a complex protein-basis for glycocalyx dysfunction in diabetes.

Alemtuzumab treatment in Denmark: A national study based on the Danish Multiple Sclerosis Registry.

OBJECTIVE: To investigate clinical outcomes in a real-world setting in the complete population-based cohort of alemtuzumab-treated MS patients in Denmark. METHODS: Data were retrieved from The Danish Multiple Sclerosis Registry between 2009 and 2019. Demographic and disease-specific patient parameters related to treatment history, efficacy, and safety outcomes were assessed at baseline and during follow-up visits. RESULTS: A total of 209 patients (78% female) started treatment with alemtuzumab during the study period with 3.1 ± 1.4 years follow-up. After 2 years, 75% of patients were relapse-free compared to 48% the year before alemtuzumab (p < 0.001). The annual number of relapses was reduced by 69% in year 4 compared with the year prior alemtuzumab. More active disease before alemtuzumab increased the annual hazard rate for relapse (HR: 2.88, p < 0.001). The Expanded Disability Status Scale (EDSS) score remained stable or improved in 81% of patients after 2 years. The need for an additional treatment course was associated with higher number of relapses in the year before alemtuzumab (odds ratio (OR) = 1.95, p = 0.001). CONCLUSION: In a country with primarily escalation strategy, relapse rate reduction was maintained for 5 years, and EDSS stabilized/improved in majority of patients. Higher relapse rate 1 year before alemtuzumab increased the odds for additional courses. Novel serious AEs were not observed.

An Extension of the Bland-Altman Plot for Analyzing the Agreement of More than Two Raters.

The Bland-Altman plot is the most common method to analyze and visualize agreement between raters or methods of quantitative outcomes in health research. While very useful for studies with two raters, a limitation of the classical Bland-Altman plot is that it is specifically used for studies with two raters. We propose an extension of the Bland-Altman plot suitable for more than two raters and derive the approximate limits of agreement with 95% confidence intervals. We validated the suggested limit of agreement by a simulation study. Moreover, we offer suggestions on how to present bias, heterogeneity among raters, as well as the uncertainty of the limits of agreement. The resulting plot could be utilized to investigate and present agreement in studies with more than two raters.

STEMI, Cardiogenic Shock, and Mortality in Patients Admitted for Acute Angiography: Associations and Predictions from Plasma Proteome Data.

AIM: Acute myocardial infarction (AMI) remains a major cause of mortality and morbidity, and cardiogenic shock (CS) a major cause of hospital mortality after AMI. Especially for ST elevation myocardial infarction (STEMI) patients, fast intervention is essential.Few proteins have proven clinically applicable for AMI. Most proposed biomarkers are based on a priori hypothesis-driven studies of single proteins, not enabling identification of novel candidates. For clinical use, the ability to predict AMI is important; however, studies of proteins in prediction models are surprisingly scarce.Consequently, we applied proteome data for identifying proteins associated with definitive STEMI, CS, and all-cause mortality after admission, and examined the ability of the proteins to predict these outcomes. METHODS AND RESULTS: Proteome-wide data of 497 patients with suspected STEMI were investigated; 381 patients were diagnosed with STEMI, 35 with CS, and 51 died during the first year. Data analysis was conducted by logistic and Cox regression modeling for association analysis, and by multivariable LASSO regression models for prediction modeling.Association studies identified 4 and 29 proteins associated with definitive STEMI or mortality, respectively. Prediction models for CS and mortality (holding two and five proteins, respectively) improved the prediction ability as compared with protein-free prediction models; AUC of 0.92 and 0.89, respectively. CONCLUSION: The association analyses propose individual proteins as putative protein biomarkers for definitive STEMI and survival after suspected STEMI, while the prediction models put forward sets of proteins with putative predicting ability of CS and survival. These proteins may be verified as biomarkers of potential clinical relevance.

Gonadal sex and animal experimentation: Perfection vs. 3R principle?

Replicability of experimental results and optimal use of experimental animals are everybody's concern. Current efforts towards increased replicability include guidelines and checklists as tools for experimenters, referees, editors and publishers. Guidelines are also provided for appropriate use of animals. To ensure the quality of experimental results, the number of animals must be adequate, that is, sufficiently large, for the purpose of the given experiment. To comply with current ethical recommendations, the use of animals should be reduced as much as possible. Therefore, determination of the number of animals for a given scientific objective includes contrasting considerations. Current guidelines for animal experimentation, notably from the National Institute of Health, mandate (with very few exceptions) inclusion of animals of both sexes in experimental designs statistically powered to address the difference between the two groups. Notably, absence of evidence for sex differences between the organ or system functions under study does not qualify as an exception. Mandatory, equal representation of both sexes raises several questions including ethical ones. Other guidelines, by public regulators and major publishers, do not seem to have a similar selective focus on sex differences. In summary, current concerns about replicability of scientific results are justified. Concomitantly, the knowledge of sex differences also between non-reproductive, non-endocrine organ functions is increasing. In principle, sex matters in any experimental context. However, an indiscriminate demand for inclusion of both sexes in all experimental protocols seems a waste of animals, money and time, violating traditional principles of animal experimentation, particularly that of reduction.

Embolization of the middle meningeal artery in patients with chronic subdural hematoma-a systematic review and meta-analysis.

BACKGROUND: Chronic subdural hematoma (CSDH) remains a neurosurgical condition with high recurrence rate after surgical treatment. The primary pathological mechanism is considered to be repeated microbleedings from fragile neo-vessels within the outer hematoma membrane. The neo-vessels are supplied from peripheral branches of the middle meningeal artery, and embolization of MMA (eMMA) has been performed to prevent re-bleeding episodes and thereby CSDH recurrence. OBJECTIVE: To evaluate the published evidence for the effect of eMMA in patients with recurrent CSDH. Secondarily, to investigate the effect of eMMA as an alternative to surgery for primary treatment of CSDH. METHOD: A systematic review of the literature on eMMA in patients with recurrent CSDH was conducted. PubMed, Embase, and Cochrane databases were reviewed using the search terms: Embolization, Medial Meningeal Artery, Chronic Subdural Haematoma, and Recurrence. Furthermore, the following mesh terms were used: Chronic Subdural Haematoma AND embolization AND medial meningeal artery AND recurrence. Eighteen papers were found and included. No papers were excluded. The number of patients with primary CSDH and the number of patients with recurrent CSDH treated with eMMA were listed. Furthermore, the number of recurrences in both categories was registered. RESULTS: Eighteen papers with a total of 191 included patients diagnosed with CSDH treated with eMMA for primary and recurrent CSDH were identified. Recurrence rate for patients treated with eMMA for recurrent CSDH was found to be 2.4%, 95% CI (0.5%; 11.0%), whereas the recurrence rate for patients treated with eMMA for primary CSDH was 4.1%, 95% CI (1.4%; 11.4%). CONCLUSION: eMMA is a minimally invasive procedure for treatment of CSDH. Although this study is limited by publication bias, it seems that this procedure may reduce recurrence rates compared with burr hole craniostomy for both primary and recurrent hematomas. A controlled study is warranted.

A meta-analysis of genome-wide association studies identifies multiple longevity genes.

Human longevity is heritable, but genome-wide association (GWA) studies have had limited success. Here, we perform two meta-analyses of GWA studies of a rigorous longevity phenotype definition including 11,262/3484 cases surviving at or beyond the age corresponding to the 90th/99th survival percentile, respectively, and 25,483 controls whose age at death or at last contact was at or below the age corresponding to the 60th survival percentile. Consistent with previous reports, rs429358 (apolipoprotein E (ApoE) ε4) is associated with lower odds of surviving to the 90th and 99th percentile age, while rs7412 (ApoE ε2) shows the opposite. Moreover, rs7676745, located near GPR78, associates with lower odds of surviving to the 90th percentile age. Gene-level association analysis reveals a role for tissue-specific expression of multiple genes in longevity. Finally, genetic correlation of the longevity GWA results with that of several disease-related phenotypes points to a shared genetic architecture between health and longevity.

Epigenome-wide exploratory study of monozygotic twins suggests differentially methylated regions to associate with hand grip strength.

Hand grip strength is a measure of muscular strength and is used to study age-related loss of physical capacity. In order to explore the biological mechanisms that influence hand grip strength variation, an epigenome-wide association study (EWAS) of hand grip strength in 672 middle-aged and elderly monozygotic twins (age 55-90 years) was performed, using both individual and twin pair level analyses, the latter controlling the influence of genetic variation. Moreover, as measurements of hand grip strength performed over 8 years were available in the elderly twins (age 73-90 at intake), a longitudinal EWAS was conducted for this subsample. No genome-wide significant CpG sites or pathways were found, however two of the suggestive top CpG sites were mapped to the COL6A1 and CACNA1B genes, known to be related to muscular dysfunction. By investigating genomic regions using the comb-p algorithm, several differentially methylated regions in regulatory domains were identified as significantly associated to hand grip strength, and pathway analyses of these regions revealed significant pathways related to the immune system, autoimmune disorders, including diabetes type 1 and viral myocarditis, as well as negative regulation of cell differentiation. The genes contributing to the immunological pathways were HLA-B, HLA-C, HLA-DMA, HLA-DPB1, MYH10, ERAP1 and IRF8, while the genes implicated in the negative regulation of cell differentiation were IRF8, CEBPD, ID2 and BRCA1. In conclusion, this exploratory study suggests hand grip strength to associate with differentially methylated regions enriched in immunological and cell differentiation pathways, and hence merits further investigations.